Block 1 Flashcards
The Chemical Composition of Nucleic Acids
Basic Components
o The nitrogenous bases
♣ Purines: Adenine and Guanine (each has two aromatic rings)
♣ Pyrimidines: Cytosine, Thymine, Uracil (each has one aromatic ring)
o Ribose or Deoxyribose (the 2’ C is deoxygenated in DNA, has -OH in RNA)
o Phosphate groups
Polynucleotides
o Individual nucleotides are joined to each other at the 3’ and 5’ carbons through a phosphodiester bridge to form the sugar-phosphate backbone of DNA
o Nucleic acid chains have polarity
♣ One end of a chain has a 5’ PO4 group
♣ One end has a 3’ –OH group
♣ By convention, base sequences are written in the 5’ 3’ direction
Right handed, B-form DNA
the most common conformational form of DNA
Major and Minor Grooves:
In the most common form of the double helix (B-DNA), the glycosidic bonds of a base pair are not diametrically opposed to each other, resulting in major (12 angstrom wide) and minor (6 anstrom wide) grooves
♣ The grooves are lined with potential hydrogen bond donor and acceptor atoms
o Many DNA binding proteins bind in the major groove of DNA in a sequence specific manner
Clinical application of DNA structure (grooves)
♣ CLINICAL APPLICATIONS: Certain cancer drugs (actinomycin D) can exert their effects by intercalating into the minor groove to interfere with with RNA and DNA synthesis
• The antibiotic actinomycin D (produced by Streptomyces antibioticus) binds to DNA duplexes, thereby interfering with the action of enzymes engaged in replication and transcription
• Actinomycin D is an anti-cancer drug commonly used in the treatment of pediatric malignancies such as Wilms’ tumour, Ewing’s sarcoma, and rhabdomyosarcoma
Key features of DNA structure
o Two helical polynucleotide chains coiled around a common axis that run in antiparallel directions (resembles a twisted ladder structure)
o Bases are on the inside of the helix and phosphate sugar backbone is on outside
♣ The planes of the bases are “stacked” perpendicular to the helix axis
♣ Stacking resonance (sharing e-) helps stabilize the double helix
o Helical structure repeats after 10.5 residues at intervals of 36 anstroms
o Two chains of helix are held together by hydrogen base pairing
♣ Adenine must pair with thymine (A-T: two bonds) and guanine must pair with cytosine (G-C: three H bonds)
• A-T bonds are weaker than G-C bonds… 2 < 3
3 Forces Holding DNA Together
3 Forces holding DNA together:
- Resonance
- H-Bonding
- Ionic Bonding
Non-B Form DNA
o The classic Watson-Crick B-DNA is an average structure
o In vivo, DNA has subtle but functionally significant deviations from this average structure
Z-DNA
♣ Z-DNA (left-handed, zigzagging alternating purines and pyrimidines, may have a role in gene expression)
• Z-DNA binding proteins required for pathogenesis have been isolated from poxviruses, including variola (agent of smallpox)
♣ Additional conformations: triplexes, cruciforms, slipped structures, etc.
Properties of DNA/RNA: Denaturaton/Renaturation
o The two strands of a double helix separate when hydrogen bonds are disrupted by changes in pH or heating
♣ Ionic composition (salt concentration) of the solution will effect denaturation and annealing rates
♣ Size can play a role, mostly in extremes (super long vs super short)
o Denaturation (melting) and renaturation (annealing) can be monitored spectrophotometrically
♣ DNA melting can be monitored spectrophotometrically at 260 nm dur to a hyperchromic shift that occurs upon base stacking annealed DNA
• Bases absorb UV light better when ss VS ds
Melting Temperature
♣ The melting temperature (Tm) is defined as the temperature at which half (50%) of the helical structure is lost
• Tm depends on % GC content: DNA high in GC content melts at a higher temperature than NA with high AT content
• Tm depends on ionic strength of the solution: high salt favors duplex, low salt favors denatured
o Electrostatic repulsion caused by the charge on the phosphate backbone
Factors Affecting Tm
Inc GC = Inc Tm
Inc salt = duplex
Dec salt = denatured
B-DNA helix is flexible
o It can be locally bent, kinked, or supercoiled
o This flexibility is important for DNA compaction
DNA molecules can be: (conformations)
(1) linear (2) relaxed circular (3) supercoiled circular
o Linear and circular DNA have very different topological properties
o Also, there can be negative and positive supercoils based on direction of coiling
♣ DNA tends to be negatively coiled
Relaxed DNA has _ bp per turn of the helix
Relaxed DNA has 10.5 bp per turn of the helix
Positive supercoiling of DNA
o Positive supercoiling of DNA occurs when the right-handed, double-helical conformation (twisted in a right-handed fashion) until the helix begins to distort and “knot”
Negative supercoiling
o Negative supercoiling involves twisting against the helical conformation (twisting in a left-handed fashion), which preferentially underwinds and “straightens” the helix at low twisting stress, and knots the DNA into negative supercoils at high twisting stress
♣ Negative supercoils favor local unwinding of the DNA, allowing processes such as transcription, replication, and recombination
• DNA in cells is typically somewhat negatively supercoiled
Topoisomerases
enzymes that change the topological state of circular DNA but not its covalent state
Type I topoisomerases
o Type I topoisomerases create transient single-stranded breaks in DNA
♣ They are nicking-closing enzymes and can relax both negatively and positively supercoiled DNA
♣ The enzyme acts by:
• STEP 1: Cleaving one strand of DNA
o Active-site Tyr attacks a phosphodiester bond in one DNA strand, cleaving it and creating a covalent 5’-phosphotyrosyl protein-DNA linkage
• STEP 2: Passing a segment of DNA through the break
o Enzyme changes to an open confirmation and the unbroken DNA strand passes through the break in the first strand
• STEP 3: Resealing the break
o Enzyme in closed conformation; liberated 3’-OH attacks the 5’-phos-photyrosyl protein-DNA linkage to religate the cleaved DNA strand• DO NOT need energy donors (ie, ATP-independent)
• At each step, one high-energy bond replaces another
Type II topoisomerases
o Type II topoisomerases create transient double-stranded breaks in DNA
♣ The enzyme acts by:
• STEP 1: Two strands are cleaved
o The multisubunit enzyme binds a segment of DNA molecule, and a second segment of the same DNA molecule is bound at the N gate
o The second segment of DNA is trapped, and the segment is cleaved on both strands to form two 5’-phosphotyrosyl linkages to the enzyme
• STEP 2: The DNA is passed through the break
• STEP 3: The break is resealed
o The broken DNA is religated, and the second DNA segment is released through the C gate
• DOES require two ATPs to complete a reaction cycle
CLINICAL IMPLICATIONS of Superhelicity
• Topoisomerases are targets for antibiotics
o Coumarins (novobiocin, coumermycin A1)
♣ Inhibit bacterial type II topoisomerases and DNA gyrase from binding ATP; not often used to treat infections in humans
o Quinolones (nalidixic acid; ciproflaoxacin, Cipro)
♣ Inhibit the last step of topo reaction, which is resealing the DNA strand breaks
♣ Wide-spectrum and mostly selective for bacterial enzymes
• Topoisomerase inhibitors used as chemotherapy agents
o Targets cancer because most rapidly growing cells (tumors, others) express topoisomerases
♣ Rapidly replicating cells such as cancer cells have elevated levels of Type II topoisomerases, and are therefore more likely to incur lethal DNA damage through inhibition of Type II topoisomerases than slow growing cells
o Eukaryotic Type I topoisomerase inhibitors
♣ Captothecin, irinotecan (Campto), topotecan (Hycamtin)
♣ Trap the enzyme-DNA complex in its cleaved state
o Eukaryotic Type II topoisomerases inhibitors
♣ Doxorubicin (Adriamycin), etoposide (Etopophos), ellecticine
♣ Can block the binding of ATP
Chromatin
The genomic DNA within the nucleus in complex with its nuclear proteins – the mode by which the genes are complexed in chromatin determines which genes are activated and which are repressed
Heterochromatin:
highly condensensed, usually (not always) transcriptionally inactive (about 10% of the chromatin)
Euchromatin:
the remaining, less condensed chromatin; some, but not all, is transcriptionally active
The Nucleosome:
- The Nucleosome: the fundamental and smallest unit of chromatin packaging
a. There are millions of nucleosomes in each nucleus, each shaped like a rugby ball
i. Using electron microscopy, these can be seen as beads along a string (string being linear DNA)
ii. As in bulk chromosome, each nucleosome is half protein, half DNA
iii. The protein component of each beat consists of an octomer (8) of histone molecules, plus a few non-histone molecules
The Histones
i. Grouped within 5 related protein “families”
a. H1 family: termed “linker” histones because they are located between the nucleosomal beads
b. H2A, H2B, H3, H4: termed “core” histones
i. Two molecules of each core histone make-up each nucleosome; hence each nucleosome contains an octamer (8) of histone molecules
ii. All the histones are highly basic due to the enrichment of lysine+ and arginine+ residues, and are evolutionarily highly conserved, indicating their vital cellular function
Histone Function:
histones function to inhibit transcription, globally repressing the genome
Histone Acetylation
i. In order for transcription to occur, the histones must be modified via acetylation of the lysine+ and arginine+ residues to neutralize the positive charges
1. This results in repulsion of histones from DNA gene sites causing “chromatin opening,” a process that allows entry of transcriptional machinery
Histone Methylation
i. In contrast, methylation of histone H3 causes formation of the non-transcribable ‘hetero’-chromatin by serving as a binding surface for the non-histone protein HP1 (heterochromatin protein 1)
1. HP1 is bound, other proteins (i.e., transcription factors) can’t
Histone Phosphorylation
i. Phosphorylation of serine and threonine is usually associated with the repression of transcription that occurs during condensation of chromatin into recognizable chromosomes as the cell enters M-phase (mitosis) of the cell cycle
Posttranslational modifications of histones
Methylation and phosphorylation INHIBIT transcription
Acetylation ALLOW transcription
The posttranslational modifications of histones occur primarily at….
a. The posttranslational modifications of histones occur primarily at specific residues located within the histone amino-terminal tails protruding from the surface of the nucleosome
i. Histone modifications impact chromosome function though at least two distinct mechanisms:
1. Histone modifications alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA
a. This is exemplified by histone acetylation/deacetylation
2. Histone modifications provide binding sites for regulatory proteins containing protein domains that recognize specific histone modifications
a. HP1 uses its chromodomain to bind methylated lysines residues on histone H3 during heterochromatin formation
ii. Histone Modifications are carried out by specific histone modifying enzymes (histone acetyltransferases, methyltransferases, etc)
epigenetic modifications
i. Epigenetics modifications produce heritable changes in gene function that occur without a change in the sequence of DNA
DNA Methylation
- Another type of epigenetics modification is methylation of DNA, which is when a methyl group is added to the 5’ position of the pyrimidine ring in cytosine residues in CpG dinucleotides
a. DNA methylation is carried out by DNA methyltransferases (DNMTs)
b. This represses gene expression by blocking binding of TFs that normally activate genes
c. Can synergize with repressive histone modifications to promote heterochromatin formation
i. Methylated CpG in DNA is bound by methyl CpG binding proteins (MBPs)
ii. MBPs recruit histone deacetylases (HDACs)
iii. MBPs recruit histone methyltransferases (HMTs) - Methylated histones are recognized and bound by HP1, completely silencing gene
Clinical Relevance of DNA Methylation
a. CLINICAL RELEVANCE: Importance of epigenetic changes in diagnosis and treatment of cancer
i. Epigenetic modifications of DNA and histones can combine to suppress expression of tumor suppressing genes, whose products control cell growth by regulating the processes of mitosis and cell division
ii. DNMT inhibitors and HDAC inhibitors have been tested for use as anti-cancer agents
1. HDAC inhibitors directly inactivate HDAC enzymatic activity
2. DNMT inhibitors incorporate into DNA in the place of Cytosine, rendering CpG dinucleotides incapable of methylation by DNMT
Nucleosome Structure
a. Nucleosomes are approx. 50% (histone) protein, 50% DNA
i. Each nucleosome is considered to contain approx. 200 pb DNA
ii. Each nucleosome forms by combining two histone dimers (H2A/H2B) with one histone tetramer (2x H3/H4) to form the histone octamer
iii. Non-Histone: any chromatin protein that is not a histone (DNA repair enzymes, DNA and RNA polymerases, and TFs)
b. Transcribing chromatin does have nucleosomes!
i. Modifications during gene activation
1. The dissolution of one or two nucleosomes from the gene promoter and of histone H1 from linker DNA
2. Histone acetylation by histone acetyl transferases (HATs)
The 30nm Fiber
- The 30-Nanometer Fiber:
a. Six 200 bp nucleosomes are packed into a solenoid arrangement as one 1200 pb fiber with the help of Histone H1
i. Solenoid: 6 nucleosomes per turn
Loops
- Loops:
a. An average of 50 1,200 bp 30 nm fibers are coiled into a loop containing 60,000 pb of DNA
b. The bases of each loop are attached to a proteinaceous structure termed the nuclear matrix, which contains important structural and gene regulatory machinery
c. Looping circularizes the DNA, enabling localized genes to become supercoiled, which is required for transcription
d. Genes located in one loop can interact with up- and downstream regulatory sequences in adjacent loops, such as enhancers
Minibands
- Minibands:
a. Tandems of loops that encircle the chromosome axis
b. Proximity of loops on adjacent minibands would permit regulatory sequences in distal loops to interact
Mitotic Chromosome Structure
a. The formation of individual chromosomes at the onset of mitosis reflects hyper-condensation of chromatin, which is mediated by hyper-phosphorylation of chromatin proteins, especially histone H1
i. Each chromosome contains a single linear DNA molecule of an average 75 million bp DNA
ii. Centromere: locus of repetitive DNA in the middle of the chromosome
iii. Two telomeres: loci of repetitive DNA that cap the ends of the chromosome
Telomeres
i. Two telomeres: loci of repetitive DNA that cap the ends of the chromosome
1. Contain a repeating sequence of GGGTTA up to 15,000 bps
2. At each cell division, several repeats are lost in somatic cells causing shortening of telomeres and cellular senescence
3. Telomeres in germ-line cells are maintained at full-length via an enzyme termed telomerase, which adds on GGTTA segments after cell division
Clinical Significance of Telemeres
CLINICAL SIGNIFICANCE: telomerase is normally quiescent in somatic cells, but can be mutated and activated, causing abnormal maintenance of telomere length and cellular immortality that result in tumor formation
Nuclear Matrix
The nuclear matrix maintains the dynamic structural organization of the interphase nucleus by organizing the genome into domains that regulate gene expression and cell replication
Three Components of nuclear Matrix:
- Nuclear Envelope/Pore Complex/Nuclear Lamina
- Nucleolus
- Internal Nuclear Matrix
Three components of the Nuclear Matrix:
- Nuclear envelope/Pore Complex/Nuclear Lamina
Nuclear envelope
a. The nuclear envelope
i. A double membrane that encloses the perinuclear space, which is continuous with the lumen of the endoplasmic reticulum (ER)
ii. It is divided into segments, limited by nuclear pores
iii. The outer membrane (faces cytoplasm) contains ribosomes for protein translation
iv. The inner nuclear membrane contains integral proteins that bind the nuclear lamina, which in turn attaches to marginal heterochromatin
Nuclear Pore Complex
i. Nuclear pores between segments of the envelope are ~10 nm diameter
ii. The pore complex regulates passage of proteins into, and proteins/RNAs out of, the nucleus
1. Proteins smaller than ~40kD diffuse thru, larger proteins are under complex regulation
Nuclear Pore Structure
i. Each pore has three strata of proteins arranged as an octamer: (1) cytoplasmic stratum (2) nuclear stratum and (3) middle stratum
1. Strata contain ~100 different proteins termed nucleoporins
2. Nucleoporins in the cytoplasmic stratum serve as “docking sites” for proteins called nuclear cargo proteins that contain nuclear localization signals (NLSs)
a. NLSs are short amino acid sequences that contain positive charged basic amino acids (lysine [K] and arginine [R])
i. Cytoplasm Nucleus Import (only proteins are imported)
- Proteins larger than 45 kD must have a NLS to get in
- NLS-Receptor Proteins are in the cytoplasm
a. Example: importin, a heterodimer consisting of subunits importin- and importin-
i. Importin- binds the NLS of a nuclear cargo protein
ii. Then the complex migrates to the pore
iii. Importin- binds a nucleoporin in the pore complex
iv. Then, energy-dependent transport thru the pore
v. Once in the nucleus, the nuclear cargo protein is released from importin, which is cycled back thru the nuclear pore complex
Other mechanisms of nuclear import:
a. Proteins may have to be de-phosphorylated
b. Proteins may have to be released from a cytoplasmic masking protein to be recognized by importin
a.
ii. Nucleus Export Cytoplasm (Proteins and all RNA species are exported)
- Exportins mediate nuclear protein export
a. Exportins contain leucine-rich NESs (Nuclear Export Sequences) in their amino acid sequence
b. Many if not must nuclear proteins have both an NLS and an NES - RNA export does not depend on type of sequence of RNA
a. RNA export is mediated by RNA-BPs which may be bound to an exportin protein
i. Protein and RNA transport are not pore-specific
- The same pore can accommodate protein import as well as export of protein and RNA
Energy for nuclear import and export
i. Energy for nuclear import and export is provided by a GTP-BP termed Ran-GTP (“ran” in and out of the nucleus)
1. Within nucleus, Ran-GTP binds to both empty nuclear import receptors and cargo-bound export receptors, which migrate to the cytoplasm where Ran-GTP is hydrolyzed to Ran-GDP, releasing all bound moieties
a. Empty nuclear import receptors migrate back into nucleus
b. Nuclear import receptors bind proteins with NLSs before migrating back to nucleus
Clinical Significance of Nuclear Import/Export
i. CLINICAL SIGNIFICANCE: Drugs cannot readily penetrate the nucleus so nuclear structures are poor targets for therapeutic intervention
1. Knowing how substances are targeted to the nucleus, approaches may be designed that enable the development of drugs that can target specific nuclear structures and genes
a. The Nuclear Lamina
i. The nuclear lamina is subjacent to the inner nuclear envelope, to which it is bound
1. In interphase nuclei, the nuclear lamina maintains the nucleus as a sphere
2. During mitosis, the lamina breaks down due to hyperphosphorylation of lamina proteins
The nuclear lamina’s proteins
i. The nuclear lamina’s proteins are termed lamins A, B, and C
ii. Lamin A (LMNA) gene: Expresses Lamins A and C, which interact with the marginal heterochromatin
iii. Lamin B binds to the lamin-B receptor (LBR) on the inner nuclear membrane
1. When the nuclear envelope falls apart during mitosis, lamin B remains bound to the LBR, mediating re-formation of the nuclear envelope segments at the start of the next interphase
Clinical Significance of Nuclear lamins
i. CLINICAL SIGNIFICANCE: Laminopathies – mutations of the LMNA gene disrupt the lamina
1. Diseases range from several rare cardiac and skeletal muscular dystrophies to a severe form of premature aging (progeria), whch is caused by deletion of the part of the gene that generates the mRNA encoding lamina A
Nucleolus
a. The site of ribosomal RNA (rRNA) production, comprised of portions of ten chromosomes (each contributing ~40 rRNA genes)
i. Nucleoli do not have a limiting membrane
b. Cells need lots of ribosomes to live
i. To satisfy the cell’s demand, transcription of rRNA genes occurs constantly via RNA Polymerase I
1. Physical gene assumes the shape of a “Christmas tree”
The Internal Nuclear Matrix
- The Internal Nuclear Matrix (98% of the nuclear matrix)
a. Consists mostly of chromatin: DNA, Histones (H1, H2A, H2B, H3, H4) and non-histones (TFs, RNA and DNA polymerases, etc.)
