MGD Sessions 1-6 Flashcards
Why are amino acids classified by their R group?
Only part of molecule not disrupted upon formation of peptide bonds
Which isomer are naturally occurring amino acids found?
L
Which group of amino acids do glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine and tryptophan all belong to?
Non-polar (hydrophobic)
Which group of amino acids do serine, threonine, asparagine, glutamine, tyrosine and cysteine belong to?
Polar, uncharged (hydrophilic)
Which group of amino acids do lysine, arginine, histidine, aspartate and glutamate belong to?
Polar, charged (hydrophilic)
Which amino acids have positive R groups?
Lysine
Arginine
Histidine
Which amino acids have negative R groups?
Glutamate
As
Aspartate
If pH > pK what happens to the R group on an amino acid?
It is deprotonated
Which type of peptide bonds only are found in a protein?
Trans so side chains aren’t too close together
What is the isoelectric point?
pH at which a protein has no net charge
If pH
Protonated
If pH > pI what happens to the protein?
Deprotonated
How many amino acids in length are peptides/oligopeptides?
Few
Give three examples of basic proteins.
Chymotripsinogen
Cytochrome C
Lysozyme
Give four examples of acidic proteins.
Pepsin
Serum albumin
Urease
Haemoglobin
What is the pI of myoglobin?
7.0
What determines the conformation of the backbone of a polypeptide and hence how the protein folds?
Bond angles of peptide bonds
Why are not all bind angles available in a polypeptide?
Some will cause side group clashes
How many amino acids are there per turn in an alpha-helix?
3.6
What is the distance between amino acids in an alpha helix?
0.15 nm
What is the height of a turn in an alpha-helix?
0.54 nm
Is an alpha-helix right handed or left handed?
Right handed
Where are the side chains on an alpha-helix positioned so they do not affect the alpha-helix structure?
On the outside
What bonds allow an alpha-helix to be formed?
Carbonyl group of one residue H-binding to amine group of the residue 4 a.a. away
What type of residues are strong helix formers?
Small, hydrophobic
Why are small hydrophobic residues strong alpha-helix formers?
Keep their hydrophobic side chains out of solution
Why is proline an alpha-helix breaker?
Rotation around N-C(alpha) is impossible
Why is glycine an alpha-helix breaker?
It’s tiny R group allows for other conformations
How does a beta strand compare to an alpha-helix?
Less compact
Fully extended
What is the distance between amino acids in a beta-strand?
0.35 nm
How are the R groups of amino acids arranged in a beta-strand?
Alternate b/w opposite sides of the chain
What provides stability in the anti-parallel alignment of beta-strands?
Hydrogen bonds
How does a sheet with parallel alignment compare to a sheet with antiparallel alignment of beta-strands?
Less compact
What is used to illustrate beta-strands on a diagram?
Flat arrows
What is the function of fibrous proteins?
Provide support, shape and protection
How many types of repeating secondary structure make up fibrous proteins?
One
What is the structure of collagen?
Super helix of three alpha-helix collagen chains wrapped around each other, stabilised by hydrogen bonds
How does the structure of collagen allow it to form fibrils?
Superhelices can form covalent cross-links
What are globular proteins used for?
Catalysis
Regulation
How many secondary structures do globular proteins contain?
More than one
What are motifs?
Globular folding patterns of 1 or more secondary structure elements e.g. Beta-alpha-beta loop or beta-barrel
What are domains?
Part of a polypeptide chain that fold into a distinct shape, often with a specific functional role
Why do membrane proteins show an ‘inside-out’ amino acid distribution by having a hydrophobic exterior?
To allow it to sit in the membrane
How can covalent bonds in the tertiary and quaternary structure of a protein be broken?
Reducing agent
Why are most proteins with disulphide bonds secreted?
They are stable
What holds protein structures together?
Covalent bonds Electrostatic interactions b/w charged groups H-bonds Van der Waals Hydrophobic effect
How can proteins be denatured?
Heat
pH
Detergents/organic solvents
How does pH denature a protein?
Alters ionisation state of a.a. and disrupts ionic/H-bonds
How do detergents/organic solvents cause denaturation?
Disrupt hydrophobic interactions
What determines how a sequence is folded?
Primary structure
What may be required for a protein to fold?
Chaperone
What happens when mis folding occurs?
Molecules cluster to form accumulations of denatured proteins which disrupt cell and its function
Give three examples of degenerative neurological disorders caused by misfolded proteins.
BSE (mad cow disease)
CJD
Kuru
What can happen when soluble proteins misfold?