b. Matrix proteins are disease-specific and tissue-specific
i. Some of the nuclear matrix proteins in normal cells and cancer cells is different – of diagnostic value (used in diagnostic tests for bladder cancer and cervical carcinoma)
a. Chromosomes occupying specific locations in the interphase nucleus
a. Chromosomes occupying specific locations in the interphase nucleus
i. Telomeres stick to the nuclear envelope while centromeres stuck to the opposite side of the envelope
ii. Chromosomes occupy the same site of the interphase nucleus, generation after generation
iii. Chromosome specific territories in the nucleus have been shown using fluorescent in situ hybridization (FISH) of DNA – also called chromosome painting
DNA replication and transcription occur in the …
a. DNA replication and transcription occur in the nuclear matrix
i. Although most nuclear material is removed during preparation of the nuclear matrix, sites of transcription are retained as indicated by in situ hybridization
a. The Nuclear matrix organizes DNA replication
i. In S-phase, DNA is replicated in groups of short sequences termed “replicons” – after matrix preparation, these replicons are retained in the matrix
Phases of the Cell-Cycle:
• Duration of each cell-cycle revolution is termed the generation time (Tg)
o During each Tg the cell goes through four cell-cycle phases:
♣ (1) G1 Phase (Gap 1) – duration is highly variable
• Cells in G1 have four choices:
o Senescence (i.e., G0)
o Differentiation (also G0)
o Apoptosis cell death
o Proliferation entry into cell cycle
♣ (2) S-Phase (Synthesis [of DNA] Phase)
• Chromosomes are duplicated, 2N 4N DNA
• Histones are synthesized to make-up the new chromosome
♣ (3) G2 Phase (Gap 2)
• Preparation for mitosis occurs; late in G2 the centrosome is duplicated
• Hyper-phosphorylation of histone and non-histone proteins occurs in late G2
♣ (4) M Phase (Mitosis):
• Cells become spherical
• Nuclear membrane disintegrates
• Chromatin condenses into chromatids (2/chromosome)
• Chromosomes align on equatorial plan, then chromatids segregate into “daughter” cells
Cell-Cycle Checkpoints
External Factors
• External Factors “1o messengers”
o Secreted molecules and peptides – GFs, cytokines, hormones – which interact with a cognate receptor that is in the cell membrane (or, for steroid/retinoid hormones, in the cytoplasm)
♣ Factors that ACTIVATE the cell-cycle (examples): FGFs, IGFs, Wnts
♣ Factors that INHIBIT the cell-cycle: TGF
Cell Cycle Checkpoints Internal Factors
• Internal Factors “2o messengers”
o “Early Response” genes: c-myc, fos, and jun
♣ Rapidly (15 min) respond to growth signals
o “Delayed Response” genes: CDKs and cyclins
♣ the CDKs and cyclins comprise heterodimers termed cyclin-dependent protein kinases (Kinases are enzymes that phosphorylate target proteins)
• The CDK subunit is the catalytic subunit, which phosphorylates the target (substrate)
o There are several different CDKs (CDKs 4, 2, and 1)
o During the cell-cycle, the content of each CDK remains constant
• The cyclic subunit is the regulatory subunit, which regulates the activity of the kinase heterodimer
o There are several cyclins (Cyclins D, E, A, and B)
o During the cell-cycle, the content of each cyclin increases
Clinical Correlation of Cell Cycle Checkpoints
o CLINICAL CORRELATION: Each of the proteins described to this point – Growth Factors, Growth Factor Receptors, early response genes, and CDKs/cyclins – are termed proto-oncogenes
♣ When a proto-oncogene is mutated, the protein it encodes becomes pathologically over-active stimulating the cell-cycle to induce cancer
Restriction Point (R)
• Regulation by extracellular growth factors is prominent in early G1, diminishing in late G1 before the restriction point (R)
o In response to growth factors, intracellular cyclins regulate discrete steps during interphase
♣ The content of cyclins D, E, A, and B increases during the cell-cycle in that sequence to activate the cell-cycle phases
• These increases occur concomitant with declining content of p27, a cell-cycle inhibitor
Cyclins DEAB
G1 Phase is activated by
- G1 Phase is activated by cyclinD/CDK4 heterodimer:
a. During early G1, growth factor activity induces sustained increase in cyclin D
i. A classical example is initiated by the Wnt growth factors, which induce increased content of the cytoplasmic protein -catenin, which migrates to the nucleus to activate transcription of the c-myc gene, which in turn activates the cyclin D gene
Wnt GF cytoplasmic -catenin –migrates to the nucleus c-myc gene cyclin D genei. Other factors also activate cyclin D transcription
1. GFs induce intracellular ras, a small GTPase-binding protein, which activates the MAP kinase pathway to effect cyclin D production
Cyclin D binds …
a. Cyclin D binds CDK4 to form cyclinD/CDK4 heterodimer
CyclinD/CDK4 phosphorylates …
a. CyclinD/CDK4 phosphorylates retinoblastoma (Rb) protein, forming P~Rb
i. In the phosphorylated state, Rb releases its hold on a transcription factor protein termed E2F
ii. Rb inhibits the cell-cycle; P~Rb releases this inhibition
E2F activates genes …
a. E2F activates genes encoding cyclin E and cyclin A, increasing their content
Basic Pattern of R regulation
CyclinD + CDK4 CyclinD/CDK4 phosphorylates Rb P~Rb releases E2F activates gene for cyclin E and cyclin A
S-Phase is activated by ….
- S-Phase is activated by cyclinE/CDK2 and cyclinA/CDK2 heterodimers
a. This depends on E2F-induced activation of genes encoding cyclins E and A
i. When content of cyclin E increases, the restriction checkpoint (R) regulated by cyclinE/CDK2 is breeched - Once breeched, the cell is committed to complete the cycle, all the way through G2 phase
- This commitment ensures that the whole genome is replicated during S-phase
- Failure to replicated all of the DNA results in cancer
b. During S phase, ‘DNA replication complexes’ which are ‘poised’ at multiple origins of DNA replication, are activated via phosphorylation of cyclinA/CDK2
i. This mechanism prevents formation of new, inappropriate DNA replication complexes
ii. This prevents the DNA from being replicated more than once
c. The end result of the cell-cycle is the precise apportionment of 1.0 genome to each daughter cell
Cyclin E restriction checkpoint is breeched cell is committed to complete cycle
CyclinA/CDK2 phosphorylates (and activates) “DNA replication complexes”
M-Phase is activated….
- M-Phase is activated at the G2/M boundary upon de-phosphorylation of the cyclinB/CDK1 heterodimer
a. This de-phosphorylation is mediated by a phosphatase termed cdc25
i. After it is de-phosphorylated, the cyclinB/CDK1 dimer enters the nucleus to phosphorlate many target proteins, causing: - Nuclear envelope breakdown (NEB)
- Mitotic spindle assembly
- Metaphase arrest
Anaphase regulation
Activated at mid-metaphase via the Anaphase Promoter Complex (APC)
Tumor Supressor Genes
• Activity of cyclin/CDK heterodimers is antagonized by proteins suppressing the cell-cycle
o Under the influence of such inhibitory proteins, cells are maintained in a prolonged G1-phase termed G0
♣ The genes that encode for these inhibitory suppressor proteins are termed tumor suppressor genes
♣ Mutation of a gene encoding a tumor suppressor protein can cause deactivation of that protein, resulting in cancer
o Retinoblastoma (Rb)
♣ Binds E2F-1, preventing E2F-1 from transcribing genes encoding cyclins E and A
♣ Rb is a “classic” tumor suppressor gene because when it is mutated, tumors of the retina termed “retinoblastomas” occur
♣ Rb, as well as proteins of related function such as p107 and p130, are termed “pocket proteins”
p21
o p21: Inhibits the cell-cycle by directly inhibiting CDK-2 and CDK-4
p27
o p27: similar to p21
p53
o p53 – the most prominent tumor suppressor
♣ Inhibits the cell-cycle via two functions
• (1) p53 induces transcription of the tumor suppressor p21
• (2) p53 promotes apoptosis (cell death)
♣ Is mutated in over ½ of human cancers
♣ Is referred to as the “sentinel of the genome,” since it induces killing of cells that contain inappropriately replicated DNA resultant from mistakes made during S-phase
Cancer (definition/cause)
Cancer: What is it?
• Cancer is a genetic disease displaying:
o Loss of cellular differentiation
o Increased proliferation and invasiveness of cells
o Changes in chromosomes: re-arrangement, loss, gain
Cancer: What causes it?
• Cancer is caused by inherited mutations and/or environmental insults to DNA during aging
o As we age, our cells evolve toward malignancy
o Cancer is definitely a genetic disease
Proto-Oncogenes
- Proto-oncogenes: about 70 genes that encode proteins that activate the cell cycle
a. Genes encoding cell surface receptors, factors, cyclins, and CDKs
b. When mutated, these are termed oncogenes
i. Mutated to be over-expressed, or the proteins the they encode have intensified activity
ii. Result is up-regulation
Tumor Supressing Genes
- Tumor Suppressing Genes: inhibit the cell-cycle
a. When mutated, products of these genes no longer “work”
b. About 20 tumor suppressors have been discovered, including:
Genes that Regulate Apoptosis
a. Diseased cells must be removed before they can colonize, so apoptosis is a normal and necessary mechanism
b. Stimulation of apoptosis causes tumors to regress
c. Apoptosis also regulates embryonic development, as shape (morphology) is attained – example: digit formation of fingers
a. Sequence of apoptosis:
i. Macrophages release TNF (tumor necrosis factor)
ii. TNF binds TNFR cell membrane receptor
iii. A balance between pro-apoptotic factors + anti-apoptotic factors is established
iv. Pro-apoptotic signals induce “leakiness” of the outer cell membrane of mitochondria
v. Cytochrome-C escapes to the cytoplasm
vi. Cytochrome-C activates Caspase, a protease that destroys chromatin, which fragments into 200 bp (i.e. nucleosomal) pieces
vii. Blebs appear on the cell surface and the cell defoliates (explodes)
Relationship of Apoptosis to Cancer
a. Relationship to cancer: In B cell lymphoma, a gene that normally inhibits apoptosis (bcl-2) is mutated
i. bcl-2 is an anti-apoptotic factor, but when mutated it is abnormally active
ii. Too much inhibition of apoptosis occurs, resulting in tumor formation
Telomerase
- Genes that induce cellular immortality: Telomerase
a. “Telomere hypothesis” – upon attaining a given length, the shortened telomere signals the cell to become senescent
i. This is a major regulatory mechanism in the aging process
b. In embryonic stem cells, and germ cells (sperm/egg), the telomere is restored to its original length after each cell division via an enzyme: telomerase
i. Telomerase is normally inhibited during aging of somatic cells so they normally become senescent after a critical number of cell divisions
ii. If the gene encoding telomerase is mutated, telomerase becomes active in somatic cells, rendering them immortal – this can cause carcinogenesis
Genes that repair DNA
a. Normally DNA damage causes the cell-cycle to stop until DNA repair enzymes fix the damaged DNA
b. If genes that encode DNA repair proteins are mutated, the proteins are inactivated, allowing clones of abnormal cells to arise
i. Recently documented in the instance of colo-rectal cancer
The Basic Properties of DNA Replication
• DNA synthesis is semiconservative
o Each daughter DNA molecule ends up with one of the original strands and one newly synthesized strand
• DNA is synthesized in the 5’ 3’ direction
• A 3’-OH primer and a template are required
o DNA polymerase I activity requires a single unpaired strand to act as a template and a primer strand to provide a free hydroxyl group at the 3’ end, to which a new nucleotide unit is added
o The catalytic mechanism of elongation likely involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases
• DNA replication is semi-discontinuous
o There is a leading strand and a lagging strand
♣ The leading strand is transcribed in the direction of movement of the replication fork
♣ The leading strand is transcribed in the opposite direction, away from the movement of the replication fork
• Still transcribed 5’ 3’, the lagging strand uses an RNA primer to provide the free 3’ OH
• It is transcribed in segments called Okazaki fragments
DNA Replication has 4 Basic Steps:
- Separation of the two complementary strands at an origin of replication (a specific place(s) in the genome)
- Formation of the replication fork (primers and Okazaki fragments)
- Chain Elongation
- Removal of the primers
Properties of E. coli DNA Polymerase I
• One protein, 3 catalytic activities o 5’ 3’ polymerase o 5’ 3’ exonuclease o 3’ 5’ exonuclease Exonuclease: Proofreading activity: removes incorrectly base paired nucleotides
Error correction by the 3’ 5’ exonuclease activity of DNA polymerase I
- Polymerase mispairs dC with dT, which impedes translocation of DNA polymerase I to the next site
- Polymerase repositions the mispaired 3’ terminus into the 3’ 5’ exonuclease site which is behind the polymerase activity when the enzyme is oriented in its movement along the DNA
- Exonuclease hydrolyzes the mispaired dC
- The 3’ terminus repositions back to the polymerase site
- Polymerase incorporates the correct nucleotide, dA
DNA replication in Prokaryotic vs Eukaryotic organisms
• Process of DNA replication is functionally conserved between pro and euk organisms
o Many of the proteins are different but they function in an analogous manner
o A major difference in the replication process is that in prokaryotic replication this is one origin of replication and in eukaryotic replication there are multiple
Proteins of the E. Coli Replisome and their function(s)
- SSB – binding to single-stranded DNA
- DnaB protein (helicase) – DNA unwinding; primosome constituent
- Primase (DnaG protein) – RNA primer synthesis; primosome constituent
- DNA polymerase III – New strand elongation
- DNA polymerase I – Filling of gaps; excision of primers
- DNA ligase – Ligation
- DNA gyrase (DNA topoisomerase II) – Supercoiling
Telomeres
• Structures at the ends of eukaryotic chromosomes
• Have tandem repeats usually of T1-4G1-4 (with A-C on opposing strand)
• Can be tens of thousands of bp long in mammals
• TG strand is longer than its complement, leaves a 3’-overhang of several hundred bases
• Telomerase is an RNA-dependent DNA polymerase (reverse transcriptase) that carries its own RNA template
o The internal template of RNA of telomerase binds t and basepairs with the TG primer of DNA
o Telomerase adds more T and G residues to the TG primer, then repositions the internal template RNA to allow the addition of more T and G residues that generate the TG strand of the telomere
o The complementary strand is synthesized by cellular DNA polymerases after priming by RNA primase
Major Sources of DNA Mutation:
• Mis-Incorporation of nucleotides during DNA replication
o Adenine tautomer base pairs like Guanine
• Inherent chemical instability of bases (NONEZYMATIC)
o Deamination of CU; 100 bp/day
o Hydrolysis of N-glycosidic bond – depurination; 5000 bp/day
• Environmental Mutagens
o Deaminating agents (CU; GX)
♣ Nitrous acid precursors
• Sodium Nitrite (NaNO2), Sodium Nitrate (NaNO3), Nitrosamine
• Ionizing Radiation
o UV light can cause pyrimidine dimers
Environmental Mutagens
o Deaminating agents (CU; GX)
♣ Nitrous acid precursors
• Sodium Nitrite (NaNO2), Sodium Nitrate (NaNO3), Nitrosamine
o Alkylating agents (adding a methyl group) – methylated G binds with T
♣ Nitrogen mustards
♣ Aflatoxin
♣ Benzo(a)pyrene
♣ AAF (N-2-acetyl-2-aminofluorene)
o Intercalating agents – lead to insertion or deletion of one or more base pairs, alter reading frames
♣ Frameshift mutations caused by flat aromatic molecules:
• Ethidium bromide, acridines
Cells have multiple DNA Repair Systems
- Mismatch Repair: Mismatches
- Base-Excision Repair: Remove abnormal/damaged bases
- Nucleotide-Excision Repair: Remove large structural damages (thymine dimers)
- Direct Repair: Systems that recognize and repair with specific damages Pyrimidine dimers (photolyase)
- Recombinational Repair: Uses sister chromatid to repair damaged region
DNA Methylation and Mismatch Repair
• Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strains
o The methylation occurs at the N6 of adenines in (5’)GATC sequences
♣ This sequence is a palindrome because present in opposite orientations on the two strands
o After a few minutes, the new strand is methylated and the two strands can no longer be distinguished
A model for early steps of methyl-directed mismatch repair:
o MutS recognizes the mismatch
o MutH recognizes the (5’)GATC
o MutS and MutL form a complex at the mismatch
o DNA is threaded through the complex such that the complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at the hemimethylated GATC sequence
o MutH cleaves the unmethylated strand on the 5’ side of the G in this sequences
o A complex consisting of DNA helicase II, SSB, and one of several exonucleases then degrades the unmethylated DNA strand from that point toward the mismatch
♣ The exonuclease that is used depends on the location of the cleavage site relative to the mismatch
o
The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase (Not shown)
Base Excision Repair
Nucleotide (BASE) Excision Repair on human DNA
• Some proteins involved in humans excision repair identified in patients with xeroderma pigmentosum (XP)
• Pathway:
o An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion
o The DNA segment –of 13 nucleotides or 29 nucleotides- is removed with the aid of a helicase
o The gap is filled in by DNA polymerase
o The remaining nick is filled in with DNA ligase
Direct Repair
• Some forms of DNA damage can be enzymatically reversed – for example, the removal of alkyl groups at the O6 position of Guanine
o Alkylation of guanine occurs from environmental mutagens and by cancer chemotherapeutic drugs
o MGMT (O-6-methylguanine-DNA methyltransferase) is the enzyme that repairs O6 methyl guanine
♣ It works by direct removal of the alkyl group
o This enzyme can interfere with some forms of chemotherapy that rely on DNA damage by alkylation
Therefore, inhibition of MGMT can facilitate cancer chemotherapy
Recombinational Repair
• Some lesions of DNA, such as double-stranded breaks, double-stranded cross-links, and lesions in single stranded regions, cannot be repaired using information from the complementary strand – Information must come from a separate homologous strand
• These types of lesions result from ionizing radiation and oxidative reactions
o Repair of these types of lesions is mediated by homologous genetic recombination called recombinational repair
Xeroderma Pigmentosum (XP
• Xeroderma Pigmentosum (XP) – a rare skin disease
o Individuals are extremely sensitive to UV light and develop skin cancer which metastasize - Patients often die before age 30
o XP results from a defect in the nucleotide excision repair mechanism for thymine dimers
♣ Recessive disease – fewer than 1,000 cases known worldwide
♣ Wide range of symptoms
• Blindness and deafness
• Blistering or freckling on minimum sun exposure
• Developmental disabilities
• Dwarfism and hypergonadism
• Increased skin and eye cancers
• Mental Retardation
Diseases Associated with Chromosomal Breakage
• Werner’s Syndrome: premature aging (adult progeria) – due to defects in a DNA helicase
• Bloom Syndrome: small body size, sun sensitivity, hypo and hyper-pigmented lesions, immunodeficiency, cancer, diabetes, and lung diseases – due to abolition of Ligase I activity needed to complete DNA repair
• Fanconi’s anemia, Ataxia telangiectasia, and Gardner’s Syndrome
o All believed to be due to defects in ligase activity
• Breast Cancer: Some forms of breast cancer are due to defects in breast cancer susceptibility genes (BRCA Genes)
o BRCA genes participate in DNA repair
(primary structure)
• Proteins spontaneously fold based on their sequence of amino acids (primary structure)
o most hydrophobic (apolar) amino acids within the core
o most hydrophilic (polar) amino acids on the surface
Secondary structures
• Secondary structures involve H-bonds between peptide bond carbonyl (C=O) groups and peptide bond amide hydrogens (>N-H) form first
o -helices, parallel and anti-parallel -pleated sheets
Basic AA structure
• There are 20 different genetically coded amino acids found in proteins
o Each aa (except proline) has a carboxyl group (-COOH), an amino group (-NH3), and a distinctive side change (R group) bonded to the -carbon atom
Chiral AA
• If the -carbon of an amino acid is attached to four different chemical groups, then that carbon is chiral and the amino acid is optically active
o Glycine is the only amino acid without optical activity because its -carbon is attached to two Hydrogen atoms
o AAs (except glycine) exist in either of two mirror image forms (enantiomers) termed the D and L, which have identical chemical properties in all respects except their solutions cause polarized light to rotate in opposite directions
♣ All amino acids found in mammalian proteins are of the L-configuration
Aliphatic amino acids
o Aliphatic amino acids possess side chains comprised of saturated carbon backbones – the subtle differences in the side chains allow for tight packing within the protein interior (7 –> GAVLIMP) – Grab All Vaginas Like Its My Pussy ♣ Glycine ♣ Alanine ♣ Valine ♣ Leucine ♣ Isoleucine ♣ Methionine ♣ Proline
Aromatic amino acids
o Aromatic amino acids possess side chains derived from either benzene (Phe and Tyr) or indole (Trp); each contains a methylene spacer (-CH2-) between the aromatic ring and the -carbon, this minimizes steric repulsion between the ring and the polypeptide chain backbone (3 –> PTT)
♣ Phenylalanine
♣ Tyrosine
♣ Tryptophan
Uncharged polar amino acids
o Uncharged polar amino acids have side chains that do not ionize at physiological pH (Met is sometimes classified in this group because of the slightly polar nature of its sulfur-containing side chain) (5 –> STAGC) Stop Touching All Girls C*nts ♣ Serine ♣ Threonine ♣ Asparagine ♣ Glutamine ♣ Cysteine
Charged polar amino acid
o Charged polar amino acids have side chains that can carry either a positive charge or a negative charge; although formally classified in other groups, the side chains of Tyr and Cys can ionize with pKa values close to physiological pH (5 –> LAHAG) Like All Hoes, Ashley Gags ♣ Lysine + ♣ Arginine + ♣ Histidine + ♣ Apartate + ♣ Glutamate +
The pKa of a group …
• The pKa of a group is the pH at which it is 50% protonated and 50% unprotonated
o At pH values lower than the pKa, there is a higher concentration of H+ in solution and >50% of the side chain group is protonated
o At pH values higher than the pKa, the solution has fewers H+ and <50% of the group is protonated
pI
• At some intermediate pH, the amino acid, peptide, or protein will have a net charge of zero; this pH is termed the isoelectric point, or pI
Sulfur-containing amino acids
• Methionine and Cysteine, the only amino acids containing sulfur (S), have unique roles and sensitivities
Significance of Met
o In newly synthesized (nascent) proteins, Met is always the first or N-terminal amino acid, but it also occurs at various positions throughout a polypeptide
o Under some conditions, the S of Met is oxidized, impairing protein function
Significance of Cys
• The thiol or sulfhydroyl group of cysteine can spontaneously oxidize to form a disulfide linking two cysteine residues within a protein
o Disulfide bonds form between specific cysteine residues because chain folding places the –SH groups in close proximity
♣ Disulfides rarely occur in intracellular proteins, but are common in extracellular proteins where their presence increases conformational stability of the structure
♣ Disulfide bonds are important in stabilizing the circulating peptide hormone insulin
Metamorphic proteins
• Metamorphic proteins exist in an ensemble of distinct structures of approximately equal energy that are in equilibrium (envision a thermodynamic folding funnel with two wells of similar depth)
alpha-Keratin
• -Keratin – a primary component of hair and nails
o Consists of two right-handed -helices that are intertwined in a left-handed supercoil called an -coiled coil
♣ Bonding between coils is both non-covalent (hydrophobic interactions, ionic bonds, H-bonds) and covalent (disulfide bonds between adjacent cysteine residues)
♣ Moderate number of disulfide bonds allow hair to stretch but then return to its original shape
• A much higher number of disulfide cross-links makes nails (and horns, claws, and hooves) rigid
♣ Belongs to a large family of coiled coil proteins that also includes the intermediate filaments that provide internal scaffolding for cells and myosin and tropomysin, which are involved in muscle action
Collagen
• Collagen – the most abundant protein in the body (20-25% of total protein) and exists as a superfamily of molecules whose type and organization are dictated by the structural roles each plays in different organs
o Typical collagen molecule is a long, rigid structure in which three polypeptides are wound around each other in a rope-like triple helix
o The individual collagen chains ( chains) are left-handed helices (NOT left-handed helices)
♣ 3 of them are wound around each other in a right-handed triple helix
♣ Multiple triple helices interdigitate to make collagen microfibrils
Clinical significance of Pro and Gly in Collagen
o Hydroxyproline and hydroxylysine are formed through post-translational hydroxylation reactions of proline and lysine and serve to stabilize the triple-helical structure
o The enzymes that catalyze the hydroxylation reactions require ascorbate (Vit C), a deficiency of which results in scurvy
♣ The symptoms of scurvy (e.g., bleeding gums) are due in part to the decreased tensile strength of collagen
Synthesis of Collagen
o Synthesis, post-translational modification, and triple helix assembly occur inside the cell, then the resulting procollagen molecule is secreted into the extracellular matrix where specific peptidases cleave N- and C- terminal propeptides
♣ Once removed, the mature collagen triple helix (tropocollagen) self-assembles into fibrils, with subsequent cross-linking to form mature collagen fibers
Elastin
• Elastin – A connective tissue protein with rubber-like properties that is found in the lings, large arterial walls, and elastic ligaments
o Comprised predominately of small, non-polar amino acids (glycine, alanine, valine) and is also rich in proline and lysine – contains few few hydroxyl derivatives
o Elastin is synthesized from a precursor, tropoelastin, which is secreted into the extracellular space where it interacts with specific glycoprotein microfibrils
♣ Some lysyl side chains are oxidized to form allysine residues which cross-link with lysine amino groups of neighboring polypeptides to produce elastin
Role of alpha-antitrypsin (1-AT) in elastin degredation
o Role of -antitrypsin (1-AT) in elastin degredation
♣ 1-AT is a small protein that inhibits a number of proteolytic enymes in bodily fluids
♣ Crucial function of 1-AT is to inhibit elastase, which is released by neutrophils and functions to degrade elastin
• Elastase destroys the alveolar epithelium if unregulated by 1-AT
• There are both genetic and environmental causes of insufficient functional 1-AT and both can lead to emphysema
o Genetic: 1-AT deficiency is predominantly due to the inheritance of two prominent mutant alleles, Z and S
♣ The Z allele causes a more severe deficiency and is due to an E342K substitution that causes 1-AT to be retained inside the cell
o Environmental: 1-AT contains a methionine residue that is essential for binding to target proteins (i.e., elastase)
♣ Elements in cigarette smoke cause the oxidation of the Met, which renders 1-AT unable to bind and inactivate elastase
♣ Heavy smokers can develop emphysema even if they have no mutant 1-AT alleles
Hemoglobin
• Adult hemoglobin (Hb) is a tetramer of two and two subunits
o Each subunit contains a heme cofactor to which O2 can be reversibly bound on the Fe2+ atom
o Hemoglobins of other subunit compositions occur developmentally
♣ Most important: Fetal Hb (HbF): comprised of 22
• O2 binding to Hb is cooperative such that binding of each O2 molecule to the tetramer increases the affinity with which the next O2 binds
o Similarly, dissociation of each O2 decreases affinity for those remaining so they dissociate more easily
o This cooperativity results in a sigmoidal O2 binding curve and that greatly increases the ability of Hb to release O2 in tissues
The affinity of Hb for O2 is decreased by:
o (1) High concentrations of H+ (lower pH)
o (2) CO2
o (3) 2,3-diphosphoglycerate (DPG)
o (4) Increasing Temperature (Fever)
Sickle Cell Disease `
• Sickle Cell Disease is an inherited mutation of the - globin gene causing a change from Glu to Val at position 6
o Presence of Val at position 6 allows deoxyHbS to polymerize into microfibrils that distort RBC shape and cause both hemolysis and vaso-occlusive disease
The Globin Genes
• On both chromosome 16 (alpha-globin family) and chromosome 11 (beta-globin family), the genes are expressed developmentally from 5’ 3’
• Expression of embryonic alpha-globin (zeta) occurs only briefly, and then the adult form (alpha) dominates through fetal growth and into adulthood
• Each chromosome 16 has 2 alpha-globin genes (a person has 4 total); each is active and codes about ¼ of expressed alpha-globin
o Consequently, a defect in 1 or 2 alpha-gene clusters is not too serious
• Expression of embryonic beta-globin (epsilon) is also brief but followed by a fetal form (gamma) that does not disappear until after birth
o HbF is clinically important
• There are two forms of adult Hb: HbA and HbA2
o HbA2 usually accounts for only a few present of adult Hb
• Each chromosome 11 has only one functional beta-globin gene
o Beta-globin gene defects are more likely to have clinical consequences
Overview of gas transport and pH regulation in humans
• Hemoglobin is a specialized protein designed to transport oxygen from the lungs to the peripheral tissues where oxygen tension is low
o Metabolism in the peripheral tissues generates CO2 and H+ that are transported back to the lungs, in part by hemoglobin
O2 solubility in plasma
• O2 has very low solubility in plasma
o >98% of the O2 that reaches tissues is carried in RBCs bound to Hb
o Situation is different for CO2
♣ RBCs contain carbonic anhydrase, which catalyzes the rapid reversible hydration of CO2 to carbonic acid (H2CO3)
• H2CO3 then rapidly and spontaneously dissociates to bicarbonate (HCO3-) and a H+
• CO2 and HCO3- are soluble in plasma and RBC cytosol and most of the CO2 made in tissues returns to the lungs as those species
o About 14% of the CO2 made is carried bound to Hb
Tertiary Structures of Myoglobin and Hemoglobin
• Hemoglobin is a heterotetrameric protein having the subunit composition ()2
o The alpha and beta subunits have similar sequences and tertiary structures
o Both subunits are evolutionarily related to myoglobin, a monomeric protein abundant in muscle that is designed to store O2
♣ Both proteins contain a Fe2+-protoporphryin IX prosthetic group that is responsible for binding O2
• Note: Fe2+ is the ferrous form of iron that is capable of binding O2
• Fe3+ is the ferric form that cannot bind O2, and is present in an inactive form of hemoglobin called methemoglobin
Structure-function relationships in myoglobin and hemoglobin
• The partial pressure of dissolved oxygen in aqueous solution is proportional to the partial pressure in the gas phase
o Myoglobin gives a normal binding curve, which is hyperbolic in shape
o Hemoglobin shows sigmoidal cooperative binding of the oxygen that is a direct result of its more complex subunit structure
How O2 binding changes the conformation of the Hb subunit
• Without O2 bound, the heme Fe2+is pulled away from the plane of the porphyrin ring by a His residue of the polypeptide chain
o When O2 binds, it pulls the Fe2+ back into the plane of the ring and that moves the His residue and its whole section of the polypeptide chain
o That in turn causes the Hb subunits to shift relative to one another to an arrangement that favors the R-conformation
Bohr effect
o The reciprocal relationship between O2 and H+ is termed the Bohr effect, or isohydric shift
o Changes in H+ binding result from a shift in the pKa of specific residues (mostly histidines) due to charges microenvironment effects trigged by conformational changes in the hemoglobin molecule
2,3-diphosphoglycerate (2,3-DPG or DPG)
• DPG is a negative allosteric effector of O2 binding
o DPG binds to a specific site in a cleft between the beta subunits
♣ Binding stabilized mostly by ionic interactions
o Special regulatory mechanisms exist in RBCs to control the concentration of 2,3 DPG in order to fine tune the affinity of hemoglobin for O2 in response to changes in metabolism and environment
♣ This obviates the need to induce the synthesis of different hemoglobin isoforms of altered O2 affinities (which is not possible for human RBCs since they lose the capacity to synthesize protein during their terminal differentiation)
Ribonucleic acids play three well-understood roles in living cells:
- Messenger RNAs: encode amino acid sequences of the polypeptides found in the cell (5% of total RNA, most complex)
- Transfer RNAs: match specific amino acids to triplet codons in mRNA during protein syn (~15% of total RNA (and snRNA in eukaryotes)
- Ribosomal RNAs: the constituents and catalytic appropriate amino acids (~80% of total RNA, least complex)
Ribonucleic acids play several less-understood functions in eukaryotic cells
o MicroRNA: appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences
o Other functions: Ribonucleic acids act as genomic material in viruses
RNA Metabolism
• Ribonucleic acids are synthesized in cells using DNA as a template in transcription
o Transcription is tightly regulated in order to control the concentration of each protein
• Being mainly single-stranded, many RNA molecules can fold into compact structures with specific functions
o Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors
• Most Eukaryotic ribonucleic acids are processed after synthesis
o Elimination of introns; joining of exons
o Poly-adenylation of the 3’ end
o Capping the 5’ end
Transcription in Prokaryotes
• Basic Reaction:
o Nucleoside triphosphates added to the 3’ end of the growing RNA strand
o Chemically the RNA polymerization reaction is very similar to DNA synthesis, occurring in the 5’ to 3’ direction
o The 3’ hydroxyl group of the existing RNA chain undergoes a nucleophilic attack on the alpha-phosphate of the incoming nucleotide
o The growing chain is complementary to the template strand in DNA
o The synthesis is catalyzed by an enzyme (RNA polymerase)
♣ Two Mg2+ ions are used as cofactors among many Asp residues
Basic Properties of RNA Polymerases
• In prokaryotes, a single RNA polymerase synthesizes mRNA, rRNA, and tRNA
• Prokaryotic RNA polymerase is a multisubunit enzyme
o RNA polymerase holoenzyme has five core subunits of 2’ plus a sixth,
o The core enzyme is responsible for polymerization but it lacks specificity and cannot recognize the promoter
♣ The “sigma” factor allows the holoenzyme to recognize promoter regions on the DNA
o RNA pol lacks 3’ 5’ exonuclease, so it has a high error rate of 1/104-1/105
♣ You don’t have to worry about inheriting them because they are largely unstable, so it doesn’t matter
o RNA binds to promoter regions to initiate transcription
Anatomy of a bacterial gene
- Promoter: site for binding RNA polymerase
- Operator: binding sites for repressor of activator
- Structural gene: often times many related genes transcribed as a single unit
- Together they are called an operon
Initiation of transcription requires several steps generally divided into two phases:
o (1) Binding phase – the initial interaction of the RNA polymerase with the promoter leads to formation of a closed complex, in which the promoter DNA is stable bound but not unwound
♣ The sigma subunit helps provide specificity
♣ A 12 to 15 bp region of DNA is then unwound to form an open complex
o
(2) Initiation phase – encompasses transcription initiation and promoter clearance
Elongation
• Elongation – once commenced, the sigma subunit is released and it replaced by the protein NusA
o The polymerase leaves the promoter and becomes committed to elongation of the RNA
Termination
• Termination – once transcription is complete, the RNA is released, the NusA protein dissociates, and the RNA polymerase dissociates from the DNA
DNA Template Strand:
• DNA Template Strand: serves as template for RNA polymerase
DNA Coding Strand:
• DNA Coding Strand: the non-template strand; has the same sequence as the RNA transcript
The Sigma Subunit of the Holoenzyme
The Sigma Subunit of the Holoenzyme Enables RNA Polymerase to Recognize Promoters – Not All Promoters Are Created Equal
• Different sigma factors recognize different promoters
• Sigma increases specificity of RNAP, but decreases affinity
• Strong promoters cause frequent initiation and tend to conform closely to the consensus
• Different sigma factors lead to differential gene expression: Change in sigma utilization can be used to regulate developmental changes in gene expression
Transcriptional Elongation
• Elongation proceeds in the 5’ to 3’ direction partially through the energy released by cleavage of the phosphate bond in the incoming ribonucleotide, and by subsequent hydrolysis of pyrophosphate to inorganic phosphate
o The sigma factor dissociates from the holoenzyme complex immediately after elongation begins
• Multiple RNA polymerase complexes load onto the promoter region in sequential fashion, allowing the gene to be transcribed continuously
o The rate of elongation by E. coli polymerase is about 30 nt/second
o Elongation is extremely processive
♣ Once sigma dissociates, a single RNAP can synthesize thousands of nucleotides before it dissociates
transcription bubble
• A transcription bubble forms during the process of elongation