Create insoluble form
What causes Alzheimer’s disease?
Amyloid beta-protein tail misfolding to form a highly ordered structure w/a high degree of beta-sheet which disrupts the rest of the protein formation
What is the inter-chain assembly in Alzheimer’s disease stabilised by?
Hydrophobic interactions b/w aromatic a.a.
What does synthetase require to function?
ATP
Does synthase require ATP?
Nope
How does phosphatase work?
Uses water to remove phosphoryl group
How does phosphorylase work?
Phosphate breaks bond and makes phosphorylated products
What does dehydrogenase accept?
Electrons
Are the oxygen atoms in the substrate for an oxidase?
Nope
Where are the oxygen atoms in oxygenase?
One or both in substrate
What are ribozymes?
RNAs which act like enzymes to catalyse cleavage and synthesis of phosphodiester bonds
What is Kcat?
Turnover number
Molecules of substrate to product pre enzyme per second
If the additional molecule needed for an enzyme to function is present, what name is given to the enzyme?
Holoenzyme
If the additional molecule an enzyme needs to function is not present, what name is given to the enzyme?
Apoenzyme
What is a cofactors?
Metal ion needed for enzyme function
What is a coenzyme?
Small organic molecule
What is a co substrate?
Co enzyme that makes a transient association and dissociates from the enzyme in an altered state
What is the name given to a coenzyme permanently associated with an enzyme which is returned to its original form?
Prosthetic group
Where are coenzymes often derived from?
Vitamins
What is the free energy of activation?
Peak energy difference b/w product and high energy intermediate
Does an enzyme change the equilibrium of a reaction?
No, just reached it quicker
What is transition-state stabilisation in enzyme action?
Enzyme stabilises structure in which the bonds are neither product or substrate and therefore greatly increase conversion to product
Can the transition state caused by enzymatic action be isolated?
Nope
How can the presence of catalytic groups increase enzyme action?
Enhance probability of transition state formation
What happens in visualisation of transition state during enzyme action?
ES formed –> conform into product shape –> product shape formed
What is Vmax?
Maximal velocity - when all available active sites are blocked
What kind of curve is formed by an enzyme that follows Michaelis-Menten kinetics when you plot V0 versus [S]?
Hyperbolic
What kind of curve is created by an allosteric enzyme when V0 is plotted against [S]?
Sigmoid all
What is Km?
Affinity of an enzyme for its substrate
[S] at 1/2 Vmax
Does Km vary with [E]?
Nope
What do a small and large Km indicate?
Small = high affinity Large = low affinity
When [S]»_space; Km, what can be said about the reaction kinetics?
0 order w.r.t. [S]
What is used to graphically calculate Km, Vmax and mechanism of action of enzyme inhibitors?
Lineweaver-Burk plot
What does the y-intercept of a Lineweaver-Burk plot give?
1/Vmax
What does the gradient of a Lineweaver-Burk plot give?
Km-Vmax
What are suicide inhibitors?
Conversion of molecule to form which covalently bonds to active site of enzyme by the enzyme itself
Is competitive inhibiting reversible?
Yes
What kind of bonds are formed in competitive inhibition?
Non-covalent
What is observed at high [S] in competitive inhibition?
Vmax
What happens to Km in competitive inhibition?
Increases
What effect does competitive inhibition have on the Lineweaver-Burk plot?
Rotates anti-clockwise
How does non-competitive inhibition affect Vmax and Km?
Decreases Vmax
Km unaffected
What affect does non competitive inhibition have on the Lineweaver-Burk plot?
Rotates clockwise
How do you distinguish between competitive and non-competitive inhibition by using a graph?
Plot 1/V0 v.s. 1/[S] and see Vmax decrease and Km remain constant if non-competitive inhibitor
How do captopril, enalapril and lisinopril cause vasodilation?
Block conversion of angiotensin I to II
What action does aspirin have?
Irreversibly inhibits prostaglandin and thromboxane synthesis
What are allosteric enzymes regulated by?
+ve and -ve effectors which bind noncovalently to an allosteric site
What can binding of an effector do to an allosteric enzyme?
Affect affinity of enzyme for its substrate
Modify Vmax
Both
What do allosteric enzymes often catalyse?
Early committed step early in a pathway
What form are amino acids always written in?
Ionised
What is the structure of haemoglobin?
Tetramic - 2 alpha and 2 beta subunits
Why does haemoglobin have a sigmoidal binding curve?
Has cooperativity
What conformational change occurs between the T and R states of haemoglobin?