o Negative superhelicity in the double-stranded DNA facilitates the melting of the two strands of DNA and the formation of this bubble
o The RNA transcript is only base paired with the DNA template in this region, leaving a growing chain behind as the DNA helix reforms behind the elongation complex
Termination of Transcription
• Bacteria termination of transcription occurs by protein-dependent or protein-independent mechanisms, both of which rely on a hairpin loop structure
Rho-Independent termination
♣ Active signals for termination lie in nascent RNA chain – information is in the structure
• Palindromic GC-rich region followed by AT
o The hairpin is formed at the palindromic sequence, reducing the length of the RNA-DNA hybrid
• RNAP pauses at GC
• dA-rU form weak hybrid A-T, therefore DNA duplex reforms
• Core RNAP has less affinity for dsDNA than ssRNA therefore complex dissociates
Rho-Dependent termination
♣ Due to the interaction of a terminator protein (Rho) with the RNA polymerase elongation complex as it pauses at a hairpin loop in the nascent transcript
• The Rho protein is an ATP-dependent RNA-DNA helicase that disrupts the RNA-DNA hybrid, leading to disassembly of the elongation process
o Rho binds to rut site, a common CA-rich sequence (Rho utilization element)
o Rho processes in an ATP dependent manner until termination site is reached
There are 3 different eukaryotic RNA polymerases – each synthesizes a different kind of RNA
o (1) RNA polymerase I – Synthesizes pre-ribosomal RNA
♣ Precursors for 28S, 18S, and 5.8 rRNAs
o (2) RNA polymerase II – responsible for synthesis of mRNA
♣ Very fast (500-1000 nucleotides/sec)
♣ Specifically inhibited by mushroom toxin -amanitin
♣ Can recognize thousands of promoters
o (3) RNA polymerase III – makes tRNAs and some small RNA products
o Mitochondria have their own RNA polymerase
Inhibitors of transcription – Used as drugs, some can distinguish between bacterial and eukaryotic systems
- Rifampicin: Inhibits bacterial RNAPS, important in treatment of tuberculosis
- -amanitin: Mushroom toxin, potent inhibitor of RNA polymerase II
- Actinomycin D: Intercalates between two G-C base pairs in DNA
- Daunorubicin: Intercalates between base pairs
- Cordycepin: Chain terminator that lacks 3’ OH
Addition of the 5’ cap
o Enhances stability: protection from nucleases
o Enhances translation efficiency
o Modifications:
♣ 7-methyl-guanosine added by guanylyltransferase
♣ methyl groups sometimes added to riboses by methyl transferases using S-Adenosyl Methionine (SAM) as a donor
o Note: only mRNA is modified with a cap, not tRNA or rRNA
Addition of 3’ Poly-A tail
o Important for mRNA stability
o Helps in translation
o Added downstream of polyadenylation signal in 3’ UTR in a context-dependent manner
♣ 3’ UTR contains cleavage and polyadenylation signals
o Nascent mRNA is considerably longer than length of RNA preceding poly A site
Splicing
o Genes of nucleated cells are fragmented
o Some genes contain more than 90% intronic sequences
o Splicing usually occurs in order from the 5’ to 3’ end of the mRNA precursor, but not always
Cystic Fibrosis
o CFTR encodes the chloride channel which when mutated causes cystic fibrosis (CF)
♣ CFTR gene is composed of 27 exons covering over 189,000 base pairs of DNA
♣ The processed mRNA (introns removed) is approximately 6,000 nucleotides
• only 3% of the gene is finally seen in the mRNA that is translated into protein
Mechanism of mRNA Splicing in Eukaryotes
• The intron is spliced out in the form of a lariat
o Steps: 2 transesterifications
♣ (1) 2’ OH attacks 5’ splice site
♣ (2) Newly formed 3’ OH attacks 3’ splice and exons
♣ (3) Lariat form of intron with 2’-5’ phosphodiester bond formed
MARFAN Syndrome
Disease of splicing • Aortic aneurysm – weakened and bulging area in the aorta
• Arachnodactyly – fingers are abnormally long and slender in comparson to the palm of the hand
• Dural ectasia – the brain and the spinal cord are surrounded by fluid contained by a embrane called the dura, which is primarily connective tissue
o The enlargement of this membrane is reffered to as “dural ectasia”
• Proposed Mechanism: FBN1 mutations resulted in the production of abnormal fibrillin protein that, when incorporated into microfibrils along with normal fibrillin, resulted in structurally inferior connective tissue
o This is consistent with aortic aneurism, a dural ectasia, but not arachnodactyly
• Revised Mechanism: Fibrillin was homologous with the family of latent transforming growth factor (TGF-) binding proteins (LTBPs), which serve to hold TGF- in an inactive complex in various tissues, including the extracellular matrix
o Researchers showed that fibrillin can bind TFG-
o Hypothesis: abnormal fibrillin, or reduced levels of fibrillin, in connective tissue might result in an excess of active TGF-
• New therapy?
o It has been found that blocking TGF- with neutralizing antibodies leads to the normalization of lung development in affected mice
Other Diseases of splicing
• Systemic lupus erythematosus: an autoimmune disease in which serum antibodies to U1 are formed, blocking proper splicing from occurring
o Other connective diseases also exhibit serum antibodies to U1
• Globinopathies: many diseases of globin are caused by splicing defects
• Spinal Muscular Atrophy (SMA): a mutation in exon 7 of the SMN-1 gene leads to decreased survival of motor neurons
• Becker Muscular Dystrophy (BMD): a mutation in exon 27 of the dystrophin gene affects the dystrophin protein and muscle fiber formation
• Frontotemporal Dementia with Parkinsonism (FTDP-17): a mutation in exon 10 of the Tau microtubule associated protein gene which promotes a more stable protein
o Mutant Tau proteins form abnormal filamentous structures in the brains of FTDP-17 patients
Operon
- Functionally related genes in prokaryotes are organized into operons
- The genes of an operon are coordinately controlled: an operon is a coordinated unit of gene expression
Regulatory Elements in Prokaryotic Transcription
o A regulatory gene: example, LacI gene codes for repressor protein
o A promoter: nucleotide sequence that serves as a recognition motif for RNAP binding
o An operator: nucleotide sequence that binds repressor protein
Inducers
• Inducers bind to the repressor to weaken affinity for operator
o In the lac operon, the inducers are allolactose (side product of B-Galactosidase) and IPTG (gratuitous inducers)
Lac Repressor
• Lac Repressor binds tightly to the operator as a tetramer in a tethered dimer configuration: searches DNA in one-dimension to find operator
o It is constitutively expressed from its own promoter
Negative Regulation
• Common patterns of regulation of transcription initiation
o (1) Repressor binds to the operator in the absence of the molecular signal
♣ The external signal causes dissociation of the repressor to permit transcription
o (2) Repressor binds in the presence of the signal
♣ The repressor dissociates and transcription continues when the signal is removed
Positive Regulation
• Common patterns of regulation of transcription initiation
o (1) Activator binds in the absence of the molecular signal and transcription proceeds
♣ When the signal is added, the activator dissociates and transcription is inhibited
o (2) Activator binds in the presence of the signal
♣ It dissociates only when the signal is removed
Glucose Effect
• Glucose is used up first in E. coli, then other carbon sources are utilized
o When glucose goes down, cAMP goes up
o CAMP binds to CAP (Catabolite Activator Proteins aka CRP)
o cAMP-CAP binds to promoter, binding facilitates RNAP binding to promoter
♣ DNA binding properties of CAP
• CAP binding site shows two-fold rotational symmetry
• CAP binding creates additional sites for RNAP interaction
• So glucose is absent, the lac operon transcription is stimulated by CRP-cAMP
• When lactose is absent ~no transcription
o Whether [glucose] is high or low, if lactose is absent, the repressor stays bound
♣ No transcription even when CPR-cAMP binds
o When [glucose] is high, cAMP is low, and lactose is present, there will be LOW levels of lac operon expression
o When [glucose] is low, cAMP is high, and lactose is present, there will be HIGH levels of lac operon expression
Two requirements for strongest induction of the lac operon
- Lactose must be present to form allolactose to bind to repressor and cause it to dissociate from operator (reducing repression)
- [Glucose] must be low so that cAMP can increase, bind to CRP, and the complex can bind near the promoter
• When lactose is low, repressor is bound inhibition
• When lactose is high, repressor dissociates permitting transcriptionally
• When glucose is high, CRP is not bound transcription is dampened
• When glucose is low, cAMP is high and CRP is bound activation
Differences in the strategies for gene regulation in prokaryotes and eukaryotes:
• Differences in the strategies for gene regulation in prokaryotes and eukaryotes: o Prokaryotes ♣ Polycistronic mRNAs ♣ Coupled Transcription/Translation o Eukaryotes ♣ Monocistronic mRNAs ♣ Uncouple Transciption/Translation
Transcription factors may be useful drug targets:
• Transcription factors may be useful drug targets:
o Human cancers are dependent on inappropriate activity of oncogenic transcription factors
o New strategies of targeting transcription factors include disrupting protein-protein interactions and blocking binding at the epigenetic level by modulating chromatin accessibility
RNA polymerase II binding to eukaryotic genes:
• Requires five types of proteins
o (1) Transcription activators (enhancer binding proteins)
♣ Proteins that bind to upstream activator sequences (UAS) that are often palindromic
o (2) Coactivators
♣ Act indirectly (with other proteins, not with DNA)
o (3) Basal Transcription Factors
o (4) Architectural regulators to facilitate DNA looping
o (5) Chromatin modification/remodeling proteins
Assembly of pre-initiation complex
o (1) Pol II is recruited to the DNA by TFs
o (2) The transcription bubble forms
o (3) The CTD is phosphorylated during initiation
♣ The polymerase escapes the promoter
o (4) Transcription elongation is aided by elongation factors after TFIIE and TFIIH dissociate
o (5) Elongation factors dissociate
♣ The Carboxyl Terminal Domain (CTD) is dephosphorylated as transcription terminates, a process facilitated by transcription termination factors
RNA Polymerase
•
RNA polymerase II and its associated basal transcription factors form a preinitation complex (PIC) at the TATA box, a process that is facilitated by transcriptional activators acting through co-activators (Mediator and/or TFIID)
o Composite promoter with typical sequence elements and protein complexes found in eukaryotes
♣ The carboxyl terminal domain (CTD) of pol II is an important point of interaction with Mediator and other protein complexes
♣
Transcriptional activators bind to enhancer sequences (UAS) and coactivators
♣ HMG proteins are architectural regulators that facilitate DNA looping to allow machinery at a distance access to transcriptional start site
o Transcriptional repressors function by many mechanisms, including binding to DNA to displace activator proteins, or interacting with other protein complexes to disrupt initiation
Nucleoside
• A pentose sugar may be attached in an N-glycosidic linkage to the purine or pyrimidine base, in which case the compound is called a nucleoside
Nucleotide
o A phosphate may be esterified to one of the OH groups, typically attached to the C-5’ of the nucleoside pentose, in which case the compound is called a nucleotide
Hyperuricemia
• Uric acid is the least soluble purine base and exists at or near saturation levels in most individuals
• (1) Primary gout is characterized by hyperuricemia due to a variety of inherited metabolic abnormalities
• (2) Secondary gout is characterized by excessive uric acid production due to a coexisting acquired condition
♣ Long term treatment of gout involves administration of allopurinol, which inhibits the rate of uric acid production
Xanthinuria
Disease of nucleotide metabolism
♣ Xanthine is the second most insoluble purine base and can also precipitate when overproduced, a condition known as xanthinuria
♣ Xanthine can results from a variety of abnormalities, the most common of which is a deficiency of xanthine dehydrogenase, estimated to occur in 1 in 6,000-60,000 individuals
Immunodeficiency Diseases (caused by nucleotide metabolism)
♣ Adenosine deaminase deficiency
• A deficiency of adenosine deaminase (ADA) is associated with a severe combined immunodeficiency involving T-cell and B-cell dysfunction
• The mechanism is not completely understood, but it is likely due to accumulation of dATP, particularly in lymphocytes
o dATP is an effective inhibitor of ribonucleotide reductase and, consequently, of DNA synthesis and cell division
♣ Purine nucleoside phosphorylase (PNP) deficiency
• A deficiency of purine nucleoside phosphorylase (PNP) is associated with impairment of T cell function
o PNP-deficient individuals under excrete uric acid and over excrete PN subtrates
o Accumulation of dGTP in T-cells with resulting inhibition of ribonucleotide reductasae and DNA replication has been proposed as an explanation for immune dysfunction in this disorder
Lesch-Nyhan Syndrome
• Lesch-Nyhan Syndrome – an inherited disorder of salvage synthesis
o HPRT is lacking or is present at very low levels (<1% of normal) in these individuals
♣ The HPRT locus is located on the X chromosome, but because this disorder is very debilitating, affected individuals do not reproduce – therefore, males are almost exclusively affected
o This syndrome is associated with aggressive behavior, self-mutilation, and mental retardation
o Because of the disruption in salvage synthesis, these individuals also display high levels of uric acid and gout
o High levels of PRPP and increased rates of de novo synthesis are observed because reduced synthesis of purine nucleotides by the salvage pathway results in inadequate feedback inhibition of PRPP synthetase and ATase
♣ Increased levels of PRPP lead to an increase in de novo synthesis and a resulting overproduction of uric acid
o Identified mutations in the HPRT gene have variable effects on enzyme activity (0.1 to ~10% residual)
♣ Severity of enzyme deficiency is correlated with the degree of neurological complications
o Although administration of allopurinol to Lesch-Nyhan patients relieves the complication of gout, there is no known treatment for the neurological problems
♣ The brain has 10-20 times the HPRT enzymatic activity found in other tissues, thus it is possible that high levels of purine catabolites are toxic to the developing brain, or perhaps the lack of HPRT leads to an imbalance in purine nucleotides at critical times during development
Inherited disorders of pyrimidine nucleotide metabolism
• Orotic aciduria-UMP synthase deficiency
o Characterized by an excessive amount of orotic acid in the urine and accompanying metaloblastic anemia
♣ An apparent defect in the UMP synthase gene renders both OPRT and OMP decarboxylase dysfunctional, although it is the deficiency of the former which would result in the accumulation of orotic acid
♣ Lethal, unless treated by pyrimidine replacement, i.e., uridine
• Uracil is ineffective as therapy
• Pyrimidine 5’-nucleotidase deficiency
o Characterized by vastly increased levels of erythrocyte pyrimidine ribonucleotides (particularly cytidine nucleotides) and an accompanying hereditary anemia
• Dihydropyrimidine dehydrogenase (also called dihydrouracil dehydrogenase) deficiency
o Characterized by elevated levels of uracil and thymine in body fluids
o Convulsive disorder characterized by microencephaly
• Dihydropyrimidinase deficiency
o Characterized by elevated levels of dihydropyrimidines in body fluids
o Variable neurological symptoms
Basic Components of Translation
Amino Acid, tRNA, mRNA, Amino Acid tRNA synthetases, Ribosomes, Initiation, Elongation, and Termination Factors, ATP and GTP
Amino Acids in Translation
• Amino acids: All amino acids in a protein
o Must be present at the time of protein synthesis
o Essential amino acids
tRNAs in Translation
• tRNAs: a.k.a. adaptor molecules with highly ordered structure o At least 1 per amino acid o Actually >50 in humans o Contain two important sites ♣ (1) amino acid attachment site ♣ (2) anticodon sequence
mRNAs in Translation
• mRNAs: Translated in the 5’ 3’ direction
o In E. coli, transcription and translation are closely coupled regulation of protein expression is largely controlled at transcriptional level
Amino Acid tRNA synthetases
Amino Acid tRNA synthetases: Family of enzymes that attach amino acids to corresponding tRNAs
Ribosomes in Translation
• Ribosomes: Ribonucleoprotein complexes; consist of two subunits:
o Prok – 30S + 50S = 70S
o Euk – 40S + 60S = 80S
ATP and GTP in Translation
• ATP and GTP: energy sources
o Cleavage of 4 high energy bonds (ATP and GTP) are required for the addition of one amino acid
o Initiation and termination require additional ATP and GTP
Ribosomes in Eukaryota and Prokaryota
Ribosomes:
• Overall, Euks and Proks are very similar
• Both have two subunits with mRNA running between
• In eukaryotes, larger (80S), more complex, contain > 80 proteins
• Chloroplasts and mitochondria have ribosomes simpler than those in bacteria
• Ribosomal subunits are identified by their S (Svedberg unit) values, sedimentation coefficients that refer to their rate of sedimentation in a centrifuge
The genetic code:
The genetic code:
• There are 20 common, genetically encoded amino acids
• The genetic code for proteins consists of triplets of nucleotides
o A four-letter code in groups of two is insufficient (16)
The code is read as a sequence of triplets called codons
o There are 64 different combinations of 3 base codons (43)
o 61 of these code for the 20 amino acids (then there are 3 stop codons)
5’ … CCGUAGU AUG CGA GGU GGA CUA UAA GGAUAAC … 3’
• The code has the following properties:
o It is specific – a specific codon always codes for the same amino acid
o It is essentially universal (mito is slightly different)
o It is redundant (or degenerate) – a given amino acid may have more than one codon (synonyms, mostly differ in 3rd position)
o It is non-overlapping and comma-less
♣ The code is read from a fixed starting point as a continuous sequence of bases, three at a time
♣ Frameshift mutations occur if the reading frame is altered
Additional features off the genetic code
• The code is written in the 5’ 3’ direction
• Third base is less important in binding to tRNA
• First codon establishes the reading frame
o If the reading frame is thrown off by a base or two, all subsequent codons are out of order
• 61/64 codons code for amino acids
• Three are termination codons: UAA, UGA, UAG
• AUG = initiation codon (as well as Met codon)
Termination codons:
UAG, UGA, UAA do not code for amino acids (no tRNAs)
Silent mutations
o Silent mutations – codon base is changed to codon that codes for same a.a.
UCA = Serine
UCU = Serine
Missense mutations
o Missense mutations – codon is changed to codon that codes for a different a.a.
UCA = Serine
CCA = Proline
Nonsense mutations
o Nonsense mutations – codon is changed to become a termination factor
UCA = Serine
UAA = Terminate
Degeneracy of the Genetic Code
- Most amino acids have more than one codon (Arg, Leu, and Ser have 6)
- Only Met and Trp have a single codon
- Some codons are less subjective to causing a mutation in an amino acid sequence because of degeneracy or because of the abundance of such tRNAs
Codon recognition by tRNA
• tRNA anticodons recognize codons
o Binding follows the rules of complementary base pairing
o mRNA is read 5’ 3’ by a flipped anticodon
Molecular Recognition of Codons in mRNA by tRNA
Molecular Recognition of Codons in mRNA by tRNA
• tRNA anticodons recognize codons
o Binding follows the rules of complementary base pairing, mRNA is read 5’ 3’ by a flipped anticodon
• Wobble Hypothesis
o tRNAs can recognize more than one codon for a specific amino acid due to “wobble” at the 5’ end of the tRNA anticodon, forming non-traditional base pairs
o The result is that there need not be 61 tRNAs to read 51 codons
• Suppressor tRNAs
o Nonsense suppressors are tRNAs whose anticodons have been mutated such that they incorporate an amino acid at termination codons (i.e., they suppress the normal termination of a protein)
• The codon sequence is * with the anticodon sequence
complementary
• The codon in mRNA base pairs with the ** in the mRNA via **
anticodon;
hydrogen bonding
The alignment of two RNA segments is ***
Antiparallel
The Genetic Code is Resistant to Mutation Due to Degeneracy
• Wobble Hypothesis helps to explain degeneracy of the code:
o Degenerate code allows certain mutations to still code for the same amino acid
♣ “Silent” mutations – different nucleotide in DNA but same amino acid in protein
• Mutation in first base of a codon usually produces a conservative substitution
o Example: GUU Val but AUU Leu
Protein Synthesis Involves Five Stages:
- Activation of amino acids
a. tRNA is aminoacylated - Initation of translation
a. mRNA and aminoacylated tRNA bind to ribosome - Elongation
a. Cycles of aminoacyl-tRNA binding and peptide bond formation until a STOP codon is reached - Termination and ribosome cycling
a. mRNA and protein dissociate, ribosome recycled - Folding and post-translational rocessing
a. Catalyzed by a variety of enzymes
Activation of Amino Acids:
- Charging tRNA with amino acids
- Requires Aminoacyl-tRNA Synthetase, ATP, Mg2+
Amino Acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi
• Aminoacyl-tRNA Synthetase
o Each enzyme binds a specific amino acid and the matching tRNA
o Most cells contain 20 different aminoacyl-tRNA synthetases, one for each amino acid
o Some cells contain less than 20 synthetases; in this case, one amino acid is converted to another after charging the tRNA
o Aminoacyl-tRNA synthetases must be specific for both amino acid and tRNA
♣ Matching each amino acid with correct tRNA can be viewed as the “second genetic code”
♣ The “code” is in molecular recognition of a specific tRNA molecule by a specific synthetase
o Only a few nucleotides in tRNA confer the binding specificity: Anticodon region
Initiation
• Prokaryotes often produce polycistronic mRNAs
o The RNA component of the ribosome (16S rRNA) is involved in the correct positioning of the ribosome at the translation start site
o The Shine-Dalgarno sequence pairs with the 16S rRNA – positions ribosome in the right place to start translation
• Eukaryotes produce monocistronic mRNAs
• Initiation Codon: recognized by special initiator tRNA
o Facilitated by:
♣ IF-2 in E. coli
♣ eIF-2 in humans
o In bacteria and mitochondria, initiator tRNA carries N-formyl methionine
o In humans there is no formylation
• fMet or Met is the first amino acid in a peptide
o First codon is AUG
o All organisms have two tRNAs for Met
♣ Even eukaryotes, whose proteins begin with Met, not fMet, still use a special tRNA for initiaton
Elongation
- Peptidyl transferase catalyzes peptide bond formation
* Ribosome moves one codon using energy from hydrolysis of GTP bound to EF-G
Coupling of transcription and translation in bacteria
• General idea: because of this coupling, regulation is largely controlled at the level of transcription
Eukaryotic vs Prokaryotic Protein Synthesis Mechanisms
• Generally conserved, but some steps are more intricately regulated
o Ribosomes
♣ Eukaryotic: 80S, can be dissociated into:
• 60S large subunit (5S, 28S RNAs, and unique 5.8S)
• 40S small subunit (18S RNA)
♣ Prokaryotic: 70S, can be dissociated into
• 50S large subunit (5S, 28S RNAs)
• 30S small subunit (16S RNAs)
o Initiator tRNA
♣ In eukaryotes: the initiator amino acid is methionine and it is encoded by MET-tRNAi
♣ In prokaryotes: proteins start with N-formylmethionine
o Initiation of Translation
♣ The selection of AUG (Met codon) to initiate translation differs in prokaryotes and eukaryotes
• In prokaryotes, initiation occurs at an AUG adjacent to a purine rich sequence called the Shine-Dalgarno sequence, which base pairs with the 16S RNA
•
In eukaryotes, the AUG nearest the 5’ end of the mRNA is generally utilized as the initiator in MET
o The 40S ribosome loads at the 5’ end of the message and ‘scans’
o This stepwise movement requires energy (ATP) and helicase
Eukaryotic Protein Synthesis – Initiation
eIF1A and eIF3 help keep 60S and 40S dissociated, eIF1 helps to bind ternary complex
- Ternary complex (eIF2-GTP:met-RNA) binds to 40S along with eIF5B-GTP to create the 43S preinitiation complex
- 43S complex binds eIF4F bound CAP of mRNA to form 48S complex
- 48S complex unwinds any helix structure and sans to find first AUG (eIF4 has helicase activity)
- Once AUG found, 60S ribosomal subunit binds and releases any of the initiation factors, and the 80S initiation complex is formed
Translational control by phosphorylation
• Stimulate:
o eIF4E is a subunit of eIF4, and it recognizes and binds to the 5’ cap of the mRNA and promotes binding of mRNA to the 40S subunit
o eIF4E needs to be phosphorylated to be active
o Factors that stimulate translation through eIF-4E proteins:
♣ Tumor Necrosis Facter alpha
♣ Insulin
♣ Epidermal Growth Factor
♣ Platelet Derived Growth Factor
♣ Nerve Growth Factor
♣ Interleukin 1
• Inhibit:
o Phosphorylations of eIF2 blocks formation of 43S preinitiation complex
o Polio virus causes the proteolysis of cap binding protein (eIF4E) allowing its own uncapped mRNA to be translated
Diptheria Toxin
• Diptheria Toxin: blocks eukaryotic translation by inhibiting translocation; ADP ribosylation of EF2-translocase (eukaryotic version of EF-G)
o Diptheria toxin blocks eukaryotic translation by inhibiting translocation
o ADP ribosylation of EF2-translocase inactivation of all of the host cell EF-2 molecules causes death of the cell
o Attachment of the ADP ribosyl group occurs at an unusual derivative of histidine called diphthamide
Proteolytic Processing
• Proteolytic Processing: occurs in the ER, Golgi apparatus, secretory cesicles; i.e., zymogens, inactive precursors of secreted enzymes (preforms), include many digestive proteases
o Familial Hyperproinsulinemia: abnormally processed insulin due to deficiency in enzymes that process insulin
♣ Afflicted individuals are normal in glucose metabolism
Phosphorylation
• Phosphorylation: most often hydroxyl groups of serines or threonines, sometimes tyrosine, by protein kinases; can often increase or decrease the function of a protein or allow protein:protein interaction
Glycosylation
• Glycosylation: carbohydrate chains attached to hydroxyl groups of serines or threonines (O-linked) or asparagine (N-linked); occurs in stepwise manner in ER and Golgi; often found in proteins destined for secretion or part of the plasma membrane
o I-Cell Disease: Lack glycosyl transferase therefore cannot properly target lysosomal enzymes: patients have abnormally high levels of lysosomal enzymes in their sera and other bodily fluids
♣ Disease is characterized by psychomotor retardation, coarse facial features, and restricted joint movement
♣ Usually death occurs by age 8
Hydroxylation
• Hydroxylation: i.e., proline and lysine residues of collagen
o Scurvy causes decrease in hydroxyproline synthesis
o Type IV Ehlers-Danlos syndrome is deficient in lysyl hydroxylase
Ubiquitination
• Ubiquitination: protein trafficking and protein turnover, i.e., degradation
o In ekaryotes, proteins are linked to the protein ubiquitin via activating enzyme E1, conjugating enzyme E2, and ligating enzyme E3
o Ubiquinated proteins are cleaved by the 26 protein complex
o Ubiquitin is very highly conserved
Protein Targeting
• Protein Targeting: Proteins move from the site of synthesis to:
o Exit cell
o Become part of the membrane
o Enter a subcellular compartment, etc
♣ Most have a signal sequence at or near N-terminus
What are lipids?
• Lipids, along with amino acids, nucleotides, and carbohydrates, are the building blocks from which most cellular macromolecules are constructed
• “Lipids” – a chemically diverse group of compounds defined by their insolubility in water (i.e., are hydrophobic molecules)
o Their functions are also diverse and include storage form of energy, structural elements of membranes, hydrophobic anchors for proteins, cellular signaling hormones, lipid-soluble vitamins, and emulsifying agents in the digestive tract
Role of Lipids as Structural Components of Membranes
- Each organelle has a unique lipid and protein composition
- “Fluid Mosaic” lipids and proteins move laterally in the plane of the membrane
- Membranes are not uniform (micro domains or “rafts”)
Cardiolipin
o Cardiolipin: important component of the inner mitochondrial membrane
♣ Required for function of enzymes involved in mitochondrial energy metabolism
Barth Syndrome
o Barth Syndrome: Deficient cardiolipin synthesis
♣ Infantile death
♣ Cardiomyopathy
♣ Decreased mitochondrial ATP production
Fatty Acids
• Fatty acids are carboxylic acids composed of a hydrocarbon chain with a carboxyl group at one end and a methyl group at the other end (called the -carbon)
o Most lipids contain fatty acids or are derived from fatty acids
♣ Exception is cholesterol which is synthesized from acetate
• Sterate 18:0 — Saturated/unbranched
•
Oleate 18:19 — Unsaturated/branched
Phospholipids (Glycerophospholipids)
• Glycerophospholipids contain a glycerol backbone linked to two fatty acid-derived tails by ester linkages
• The fatty acid components attached to C1 and C2 are hydrophobic
• Saturated fatty acid is attached to the C1 carbon
• Unsaturated fatty acid, such as arachidonic acid, is attached to the C2 carbon
• The molecule attached to C3 of glycerol is hydrophilic (phosphate)
• Phosphatidic acid (PA) is the precursor in the synthesis of glycerophospholipids and triacylglycerols
o Just an –H at the head-group substituent
Phosphatidic Acid (PA) Synthesis
• PA is formed by adding two fatty acids (from the respective acyl-CoA derivatives) onto glycerol-3-phosphate by acyl transferases
• Glycerol-3-phophate is primarily derived from reduction of the glycolytic intermediate dihydroxyacetone phosphate
• Phosphatidic acid can also be formed by:
o (1) Phosphorylation of diacylglycerol (DAG) by DAG Kinase
or
o (2) Phospholipase D-mediated hydrolysis of glycerophospholipid
Glycerophospholipids – Phosphatidates
• Phosphatidates are derivatives of the basic parent compound, phosphatide acid (PA)
• Polar groups esterified to the phosphate moiety
• Cytidine triphosphate (CTP) is involved in the synthesis
o The second most important role for CTP (after nucleic acid synthesis) is phospholipid synthesis
• Pulmonary Surfactant and Lung Function
o Alveolar structures
♣ Basic units for gas exchange
♣ Have large surface areas with high surface tension at the air-water interface that opposes lung function
o Pulmonary surfactant is synthesized/secreted by alveolar type II epithelial cells
♣ Secreted surfactant forms a film covering the epithelial cells
♣ Pulmonary surfactant reduces surface tension in lung alveoli, thereby decreasing their tendency to collapse during expiration
♣ Surfactant deficiency is the primary cause of neonatal respiratory distress syndrome (RDS), and surfactant supplementation in premature infants with RDS significantly reduces mortality in these patients
o Pulmonary surfactant is a complex mixture of lipids and proteins with dipalmitoylphosphatidylcholine (DPPC) being the most abundant phospholipid in the mixture
o Lipids constant ~90% of the composition of pulmonary surfactant
Phospholipases
• Phospholipases
o Cleave phospholipids
o Phospholipid remodeling
o Release of arachidonic acid
o Intracellular signaling
• Some remove fatty acids from the C1 (phospholipase A1) or C2 (phospholipase A2) position of the glycerol backbone, which permits re-esterification reactions with other fatty acyl CoAs or release of arachidonic acid (from C2) to be used to synthesize eicosanoids (paracrine hormones)
• Other phospholipases cleave the C3 phosphodiester to produce either diacylglycerol, DAG (phospholipase C) or phosphatidic acid (Phospholipase D)
Phosphatidylinositols (PIs) and Cell Signaling
• Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two products that are active in cell signaling:
o (1) Diacylglycerol (DAG)
o (2) Inositol 1,4,5-triphosphate (IP3)
♣ IP3 triggers release of Ca2+ from the ER, which activates protein kinase C (PKC) involved in cell signaling events
Anchoring Membrane-associated Proteins
• Some extracellular proteins can be associated with the outer surface of the plasma membrane by a glycosyl phosphatidylinositol (GPI) anchor
o These are covalently bound to the C-terminal residue
• Some intracellular proteins can be associated with the cytosolic side of membranes through specific interactions with:
o Long-chain fatty acids (i.e., palmitic acid 16:0, and myristic acid 14:0)
o Isoprenoids such as the polyisoprenes farnesyl (C15) and geranylgeranyl (C20)
• In contrast, integral membrane proteins span a lipid bilayer
o The region of the protein in the lipid bilayer is enriched in hydrophobic amino acids that interact with the nonpolar fatty acid side chains of the phospholipids
Sphingolipids
- Sphingolipids are found in most membranes, but are particularly abundant in cells of the central nervous system and brain
- Like glycerophospholipids, sphingolipids contain a polar head group and two nonpolar tails
- Sphingolipids do not contain glycerol, but one molecule of the long-chain amino alcohol, sphingosine
- Ceramide is the base compound of the sphingolipids (phosphatidic acid serves this role for the glycerophospholipds)
o Sphingomyelins:
♣ Phosphocholine or phosphoethanolamine as their polar group, and because of the presence of phosphate, are also classified as phospholipids
♣ Abundant in myelin, the membranous sheath that surrounds and insulates the axons of some neurons
• Glycosphingolipids:
o Have polar head groups with one (cerebrosides) or more (globosides) carbohydrates
o Found primarily on the outer surface of the plasma membrane
o Also called neutral glycolipids because they are uncharged at pH 7
• Gangliosides (e.g. GM1, GM2)
`
o Have oligosaccharides with one or more N-acetylneuraminic acid (Neu5Ac)
♣ Neu5Ac is a sialic acid
o Are negatively charged glycolipids due to the presence of sialic acid
o Found at their highest concentration in the nervous system and brain
Sphingolipidoses (Lysosomal Storage Disease)
• Defects in the recycling center (lysosome)
• Family of progressive, degenerative diseases with multi-organ involvement
o Skeletal deformities, mental retardation, heart disease, decreased life expectancies are common features)
• ~50 different diseases (e.g., Tay-Sachs, Niemann-Pick)
o Only FDA approved treatments for about 6 of them
• Genetic disorders: individually rare, altogether incidence is ~1 in 6,000 live births (as a group they are among the most common genetic disorders in children)
• Caused by deficiency of an enzyme necessary to breakdown macromolecules, resulting in accumulation of undegraded material in the lysosome
Sphingolipidoses continued…
•
Degradation occurs in lysosomes by the action of hydrolytic enzymes, including phospholipases and glycosidases
• When activity of one of the hydrolytic enzymes is decreased or absent due to a genetic error, then the substrate accumulates in the lysosomes of the tissue(s) responsible for its catabolism
• Tay-Sachs and Niemann-Pick are two examples of the lysosomal storage diseases caused by the abnormal accumulation of sphingolipids
o Patients have progressive neurodegeneration and exhibit severe neurological defects because molecules are highly enriched in the brain and CNS tissues
Eicosanoids
•
Fatty acid derivatives that are short-lived and have a variety of effects on human tissues and cells:
o Inflammation
o Fever and pain associated with injury
o Reproductive function
o Formation of blood clots
o Regulation of blood pressure
• Family of paracrine hormones that act as short-range signaling molecules, affecting tissues near the cells that produce them
o They mediate their biological effects by interacting with cell surface G-protein coupled receptors (GPCRs)
3 Classes of Eicosanoids
• Three classes: Prostaglandins (5 member ring), Thromboxanes (6 member ring) and Leukotrienes (linear)
o All eiconsanoids are synthesized from arachidonic acid, a 20-carbon polyunsaturated fatty acid
♣ The precursor to arachidonic acid is the essential fatty acid linoleic acid
o Arachidonic acid is a component of membrane phospholipids, which makes it readily available for eicosanoid synthesis following phospholipase A2 cleavage in response to hormonal or other stimuli
Dietary Linoleic Acid
Dietary Linoleic Acid is required for synthesis of arachidonic acid and eicosanoids
• Mammals cannot convert oleate to linoleate or -linolenate, which are therefore required in the diet as essential fatty acids
Synthesis of Prostaglandins and Thromboxanes by the Cyclooxygenase Pathway
• Arachidonic acid is cyclized and oxidized by the bifunctional enzyme cyclooxygenase (COX), also called prostaglandin H2 synthase
• Mammals have two isoforms of COX that differ in their expression:
o COX1: constitutively expressed in many cells
o COX2: exhibits inducible expression in selected cell types
• The resulting 5-membered ring (e.g., PGH2) distinguishes prostaglandins from other eicosanoids
• Thromboxanes are then synthesized from PGH2 by thromboxane synthase in platelets that converts the cyclopentane ring into a six-membered oxygen containing (oxane) ring
Synthesis of Leukotrienes
• Unlike the ring-containing eicosanoids, leukotrienes are linear molecules with 4 double bonds (3 of the double bonds are in series, forming a “triene”)
• Lipoxygenase converts arachidonic acid to leukotrienes and catalyze the incorporation of 2 atoms of oxygen, forming a hydroperoxy (-OOH) group on the molecule
o Leukotrienes differ from each other due to differences in the position of this peroxide group
• 5-Lipoxygenase produces the major leukotriene members, generating 5-hydroperoxyeicosatetraenoate (5-HPETE)
o 5-HPETE is converted to leukotriene A4 (LTA4), which can be further modified to generate other leukotrienes
Inhibitors of Eicosanoid Synthesis
- Corticosteroids (prednisone) – potent anti-inflamatory drugs
a. Surpress COX2 synthesis
b. Inhibit phospholipase A2 mediated release of arachidonic acid from membrane phospholipids
c. Decreased production of pro-inflammatory eicosanoids - Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) – ibuprofen/aspirin
a. Inhibit prostaglandin and thromboxane synthesis, but not leukotriene synthesis
b. Inhibit both isoforms of COX
c. Aspirin, but not ibuprofen, irreversibly inactivates the cyclooxygenase activity of both COX1 and COX2 by acetylating the enzyme and blocking the enzyme’s active site
i. Side-effect of COX1 inhibition: stomach irritation - Prostaglandins regulate gastric mucins production, which are highly glycosylated proteins of the gastric mucosa that protect the stomach
- Low dose of aspirin decreases risk of heart attack and stroke by reducing thromboxane synthesis
a. Thromboxane induces platelet aggregation, an early step in blood clotting - COX2 specific inhibitors (Celebrex, Vioxx)
a. Side effect: increased risk of heart attack and stroke, suggests an imbalance in the fine tuning expression levels of the different eicosanoids
b. Only Celebrex remains available in the U.S.
Anti-leukotriene drugs
• Several leukotrienes have pro-inflammatory effects
• Elevated levels of certain leukotrienes are associated with asthma and anaphylactic shock (induce contraction of the smooth muscle lining the airways of the lungs, thereby increasing bronchoconstriction)
• Some asthma medications target the leukotriene pathway by inhibiting:
o 5-lipoxygenase activity
o
the interaction of leukotrienes with their cell surface receptors
Functions of Cholesterol (Polyisoprenoid)
• Precursor for the synthesis of: o Steroid Hormones o Bile Acids o Vitamin D • Structural component of membranes
Cholesterol in Membranes
• Found in all plasma and intracellular membranes
• Regulates the fluidity of the lipid bilayer
• Most cholesterol in membranes occurs in an unesterified form
• Amphiphathic lipid
o Extensive nonpolar region – 4 fused rings
o Small polar region – hydroxyl group
2 Forms of Cholesterol
- Cells tightly regulate the amount of unesterified cholesterol, with excess cholesterol being esterified and packaged as lipid droplets inside cells
- Unesterified membranes Esterified lipid droplets + inside lipoprotein particles
Lipoprotein Particles
• Cholesterol exhibits low solubility in water but its concentration in human serum is high
• Cholesterol’s high solubility in SERUM is due to it being packaged into spherical particles called lipoprotein particles (e.g., LDL, HDL)
o LDL carries cholesterol to tissues/cells that need it
• Composed of outer shell of phospholipids, unesterified cholesterol, and plasma lipoproteins (e.g., Apo B 100)
o Inside: cholesterol esters and triacylglycerol
Cholesterol Synthesis
• All 27 carbons are derived from acetate
• De novo synthesis of cholesterol occurs in virtually all cells, with the greatest capacity in liver, intestine, adrenal cortex, and reproductive tissues
• The biosynthesis of cholesterol can be broken into 4 stages involving 37 steps
o The rate-limiting step is carried out by HMG-CoA Reductase
♣ Inhibitors have been designed to target this enzyme
o Blocking de novo synthesis of cholesterol is used as a means to lower serum cholesterol levels (High serum LDL correlate w increased risk of CVD and stroke)
o Statins, such as Lipitor, are competitive inhibitors of HMG-CoA Reductase
Advantages of covaently attaching various molecules (e.g., phosphate, lipids, carbohydrates) in a post-translational fashion to generate modified proteins:
• Alter protein activity/function without having to synthesize a whole new protein
• Modifications are a mechanism to increase the functional diversity of a limited gene pool
o >200 different modified amino acids have been found in proteins
o Post-translational modifications (“PTMs”) increase the number of letters in amino acid alphabet and lead to a combinatorial explosion of diversity
♣ Multiple PTMs provides cells with a means to integrate different pathways and coordinate responses to different physiological conditions
• The majority of proteins contain covalently attached molecules (i.e., proteins contain more than just amino acids)
o Carbohydrates (~1/2 of all proteins undergo glycosylation)
♣ Linkages: N-linked (Asn-X-Ser/Thr)
O-linked (Ser)
o Phosphate: (Ser, Thr, Tyr; ~1/3 of all proteins undergo phosphorylation)
o Hydroxyl: Pro; Acetyl: Lys; ADP-Ribose (Arg)
o Ubiquitin (76 aa protein; Lys)
o Fatty acids (Cys, Gly); Lipids (Cys), Glycosylphosphatidyl-inositol (GPI)
Glycosylation – addition of carbohydrate to a protein
• The most common protein modification – 50% of all proteins
• Names “glycan” and “oligosaccharide” are used interchangeably
o Polymer containing 2 or more monosaccharides joined together by O-glycosidic linkages (Glycogen)
o Carbohydrates are the building blocks to form oligosaccharides on proteins
o Carbohydrates can be joined by different linkages
Nucleotide Sugars are the Substrates
• Nucleotide sugars:
o Monosaccharides must be “activated” to high-energy compounds before they can function as immediate precursors in glycoprotein/glycolipid synthesis
• Synthesis occurs in the cytoplasm (exception is CMP-Neu5Ac)
Nucleoside triphosphate + sugar 1-phosphate nucleoside diphosphate sugar + PPi
• Examples: UDP-Gal, UDP-Glc, UDP-GlcNAc, and GDP-Man
• Key Points:
o Glucose (Glc) is the major in vivo source for the synthesis of nucleotide sugars
o ATP is required for the synthesis of the early intermediates in the pathway (i.e., sugar 1-phosphate or sugar 6-phosphate)
♣ UTP, GTP, or CTP are used at late stages in the pathway
o Glutamine is required for the synthesis of UDP-GlcNAc
Classic Galactosemia
• Cause by a deficiency of galactose-1-phosphate uridyltransferase (GALT)
o Infants deficient in GALT fail to thrive, and have hepatomegaly, jaundice, and cataracts
o Accumulation of galactose and galactose-1-phosphate, and the metabolites galactitol and galactonate contribute to symptoms
o Treatment: eliminate lactose and galactose from the diet
o Newborn screening in many states, including WI, for GALT deficiency
Nucleotide Sugars involved in a Variety of Pathways
• UDP-glucose (UDP-Glc) is used by glycogen synthase to make glycogen
• UD-glucuronic acid (UDP-GlcA) is the substrate for several families of glucouronyltransferases involved in
o Glycoprotein/Proteoglycan biosynthesis
o Drug detoxification of xenobiotics (e.g. clinical drugs, environmental toxins)
o Excretion of steroid hormones
o Heme metabolism and excretion of bilirubin (red blood cell turnover)
• Resident integral membrane proteins of the endoplasmic reticulum
• >300 different glycosyltransferases
• Specific
o (1) sugar that is transferred
o (2) acceptor substrate
o (3) type of linkage generated
• Anomeric/C1 carbon of the sugar becomes glycosidically bonded to the hydroxyl group of the acceptor
Synthesis and Trafficking of Glycoproteins
• The majority of glycosylation reactions occur in the lumen (interior) of the ER and Golgi (secretory pathway)
• Proteins move through the secretory pathway (ER, Golgi, plasma membrane) in transport vesicles
o (1) Secreted and transmembrane proteins enter the ER as they are being synthesized by ribosomes
o (2) Proteins exit the ER in transport vesicles
o (3) Proteins travel through the cisternae of the Golgi apparatus
o (4) Proteins are sorted at the trans Golgi Network (TGN)
o (5) Soluble proteins are secreted from the cell
♣ A transmembrane protein will be present in the plasma membrane with its glycans exposed to the extracellular space
2 Major Types of carbohydrate-proteins linkages
- N-Glycosidic Linkage
a. Requires the triplet sequence Asn-X-Ser(Thr)
b. Found in membrane and secretory glycoproteins - O-Glycosidic Linkage
Biosynthesis of N-linked sugars:
• Preassembly of a glycan (oligosaccharide) chain occurs on a lipid called dolichol phosphate (dolichol-P), a membrane-associated isoprenoid derivative
o Mevalonic acid serves as the precursor of dolichol-P, as well as cholesterol
o The regulation of dolichol-P is directly related to cholesterol metabolism
• Translation of protein in the rough endoplasmic reticulum
• Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of the 14 residue oligosaccharide chain (Glc3Man9GlcNAc2) from dolichol-P to the Asn residue in certain Asn-X-Ser(Thr) sequences of growing polypeptide chains
o This reaction occurs exclusively in the lumen of the ER
• SUMMARY:
o 14 different glycosyltransferases:
♣ First 7 steps occur on sytosolic face of RER
♣ Last 7 steps occur in the lumen of RER
Processing of N-linked sugars:
• The specific location of the various processing enzymes in the ER or the cis, medial, or trans Golgi cisternae provide a mechanism for controlling their sequential action on newly synthesized glycoproteins in both space and time
o The final oligosaccharide structure assembled on a glycoprotein is dictated to a large extent by the order in which that glycoprotein encounters specific processing glycosyltransferases and glycosidases
• In contrast to glycosyltransferases that function to add carbohydrates to proteins or lipids, glycosidases are hydrolytic enzymes that remove carbohydrates
o Glycosidases are grouped into families based on the type of carbohydrate they remove; (e.g., glucosidase, mannosidase)
• The initial N linked oligosaccharide structure (Glc3Man9GlcNAc2) is recognized by chaperones in the ER called calnexin and calreticulin, which are lectins (proteins that bind to a specific glycan structure) that assist newly synthesized proteins to fold into their native conformation
o These chaperones are part of the “quality control mechanism” in the ER that facilitates protein folding and recognizes mis-folded proteins that are subsequently targeted for degradation in the cytosol by the proteasome
O-Glycosidic Linkage between Xyl and Ser
• Found in proteoglycans – a glycoprotein that contains a specific type of oligosaccharide/glycan called a glycosaminoglycan
o Proteoglycans often contain large numbers of glycosaminoglycan chains
Biosynthesis of Proteoglycans
• Proteoglycans consist of a protein backbone to which glycosaminoglycan chain(s) are covalently attached
o Glycosaminoglycans are negatively charged polysaccharides composed of repeating disaccharide units
o The disaccharide unit consists of an amino sugar and an uronic acid, both of which may be sulfated, thus contributing to the polyanionic properties of the molecule
• The addition of carbohydrates occurs in a stepwise fashion in the Golgi
o The glycosoaminoglycans chondroitin sulfate, heparin, heparin sulfate, and dermatan sulfate are attached to proteins via a Xyl-Ser O-linkage
• Hyaluronic acid, which is not covalently attached to protein and is not sulfated, serves as a scaffolding platform for large numbers of proteoglycans in the extracellular matrix
• Proteoglycans are found in almost all mammalian tissues as ubiquitous components of the cell surface and extracellular matrix
o They are especially prominent in connective tissues
o In the cornea, proteoglycans serve as “spacers for collagen,” facilitating collagen’s arrangement which is critical for the transparency of the cornea
Mucins: heavily O-glycosylated glycoproteins
• Glycans linked to serine (Ser) or threonine (Thr)
• Found in mucous secretions or in the cell surface
• Synthesized by many epithelial cells and by goblet cells of the tracheobronchial, gastrointestinal, and genitourinary tracts
• Hydrate and protect epithelial cells, but have other functions as well (e.g., fertilization)
• Example of a clinically relevant mucin:
o The Cancer Antigen 125 (CA125) is found on the mucin Muc16
o CA125/Muc16 is used as a marker for the diagnosis of ovarian cancer, with the majority of ovarian cancer patients having elevated CA125 levels
o A decline in serum CA125 levels is associated with a response to therapy
Functions of Carbohydrates
• Glycosylation is critical for normal development:
o Numerous genetic defects of glycan synthesis involving defective synthesis of:
♣ N-Glycans: large family of diseases called “congenital disorders of glycosylation” (CDGs)
♣ Glycosaminoglycans (e.g., Chondrodysplasias)
♣ O-linked Glycans: -dystroglycan (at least 5 different muscular dystrophies)
• Protein folding/conformation
o Quality control mechanism in the ER
Modification of Protein Function (Erythropoietin)
• Erythropoietin (EPO) is a growth factor secreted by the kidney: stimulates the production of red blood cells (induces the proliferation and differentiation of erythroid progenitors)
• Used in the treatment of anemia caused by bone marrow suppression (e.g., after cancer chemotherapy)
o EPO is also used as a performance-enhancing drug (Lance Armstrong)
Modification of Protein function (Erythropoietin)
- Erythropoietin is a 165 amino acid protein that contains N-linked oligosaccharides, carbohydrate constitutes ~40% of the mass of this glycoprotein
- Glycans are critical for the function of erythropoietin because the nonglycosylated protein exhibits only 10% activity
Modification of Protein Function (Heparin)
• Heparin – single most widely used drug in the world today, acts as an anticoagulant
• Forms a high affinity complex with antithrombin
o Upon binding heparin, antithrombin undergoes a conformational change which increases its activity 1,000 to 10,000 fold
o Antithombin inhibits two principle procoagulant proteases, Factor Xa and thrombin, thereby decreasing the production of fibrin clots
Carbohydrates interact with carbohydrate binding proteins termed “lectins” or “receptors”
- Cellular recognition (leukocyte adhesion selectins bind immune system cells to the sites of injury in the inflammatory response potential target for new therapeutic agents to control inflammation
- Intracellular targeting (lysosomal enzymes, lysosomal storage disorders)
- Binding sites for bacterial toxins/parasites (cholera toxin, Helicobacter pylori, flu virus)
Antigenic Function of Carbohydrates: blood transfusions and ABO blood group system
• Humans can be divided into different groups according to the presence or absence of serum antibodies (against a carbohydrate structure on glycoproteins and glycolipids) that would agglutinate red blood cells isolated from other humans
• The antigens are oligosaccharides determined by allelic glycosyltransferases
• Example: Type A person receives blood from Type B person
o Type A person views type B RBCs as foreign and produces anti-B antibodies which agglutinate the transfused RBCs
o Note: Non-primate mammals express glycans not present in humans; limits using non-primate mammalian organs for transplantation into humans
• CANCER
o Abnormal Glycosylation
♣ Glycoprotein “cancer antigens” have been detected in melanomas, gliomas, neuroblastomas, and breast, pancreatic, lung, prostate, and kidney cancers
♣ Clinical diagnostic procedures are being developed to detect abnormal glycosylation on the surfaces of malignant cells
• Diabetes and Glycation
o Glycation: a non-enzymatic process that effects mostly long-lived proteins of the lens, plasma and RBCs
♣ Glucose becomes covalently attached to -amino groups of lysine residues in hemoglobin forming “glycated” hemoglobin called HbA1c
• Patients with diabetes mellitus have high concentrations of blood glucose and therefore high amounts of HbA1c
• The changes in the concentration of HbA1c in diabetic patients can be used to follow the effectiveness of insulin treatment
• Glycated proteins undergo further structural changes to generate heterogeneous compounds termed Advanced Glycation End products (AGEs) that can damage other proteins by cross-linking them, causing pathological changes
• Influenza Virus
o Influenza virus uses host glycoproteins and glycolipids which contain sialic acid as cell surface receptors, allowing the virus to infect cells
♣ Human-adapted viruses bind sialic acid 2,6 galactose found on epithelial cells of the human upper respiratory tract (URT)
♣ Avian-adapted viruses bind sialic acid 2,6 glactose found on epithelial cells of the avian upper respiratory tract [in humans, present in the lower respiratory tract (LTR)]
o Glycosylation Inhibitors as antiviral agents:
♣ Virus encodes a surface sialidase that cleaves terminal sialic acid residues from glycoproteins that helps the virus through the mucosa (rich in sialic-acid containing mucins) of the respiratory tract and in the release of the virus from infected cells
♣ Sialidase (Neuraminidase) inhibitors:
• FDA approved drugs are analogs of sialic acid
o 1) Oseltamivir (Tamiflu, tablet)
o 2) Zanamivir (Relenza, inhaler)
• Drugs do not kill the virus but slow the virus replication down to a level where the immune system can more easily destroy it
• Lysosomal Storage Diseases
o Incomplete degradation
♣ Catabolism of glycolipids, glycoproteins, and proteoglycans occurs in the lysosome
• Their carbohydrate units are degraded by the sequential action of hydrolytic enzymes (glycosidases) solely from the non-reducing end of the oligosaccharide
• Exoglycosidases
Hydroxylation (Collagen)
• 4-hydroxyl proline: Collagen is constructed of the repeating tripeptide unit Gly-X-Y, where X is proline and Y is 4-hydroxy proline
o Prolyl 4-hydroxylase requires Vitamin C (ascorbate) for its activity
• Scurvy is caused by a lack of vitamin C: decreased levels of 4 hydroxy proline destabilizes collagen resulting in degeneration of connective tissue
Acetylation
• Lysine modification: Acetylation of lysine -amino groups in histones inhibits their interactions with DNA
• Acetylation removes the positive charge on the lysine side chain, thus inhibiting the ability of histones, which are rich in lysine and arginine residues, to interact with negatively charged DNA
o HAT – Histone Acetyltransferases
o HDAC – Histone Deacetylases
• Common modification with ~2,000 proteins identified that undergo acetylation
o Nearly all enzymes involved in glycolysis, gluconeogenesis, TCA cycle, fatty acid oxidation, urea cycle, and glycogen metabolism are acetylated
• REVERSIBLE (deacetylases remove acetyl group)
Phosphorylation
• The hydroxyl side chain of Serine, Threonine, and Tyrosine can undergo phosphorylation
o ~1/3 of all proteins undergo phosphorylation
o ~500 kinases and ~500 phosphatases in the human genome
• Protein kinases use ATP as their substrate to attach phosphate to proteins
• Protein phosphatases remove the phosphate (reversible modification)
• Introduces a negatively charged group into a protein
• Can have multiple effects:
o Cause a dramatic change in protein conformation (e.g., insulin receptor) to convert an inactive protein into an active protein (“on-off switch”)
o Serve as a docking/binding site for other proteins in signaling cascades (e.g., erythropoietin signaling pathway that stimulates the formation of RBCs)
o Serve as a “rheostat” to modulate the activity of enzymes (e.g., glycogen synthase has at least 9 different phosphorylation site, and the pattern of phosphorylation modulates the activity of the enzyme)
ADP-Ribosylation by Bacterial Toxins
• Bacterial Toxins, such as diphtheria, cholera, and pertussis toxins, act by modifying crucial host cell proteins by the addition of an ADP-ribose molecule
o The reaction is catalyzed by enzymes that use NAD+ as the substrate
• Cholera toxin modifies a trimeric G protein (GS) that renders it permanently active in signaling adenylyl cyclase, resulting in high cAMP levels
o In intestinal epithelial cells, the increased cAM levels trigger massive water loss
o Severe dehydration and electrolyte loss are the major pathologies in cholera
Protein Degradation:
• Protein degradation serves two important roles in the cell:
o (1) Removes improperly folded and damaged proteins to prevent the accumulation of abnormal proteins
o (2) Maintains the appropriate levels of normal proteins and for permitting rapid changes in these levels, which allows cells to respond to changing conditions
• Two major degradation pathways in mammalian cells:
o (1) Proteosome (2) Lysosome
Ubiquitin
• Ubiquitin (Ub) tags proteins for degradation and is one of the most highly conserved proteins known (76 amino acids)
• ATP-dependent pathway involving 3 enzymes:
o E1 Ub-activating enzyme
o E2 Ub-conjugating enzyme
o E3 Ub ligase
♣ ~700 different E3s help choose which protein should be covalently modified by ubiquitin
♣ carboxyl-terminal glycine residue of ubiquitin is linked to lysine residues of the target protein
• Proteins with a polyubiquitin chain are degraded by a large complex called the proteasome that is located in the cytosol
o The proteasome hydrolyzes ATP to unfold proteins prior to their proteolysis by the inner chamber (i.e., 20S core particle has proteolytic activity) of the proteasome
o Proteasome is not membrane bound like the lysosome
Ubiquitin-Proteasome System and Human Disease
- Mutations in BRCA1 (the breast cancer susceptibility locus) are seen in ~10% of brease and ovarian cancer
- BRCA1 protein is a tumor suppressor gene (and its mutation affects DNA repair)
- BRCA1 is an E3 ubiquitin ligase whose activity is abolished by mutations found in familiar breast and ovarian cancers
• Fatty Acids
o Common function: promotes the interaction of proteins with the lipid bilayer of a membrane
♣ Palmitoylation – Palmitic Acid (C16) is linked to a protein
♣ Myristoylation – Myrisric Acid (C14) is linked to a protein
• Farnesylation
o Intermediate of cholesterol biosynthetic pathway, farnesyl pyrophosphate (C15), is covalently attached to C-terminal cysteine (Cys) residue
o Ras must be farnesylated to associate with the plasma membrane and is essential for the transforming activity of oncogenic variants of Ras
♣ Clinical trials have used farnesyltransferase inhibitor drugs
o Farnesylation and Disease
♣ Hutchinson-Gilford Progeria Syndrome
• Rapid aging disease
• Children die at ~13 years, generally from MIs or strokes
• Disease is caused by the accumulation of a mutant form of lamin A (retains a farnesyl group rather than being converted to mature lamin A which lacks a farnesyl group)
o Lamin A is a structural protein of the nuclear lamina
• Clinical trials are ongoing using farnesyltransferase inhibitor drugs
• Glycosyl phosphatidylinositol (GPI)
o GPI is attached to the C-terminus of a target protein through the phosphoethanolamine portion of the molecule
♣ The two fatty acid moieties of GPI are imbedded in the bilayer
Peripheral Blood
‘Peripheral’ = all blood that’s not in bone marrow
Functions of peripheral blood
to convey:
– nutrients & waste
– endocrine functions (hormones) – gas exchange: O2, CO2
Plasma
Plasma: fluid phase after centrifugation with heparin (prevents clotting)
– 90% water, 10% protein
– Contrast with Serum, which is the fluid phase after clotting & centrifugation to remove clotting factors & cells.
Differential Ranges of WBCs
A. Granulocytes: have specific granules
a. neutrophils: b. eosinophils: c. basophils:
34-71% 0-7% 0-1%
Differential Count
B. Agranulocytes: a. lymphocytes: b. monocytes:
19-53% 5-12%
CBC Values
RBC (varies w altitude):
male: 4.6 - 6.1 million cells/mcL female: 4.2 - 5.4 million cells/mcL
complete
WBC: 4,000 - 10,000 cells/mcL
Erythrocyte Size
7.5 microm diameter = the internal standard
Carbonic Anhydrase (CA)
Carbonic Anhydrase (CA): CO2 --> carbonic acid --> HCO3- (bicarbonate) • Then,“band-3”pumpsHCO3- outoftheRBC.
Band 3
Band 3: an integral cell membrane protein with 2 functions
• exports HCO3-
• binds ankyrin, which in turn binds to spectrins, which are structural proteins that function to maintain biconcave shape!
Spherocytes
Because the cytoskeleton is defective, spherocytes form.
• Spherocytes can respire, they are functional.
• But, they are fragile, degraded (hemolysis) in the spleen, causing anemia.
RBC Lifespan
120 days
Leukocytes
Leukocytes
• …are transient in blood; ‘work’ in the tissues. • …function in defense.
• …include “granulocytes” & “agranulocytes”.
Neutrophils
34-71% (most common WBC); ~15 μm
• nucleus highly lobulated
– polymorphonuclear (“polys”)
• Neutrophils have 2 types of granules: 1. lysosomes
2. “specific” granules that do not stain are ‘neutro’-philic. • Why are neutrophils ‘polymorphonuclear’?
to facilitate passage between cells, to enter tissue spaces
The ‘poly’ nucleus is caused by activation of caspase, which degrades nuclear lamins ( a very limited apoptosis).
Neutrophil function and lifespan
Neutrophil function: anti-bacterial
•
• mutation of NADPH oxidase causes persistent bacterial infection neutrophil lifespan: days
– –
Bacteria emit signals that attract neutrophils.
3-step attack:
1. granulereleasetodegradebacteria
2. phagocytosiseatsbacteria(microphage)
3. NADPHoxidase
• produces superoxide anions (O2-) that kill bacteria via a “respiratory burst”
Eosinophils
0-7%; ~15 μm
• bi-lobed nucleus
• Specific granules are red (i.e. eosinophilic) & large, with a crystalloid center.
– contains major basic protein, which kills parasitesEosinophils Have 3 Functions:
1. kill parasites (major basic protein)
2. phagocytize antigen-antibody complexes
3. secrete leukotrienes
– Eosinophils increase during parasitic infections & allergic reactions.
– Eosinophils in the lungs secrete leukotrienes, causing asthma.
1. BV leakiness edema
2. bronchioles constrict asthma
3. mucous glands mucous
Short-term treatment: target smooth muscle cells to dilate bronchioles.
– β2-adrenergic agonists (SMCs relax)
– ACh antagonists
Long-term treatment: inhaled corticosteroids, & and drugs that block
– leukotriene production
– leukotriene cell receptors
– eosinophil lifespan: weeks
Basophils
nucleus obscured by blue specific granules
• specific granules heparin & histamine
• similar to “mast” cellsAntigen (Ag) invades.
In response to Ag, plasma cellsIgE. IgE binds receptors on basophil/mast cell. Later in life, when the same Ag enters:
–
basophil lifespan: years (basophils have “memory”)
•
epinephrine
– β-adrenergic agonist
• •
anti-histamine steroids
• Ag binds IgE on the basophil/mast cell surface.
• In response, the basophil/mast cell…
i. granules histamine
ii. leukotrienes
Monocytes
Monocytes: 5-12%; 15-20 μm
• largest WBC
• nucleus indented, up to horseshoe-shape
In tissues, monocytes differentiate into:
• Atigen-Presenting Cells (APCs)
• Macrophages Lifespan is months.
Lymphocytes
Lymphocytes: 19-53%; ~12 μm
• B (bone marrow) & T (thymus) lymphocytes
– histologically indistinguishable
– T cells predominate in peripheral blood (several subtypes)
– B cells predominate in tissues plasma cells antibodies
Platelets
• fragments of megakaryocyte cytoplasm • 300,000/l; ~2 m • granulomere – clotting & growth factors (PDGF) • hyalomere – microtubules • clinical: thromboembolism
Overview of Protein Targeting
• The cytosol contains many of the enzymes involved in intermediary metabolism and is packed with ribosomes
• All proteins start their synthesis on ribosomes free in the cytosol
o ~50% of the proteins made on these ribosomes remain in the cytosol as permanent residents
o Actively synthesizing ribosomes that attach to the endoplasmic reticulum (ER) membrane function to remove selected proteins from the cytosol
Two types of signaling proteins
• (1) Transmembrane proteins, which are only partly translocated across the ER membrane and become inbedded in it
• (2) Water soluble proteins, which are fully translocated across the ER membrane and are released into the lumen of the ER
o These include secretory proteins and proteins destined for the lysosome
Signal Recognition Particle (SRP) Cycle
• ~50% of proteins in the cell complete their synthesis on ER-bound ribosomes
SRP: Ribonucleoprotein composed of a single 7SL RNA molecule of 300 nucleotides plus 6 different polypeptide chains
• Cycles between the ER membrane and the cytosol
• Binds to the signal sequence and to the ribosome to function in “signal recognition” and “elongation arrest” of the polypeptide chain
Signal Sequence is the tag/zip code
• No consensus sequence (primary sequence differs between signal sequences)
• Adopt a similar three-dimensional structure that is recognized by SRP
• Usually occurs at the amino terminus of a protein
o Those that occur at the amino terminus are cleaved co-translationally by signal peptidase
Tripartite domain structure:
o a hydrophilic amino-terminal domain which typically contains a net positive charge
o A hydrophobic core domain with a minimum length of 7 residues that typically forms an -helix
o A polar carboxyl-terminal domain of 4-6 residues
Signal Recognition Particle (SRP) Receptor (Docking Protein)
- Expressed exclusively in the ER
- Integral membrane protein with its SRP binding site exposed to the cytosol
- Functions to bind SRP in the SRP-nascent polypeptide chain-ribosome complex which targets the complex to the ER membrane (SRP receptor does NOT bind free SRP)
- GTP regulates this process
SRP Signal Peptidase
- Cleavage of amino-terminal signal sequence occurs co-translationally in lumen of ER
- Water soluble protein, fully translocated across the ER membrane, folded into its native conformation
Translocation Process (Import into the lumen of the ER)
- Requires ATP hydrolysis and occurs through an aqueous pore or channel called a “translocon”
- Translocation and protein synthesis are usually coupled since unfolded polypeptide chains are the preferred substrate for translocation
- Translocated polypeptide chains fold into their native conformation in the lumen of the ER with the assistance of other proteins called chaperones
Topology and Stop-Transfer Sequences
• “Stop-Transfer sequences” are hydrophobic, -helical sequences which function to anchor the protein in the membrane
o Proteins which span the membrane many times contain numerous stop-transfer sequences
• Hydrophobic interactions between the non-polar amino acids and the fatty acyl groups of the membrane lipids firmly anchor the protein in the membrane
o This is in contrast to lipid-linked membrane proteins in which the palmitoyl, myristoyl farnesyl, or acyl chains of GPI imbed only within the inner or outer leaflet of a bilayer
Targeting of proteins synthesized on membrane-bound ribosomes
• The golgi apparatus contains a collection of membrane-bound cisternae resembling a stack of plates that has two distinct sides:
o The cis face is juxtaposed to the ER
o The trans face is distended into a tubular reticulum called the trans Golgi network (TGN)
• Each cisterna is a distinct compartment with its own set of processing enzymes, and proteins are modified in successive stages as they move from compartment to compartment across the stack in a cis to trans direction
o The TGN is the site in the cell where proteins that have been synthesized on ER-bound ribosomes are sorted for transport to lysosomes, secretory granules, or the plasma membrane
Targeting to Plasma Membrane and Secretory Granules
• Proteins (soluble or membrane-bound) are transported to the cell surface by a nonselective “default pathway” unless they carry signals that direct them elsewhere
o Constitutive Pathway: common to all cells – soluble proteins are continuously secreted without intracellular storage
o Regulated Secretory Pathway: present (in addition to the constitutive pathway) only in certain cells such as exocrine cells, endocrine cells, and neurons – a subset of secretory proteins is sorted to storage organelles (called secretory granules) from which they are released only upon stimulation of the cell
Lysosome – Recycling Center of the Cell
- Lysosome has ~60 different hydrolytic enzymes used for the controlled intracellular digestion of macromolecules
- Degraded macromolecules are recycled and used in pathways for energy production (TCA Cycle) or in biosynthetic pathways (protein synthesis)
- All hydrolytic enzymes are acid hydrolases that are optimally active near the pH 5 maintained within lysosomes
- V-type ATPases are responsible for acidifying intracellular compartments; lysosomes are the most acidified compartment in mammalian cells with a pH of 5.0 or less
Functions of the Lysosome:
• Numerous functions are carried out by lysosomes including:
o (1) disposal of abnormal proteins
o (2) downregulation of cell surface signaling receptors (e.g., EGF receptor)
o (3) Release of endocytosed nutrients (e.g., cholesterol from LDL)
o (4) Degradation of pathogenic organisms (phagocytosis)
o (5) Cellular survival (autophagy)
• There are three pathways to the lysosome
o (1) phagocytosis
o (2) endocytosis
o (3) autophagy
♣ “Self-eating” – a major catabolic, energy-producing pathway that involves the lysosomal degradation of cytoplasmic proteins and organelles
♣ Housekeeping function (Quality Control mechanism) to eliminate damaged organelles and protein aggregates
♣ During starvation, autophagy provides an internal source of nutrients for energy generation and thus survival
Neurodegenerative Diseases and Autophagy
• Protein aggregates and damaged organelles accumulate within specific neurons of patients with:
o Alzheimer’s disease
o Parkinson’s disease
o Huntington’s disease
o Amyotrophic Lateral Sclerosis (ALS/Lou Gehrig’s Disease)
• Regulation of autophagic pathway is potential therapeutic target in these neurodegenerative diseases
Mannose 6 Phosphate is the Zip Code to the lysosome:
- Lysosomal enzymes (i.e., acid hydrolases) are synthesized in the ER and transported through the Golgi apparatus
- Mannose 6-phosphate receptors are the delivery trucks that recognize the “zip code” and carry the ~60 different newly synthesized mannose 6-phosphate tagged hydrolytic lysosomal enzymes (packages) from the Golgi to pre-lysosomal compartments
• Components of lysosomal enzyme targeting:
o (1) GlcNAc phosphotransferase
♣ Recognizes a protein signal on lysosomal enzymes
♣ Transfers GlcNAc-1-PO4 from the sugar nucleotide uDP-GlcNAc to specific mannose residues
o (2) -N-acetylglucosaminidase (“uncovering enzyme”)
♣ Cleaves off the GlcNAc residue, generating the mannose 6-phosphate recognition marker
o (3) Mannose 6 phosphate receptors
♣ Recognizes mannose 6-phosphate residues on lysosomal enzymes
♣ Transports the lysosomal enzymes from the Golgi to a prelysosomal compartment (i.e., endosomes)
Lysosomal Storage Diseases (LSDs) - Treatment
• Loss of any one lysosomal enzyme causes disease
o ~50 different lysosomal storage diseases (Tay-Sachs, Niemann-Pick)
• Results in accumulation of undegraded material
• FDA approved therapies exist only for a few of these diseases
• Enzyme replacement therapy (ERT) involves intravenous injection of the missing enzyme every 2 weeks
o Treatment depends upon receptor-mediated endocytosis of mannose 6-phosphate tagged recombinant acid hydrolase enzyme via cell surface mannose 6-phosphate receptors
o Annual cost to patient: $200,000-$400,000
Lysosomal Storage Disease: Mucolipidosis II (I-Cell Disease)
• Arise from defects in lysosomal enzyme targeting because of a deficiency in the enzyme (GlcNAc phosphotransferase) that generates the mannose 6-phosphate tag on proteins destined for the lysosome
o The patient’s cells lack multiple lysosomal enzymes in their lysosomes and abnormally high levels of lysosomal enzymes are present in plasma and other body fluids
o The disease is characterized by severe psychomotor retardation, hepatosplenomegaly, many skeletal abnormalities, coarse facial features, and restricted joint movement
o Symptoms are present at birth and progress until death, usually by age 10
Lysosomal Storage Disease: Pompe Disease
- Patients accumulate undegraded glycogen
* Pompe disease is also classified as a glycogen storage disease
Molecular Basis of Vesicular Transport:
• Vesicular transport between organelles involves:
o (1) a coat protein-covered vesicle buds from a donor compartment
o (2) coat proteins are released from the vesicle
o (3) the uncoated vesicle binds to a specific target compartment
o (4) fusion of the uncoated vesicle to the target membrane
SNARE proteins provide the specificity in the fusion of vesicles with target membranes
o SNAREs are a family of integral membrane proteins
♣ Each vesicle has its own v-SNARE which pairs in a unique match with a cognate t-SNARE found only at the intended target membrane
♣ This matching ensures specificity of the fusion event
•
Example: vesicles budding from the ER contain an ER v-SNARE that will only bind to a t-SNARE found on the cis-Golgi
SNAREs act as a winch to bring membranes together
• SNARE proteins provide the specificity in the fusion of vesicles with target membranes
• Many transport vesicles move through the cell along “tracks” of microtubules
o Vesicles are NOT just freely floating
Tetanus and Botulism Toxins:
• Tetanus and botulism are caused by extremely potent neurotoxin proteins that act by inhibiting the release of neurotransmitter from presynaptic nerve endings
o Tetanus and botulinal toxins are produced by certain anaerobic bacteria (Clostridium) whose spores are widely distributed in the soil
o Tetanus and botulism toxins are peptidases that cleave SNARES essential for synaptic vesicle fusion to the plasma membrane
• Botulinum blocks the release of acetylcholine and results in a flaccid paralysis
• Tetanus blocks the release of the inhibitory neurotransmitter -aminobutyric acid (GABA) and results in a spastic paralysis (“lockjaw”)
Botox:
- Botox – diluted botulinal toxin that is injected into the skin/muscle (paralysis occurs only at the site of the injection) to lessen the appearance of wrinkles
- Botox is administered for numerous conditions related to overactive muscle contraction: brow wrinkling, back pain, bladder spasms, migraine headaches, writer’s cramp, incontinence, patients with cerebral palsy
Receptor-Mediated Endocytosis
• An efficient pathway for taking up specific macromolecules from the extracellular fluid
• This process provides a selective concentrating mechanism that increases the efficiency of internalization of particular ligands more than 1000-fold so that even minor components for the EC fluid can be specifically taken up in large amounts without taking up a correspondingly large volume of EC fluid
o Nutrients (LDL, transferrin, vit B12)
o Growth Factors (EGF)
o Polypeptide hormones (Insulin)
o Viruses
o
Toxins
Fate of receptor
And Ligand ———->
Clathrin-Coated Pits and Vesicles
Clathrin-Coated Pits and Vesicles
• Clathrin: major protein component of coated pits/vesicles
o Triskelion-shaped (“3-legged”) protein complex – 3 heavy and 3 light chains
o Forms distinctive polyhedral protein baskets or cages
o Lines coated pits that occupy ~2% of the plasma membrane surface
o Recycles: dissociates from the vesicle membrane and returns to the cell surface to form a new coated pit
♣ This removal of clathrin coat is required before the vesicle can fuse to the endosome
♣ V-SNARE and T-SNARE are involved
V-Type ATPases
• The internal pH of endosomes is lowered by the presence of vacuolar ATPases (V-Type ATPases) in its membrane
o V-type ATPases are a distinct family of ATP-dependent proton pumps that function exclusively in the hydrolytic direction to generate pH and electrical gradients across membranes
o The acidic pH of the lumen of the endosome facilitates the dissociation of receptors from their ligands
Transferrin Receptor and Iron Uptake
• Transferrin cycle transports iron into cells via the transferrin receptor
o This cycle takes 16 minutes to occur and a typical liver cell can internalize 20,000 iron atoms/min
o pH regulated process
o Vesicle fusion events involve SNARES
o Apo = iron-free (Apotransferrin)
FH is characterized clinically by:
o 1) Elevated concentration of LDL in the plasma
o (2) Deposition of LDL-derived cholesterol in tendons and skin (xanthomas) and in arteries (atheromas)
o (3) Inheritance as an autosomal dominant trait with a gene dosage effect
♣ Homozygotes are more severely affected than heterozygotes
FH Mutation
• Results from mutations that affect the structure and function of the LDL receptor
Genetics of FH
• Heterozygotes number 1 in 500 persons, placing FH among the most common inborn errors of metabolism
o Heterozygotes have two-fold elevations in plasma cholesterol from birth
• Homozygotes number 1 in 1 million persons, exhibit severe hypercholesterolemia (650 to 1000 mg/dl) and coronary heart disease begins in childhood
• Treatment is directed at lowering the plasma level of LDL (direct correlation between high LDL levels and atherosclerosis)
• LDL receptor mutations (over 400 known mutations) can be divided into 5 classes based on their phenotypic effects on the receptor
o Three of these classes result in abnormal targeting
General Principles of Signal Transduction
• Signal transduction is fundamental to life
o Enables cells to communicate with each other and to respond to an alteration in their environment (changes in pH, osmotic strength, oxygen, nutrients, etc)
o Signal transduction alters cell behavior
o Coordinates homeostasis in differentiation, development, and metabolism
• Aberrant signal transduction can cause disease
o Example: mutations in Ras, such as Gly12 to Val mutation, can initiate carcinogenesis by promoting uncontrolled cell proliferation
A common structure of signal transduction pathways:
- Extracellular signal molecules mediate cell-cell communication
- Reception of the signals depends on receptor proteins
- The receptor activates one or more intracellular pathways
- Intracellular signaling pathways distribute the signals to appropriate effector proteins
Signal molecules activate receptors
• In most cases, signaling receptors are transmembrane proteins on the cell surface
o When bound by a ligand, the receptor becomes activated and generates various intracellular signals that alter the behavior of the cell
In other cases, the receptor proteins are inside the target cells and are stimulated by small/hydrophobic signal molecules that diffuse across the target cell’s plasma membrane
Signal molecules act over short or long distances:
•
Contact-Dependent: especially important during development and in immune responses
• Paracrine: occurs between different cell types; the signaling between cells of the same type is referred to as autocrine signaling
o Cancel cells often rely on autocrine signaling for their survival and proliferation
• Synaptic: coordinates behavior of cells in remote parts of the body
• Endocrine: relies on diffusion and blood flow, and it is relatively slow
Signals can act slowly or rapidly to change the behavior of a target cell
- Slow responses: increased cell growth and division require changes in gene expression and protein synthesis
- Fast responses: responses such as movement, secretion, or metabolism often involve rapid phosphorylation of the effector proteins in the cytoplasm
Different types of cells usually respond differently to the same signal
• Different receptors respond differently because the downstream activating factors are different; even if it is the same receptor, there can be different signaling cascades
Intracellular receptor-mediated signaling
• Nitric Oxide (NO) stimulates guanylyl cyclase
o For smooth muscle relaxation, autonomic nerves in the vessel wall release acetylcholine, which acts on the nearby endothelial cells by stimulating NO synthesis
♣ NO diffuses across the membrane and binds to guanylyl cyclase in the smooth muscle cells
♣ Activated guanylyl cyclase produces cyclic GMP, which induces rapid relaxation of smooth muscle cells
• Hormone-activated nuclear receptors are transcription factors
o Steroid hormone receptors translocate into the nucleus open ligand binding
o Ligand binding induces conformational changes in the nuclear receptors and affect their interaction with either inhibitory proteins or coactivator proteins
♣ This determines the function of nuclear receptors to regulate transcription either negatively or positively
Direct stimulation of early response gene expression can occur in 30 min
♣ The gene products then induce expression of other genes (delayed response, also called “secondary response”)
♣ Some of the early response gene products suppress the expression of primary response genes (an example of negative feedback regulation)
o Nuclear receptor signaling is usually specific because:
♣ Only certain types of cells have the receptors
♣ Each of these cell types contains a different combination of other cell-type-specific gene regulatory proteins that collaborate with the activated receptor to influence the transcription of specific sets of genes
Three largest classes of cell surface receptors:
o (1) Ion-Channel coupled receptors:
♣ Also known as transmitted-gated channels or ionotropic receptors) involved in rapid synaptic signaling
o (2) G-Protein coupled receptors:
♣ Indirectly regulate plasma membrane-bound enzymes or ion channels, which is mediated by a trimeric GTP-binding protein (G-Protein)
o (3) Enzyme-coupled receptors
♣ Either function as enzymes or associate directly with enzymes that they activate
From cell surface receptors to intracellular protein networks
- Molecular switches relay the signal to the next signaling component in the chain
- Scaffold proteins bring two or more signaling proteins together so that they can interact more quickly and efficiently
- Primary signals are transduced to produce large amounts of small intracellular mediators or many copies of a downstream signaling protein, which amplify the original signal
a. A small number of EC signal molecules can evoke a large intracellular response
b. When there are multiple amplification steps in a relay chain, the chain is often referred to as a signaling cascade - A coincidence detector receives signals from two or more signaling pathways and integrates them before relaying a signal onward
- Signals can spread from one signaling pathway to another, creating branches in the signaling stream, thereby increasing the complexity of the response
- Some proteins anchor one or more signaling proteins in a pathway to a particular structure in the cell where the signaling proteins are needed
Some proteins modulate the activity of other signaling proteins and thereby regulate the strength of signaling along a pathway
• Phosphorylation vs GTP-binding
o Many intracellular signaling proteins behave like molecular switches
♣ Two important mechanisms that control “active” and “inactive” status of proteins are phosphorylation and GTP-Binding
o Other mechanisms are available, including cAMP-binding, Ca2+-binding, and protein modifications such as ubiquitlyation
o About 30% of human proteins contain covalently attached phosphate on Ser/Thr or Tyr
♣ The human genome encodes 518 protein kinases and 137 protein phosphatases
• Monomeric vs trimeric GTP-binding proteins
o GTP binding proteins switch between an “on” state when GTP is bound and an “off” state when GDP is bound
♣ In the “on” state, they have intrinsic GTPase activity and shut themselves off by hydrolyzing their bound GT to GDP
o Two types of GTP-binding proteins are present in cells:
♣ (1) Monomeric GTP-binding proteins (Small GTPases)
♣ (2) Trimeric GTP-binding proteins (G-Proteins)
Guanine nucleotide exchange factors
(GEFs) promote the release of bound GDP in exchange of GTP to activate small GTPases
GTPase-activating proteins
(GAPs) increase the rate of GTP hydrolysis of small GTPases
Mediators of signaling complex formation
• Scaffold proteins: keep signaling proteins in close proximity and regulate the specificity of signaling
• Many receptor tyrosine kinsases generate specific docking sites on themselves to interact with signaling proteins
o This occurs transiently in response to extracellular signal
• Phospholipid molecules (Phosphoinositides) can also generate docking sites in the plasma membrane to recruit intracellular signaling proteins
Modular interaction domains
• The assembly of signaling complexes depends on various highly conserved, small interaction domains, which are found in many intracellular signaling proteins
o Src homology 2 (SH2) domains and phosphotyrosine-binding (PTB) domains bind to phosphorylated tyrosine
o Src homology 3 (SH3) domains bind to short proline-rich amino acid sequences
o Pleckstrin homology (PH) domains bind to the charged head groups of specific phosphoinositides
Feedback Regulation
• Positive feedback loop sustains signaling even after the signal strength drops
o This can contribute to prolonging the effects of a stimulus
• Negative feedback loop counteracts the effects of a stimulus and thereby abbreviates and limits the level of the response
Desensitization/adaptation (termination of signaling)
• Prolonged exposure to a stimulus decreases the cell’s response to the level of stimulus
• Desensitization can occur via diverse mechanisms that regulate either receptors or intracellular signaling proteins, or the production of inhibitory proteins
o Receptor Sequestration (use of endosome)
o Receptor Down-Regulation (use of lysosome)
o Receptor Inactivation
o Inactivation of Signaling Protein
o Production of Inhibitory Protein
• All GPCRs share a structural similarity
o A single peptide chain that threads back and forth across the lipid bilayer seven times
G proteins are molecular switches for GPCRs
• GPCR activates a trimeric GTP binding protein (made of , , and subunits) by acting as a guanine nucleotide exchange factor (GEF) for the G-protein
• The subunit has GTPase activity which is enhanced by a specific regulator of G-protein signaling (RGS)
o RGSs are the counterparts of GAPs that regulate small GTPases
o There are about 25 RGS proteins encoded in the human genome
• Cyclic AMP (cAMP) is a 2nd messanger
o Normal [cAMP] in the cytosol is about 10-7
o A nonomolar signal that activates adenylate cyclase can increase [cAMP] twenty fold in seconds
• Levels of cAMP are also regulated by cAMP phosphodiesterases that hydrolyze cAMP to adenine 5’ Monophophate
• In most animal cells, cAMP exerts its effects mainly by activating the Ser/Thr kinase, cAMP-dependent protein kinase (PKA)
cAMP-dependent protein kinase (PKA)
o PKA consists of two regulatory and two catalytic subunits
♣ cAMP binds to the regulatory subunits and induces their dissociation from the catalytic subunits
o PKA mediates short term as well as long term responses
♣ Long term responses require gene transcription, which is mainly regulated through cAMP Response Element (CRE)
Cholera toxin
o Cholera toxin transfers ADP ribose from NAD+ to the subunit of GS, inhibiting GTP hydrolysis
♣ This sustains active conformation of GS and stimulation of adenylyl cyclase
♣ The resulting prolonged cAMP production in the intestinal epithelial cells causes a large efflux of Cl- and water into the gut, thereby causing diarrhea that characterizes cholera
Pertussis toxin
o Pertussis toxin (whooping cough) catalyzes ADP ribosylation of the subunit of GI, preventing the protein from interacting with GPCRs
♣ As a result, the G protein remains in GDP-bound form and is unable to regulate its target proteins
♣ Because GI can’t inhibit AC, there is an increase in cAMP
IP3 mediates GPCR signaling via Ca2+ and protein kinase C
• Phospholipase C generates IP3 and diacylglycerol (DAG) from PIP2. IP3 is a water soluble molecule that diffuses into the cytosol and acts as a small intracellular mediator
Desensitization of GPCR
• There are there general modes of desensitization of GPCR:
o (1) Receptor inactivation: GPCR is altered and cannot interact with G proteins
o (2) Receptor sequestration: GPCR is temporarily internalized
o (3) Receptor downregulation: GPCR is destroyed in lysosomes after internalization
• In each case, GPCRs are phosphorylated by PKA, PKC, or GPCR Kinases (GRKs), indicating the presence of feed back regulation
• Arrestin prevents phosphorylated GPCR from interacting with G proteins
o It also couples GPCR to the clathrin-dependent endocytosis machinery
Receptor Tyrosine Kinases (RTKs)
• Many extracellular signal proteins act through receptor tyrosine kinases (RTKs)
o RTK has only one transmembrane segment
• About 60 human genes encode RTKs, which can be classified into more than 16 structural subfamilies, each dedicated to its complementary family of protein ligands
• **RTK activations requires the receptor chains to dimerize, bringing the kinase domains of two receptor chains together so that they can become activated and cross-phosphorylate each other on multiple tyrosines (transautophosphorylation) **
• Exception: the receptors for insulin and IGF1 are ()2 dimers, and ligand binding rearranged their transmembrane receptor chains, moving the two kinase domains in the subunits close together
RTKs serve as docking sites for signaling proteins
• Transautophosphorylation of RTK contributes to the receptor activation process in two ways:
o (1) Tyr phosphorylation within the kinase domain increases kinase activity
o (2) Tyr phosphorylation outside the kinase domain creates high-affinity docking sites for binding of specific intracellular signaling proteins that contain SH2 domains or PTB domains
• There are about 115 SH2 domains and about 295 SH3 domains encoded in the human genome
• Exception: The receptors for insulin and IGF1 do not generate docking sites on the receptor but use a specialized docking protein called insulin receptor substrate 1 (IRS1)
Ras: Small GTPase –> Key molecular switch for RTK Signaling
• The Ras superfamily consists of various families of monomeric GTPases
o Only the Ras and Rho families relay signals from cell-surface receptors
o A Ras or Rho family member can coordinately spread the signal along several distinct downstream signaling pathways, thereby acting as a signaling hub
• H-, K-, and N-Ras are the three major Ras proteins in humans and they work in almost the same way
• Ras transduces cell extrinsic stimuli to a variety of cellular targets mainly through the Raf/MEK/extracellular sign-related kinase (ERK) pathway
o Raf/MEK/ERK pathway is a three layered signaling cascade
o Plays a pivotal role in controlling cell growth and differentiation
o Its dysregulated signaling can lead to disease
• In Human cancers
o 30% of human tumors have hyperactive mutant forms of Ras (mainly Gly12 to Val mutation), which contribute to the uncontrolled proliferation of cancer cells
Targeting RTK/Ras/MAPK Signaling
- Many small molecule inhibitors or therapeutic antibodies that target RTK/Ras/MAPK signaling have been developed and tested for their efficacy
- Kinases are a major therapeutic target because they can be effectively inhibited by a specific ATP analog or an allosteric inhibitor
The PI3K-AKT pathway is a key mediator of RTK signaling
• Phosphotydylinositol (PI) undergoes reversible phosphorylation at multiple sites on its inositol head group to generate a variety of phosphorylated PI lipids called phosphoinositides
• Phosphoinositide 3-Kinase (PI3K) phosphorylates inositol phospholipids
o PI3K class 1a – activated by RTKs
o PI3K class 1 b – activated by GPCRs
• PI3,4,5-triphoshate (PI(3,4,5)P3) serves as a lipid docking site for a small subset of proteins that contain pleckstrin homology (PH) domain
•
The PI3K-AKT pathway is the major pathway activated by insulin, and plays a central role in promoting cell survival and growth in response to many other signals
the JAK-STAT pathway
• Cytokine receptors do not have kinase activity and, thus, mediate their signaling in association with the cytoplasmic tyrosine kinase Janus kinase (JAK)
o More than 30 cytokines and hormones, including interferons, erythropoietin, growth hormones, etc. bind to the cytokine receptors to activate the JAK-STAT pathway
o There are four known JAKs and at least six STATs in humans
Receptor Ser/Thr kinases (the TGF/SMAD pathway)
• Transforming Growth Factor- (TGF) superfamily consists of a large number of structurally related secreted dimeric proteins
o TGF/activin family and the larger bone morphogenetic (BMP) family
• TGF family members mediate development, immune responses, and cell proliferation and death
Protein phosphatases reverse protein kinase signaling
• Human genome contains 518 putative protein kinase genes that are either membrane associated or intracellular
o 90 Tyr Kinases
o 428 Ser/Thr Kinases
• Phospho-Ser, Phospho-Thr, and Phospho-Tyr account for 86.4%, 11.8%, and 1.8%, respectively, of the phosphorylated amino acids
o Tyr phosphorylation is used for signaling of high specificity
• Protein phosphorylation used in signal transduction is reversed by dephosphorylation, which is mediated by either Protein Tyr Phosphatases (PTPs) or Ser/Thr Phosphatases
o 107 protein Tyr phosphatases are encoded in the human genome, including some dual-specificity phosphatases that also dephosphorylate Ser and Thr
o Only 30 Ser/Thr phosphatases are encoded, further supporting that Tyr phosphorylation is utilized more specifically than Ser/Thr phosphorylation for cell signaling
Signaling regulated by proteolysis: Notch
• Notch cleavage (3 cleavage sites) is mediated by -secretase
o One of its essential subunits is Presenilin
o Mutations in Presenilin are a frequent cause of early onset of familial Alzheimer’s disease, a form of presenile dementia
o -secretase containing mutant Presenilin contributes to Alzhemier’s disease by generating amyloid plaques, which may injure nerve less (-secretase loses its specificity)