Twisted through 15 degrees
How does binding of an oxygen molecule affect the affinity of haemoglobin for oxygen?
Increases it
What does 2-3 bisphosphoglycerate do to a molecule of haemoglobin?
Sits in the middle of the 4 subunits to interact with histidine and lysine residues
How does 2-3BPG affect the oxygen binding curve for haemoglobin?
Shifts it to the right
When is 2-3BPG released into the tissues?
High altitude
What action does 2-3BPG have on the affinity of haemoglobin for oxygen?
Lowers it
How can the charge on 2-3BPG be described?
V. negative
What change in residues is seen in sickle cell anaemia?
Glutamate is replaced by valine in the beta-global chain
What does the ‘sticky’ hydrophobic pocket created in SCA do?
Polymerises deoxygenated HbS
What is beta-thalassaemia?
Decreased or absent beta globin chains
Why are beta-thalassaemia patients symptomatic after birth?
Alpha globin chains cannot form stable tetramers
What is alpha-thalassaemia?
Lack of alpha globin chains
What is the function of the Bohr effect?
Ensure delivery is coupled to demand
What causes the Bohr effect?
Local increase in hydrogen ions and carbon dioxide
What does the Bohr effect cause?
Reduction in affinity for oxygen therefore release of oxygen into metabolising tissues
Why is carbon monoxide toxic?
Binds 250x more readily to haemoglobin than oxygen and upon binding increases the affinity of haemoglobin for oxygen so it is not released
What effect does carbon monoxide have on the oxygen binding curve?
Shifts left
How can haemoglobin be used to measure diabetes?
Measure HbA1c levels
Which type of haemoglobin has the highest affinity for oxygen?
Foetal
How many amino acids form a molecule of myoglobin?
153
What percentage of a myoglobin molecule is alpha-helical
75
How many polypeptide chains make up a myoglobin molecule?
1
Where is iron located in deoxymyoglobin?
Slightly below plane of ring
How is deoxymyoglobin transformed into its ferric from?
Binding of oxygen moves iron into the plane of the ring
What shape is the binding curve of myoglobin?
Hyperbolic
What happens to myoglobin in the presence of carbon monoxide?
Combines which blocks oxygen transport
What does the saturation of myoglobin depend on?
Partial pressure of oxygen
What structural changes to the myoglobin molecule cause it’s change into the ferric form?
Movement of His F8
Small overall conformation change
How do allosteric activators and inhibitors affect V v.s. [S] curves for allosterically controlled enzymes?
Activator: shifts to left
Inhibitor: shifts to right
Where can phosphate groups be added to enzymes?
-OH of serine, threonine and tyrosine
What is addition of a phosphate group catalysed by?
Kinase
What is removal of a phosphate group catalysed by?
Phosphatase
What are zymogens?
Inactive precursor enzymes
How are zymogens activated?
Removal of part of the polypeptide chain
What do zymogens allow?
Safe transportation of inactive form without causing premature effects e.g. preventing premature proteolytic cleavage by proteases
How is the rate of enzyme synthesis regulated?
Increase or decrease in the rate of mRNA transcription
What is the function of ubiquitin?
Added to proteins to tag them for destruction
What is feedback inhibition?
End product inhibits enzymes earlier in pathway
What is feedforward activation?
Increase in initial substrate increases first pathway step
What is counter regulation of opposing metabolic pathways?
Catabolic pathway breaking down A inactivates anabolic pathway making A e.g. glycogenolysis and glycogenesis
What are the main mechanisms which regulate the blood clotting cascade?
Inactive zymogens at low conc
Amplification of initial signal
Localisation of clotting factors to site of damage
Feedback activation of thrombin
Termination of clotting by multiple processes
What does activated thrombin enhance activation of?
Factors V, VIII and XI
How are clotting factors localised to the site of damage?
Gla domains on factors bind to damaged endothelial cell lining and allow rapid activation of downstream effectors
What is heterochromatin?
Solenoid 30 nm fibres which are not used in gene expression
What is euchromatin?
‘Beads on a string’ DNA used for gene expression
Describe the structure of chromosomes.
Double DNA strand –> nucleosomes (around histones) –> sole lids –> hierarchical loops
What is a nucleoside?
Base+sugar
What is a nucleotide?
Base + sugar + phosphate
What is DNA a polymer of?
Deoxyribonucleotides
What is RNA a polymer of?
Ribonucleotides
What secondary structure does DNA form?
RH double helix
What secondary structure does RNA form?
Stem loops
What is at the 5’ and 3’ ends of a single stranded chain of polynucleotides?
5' = phosphate 3' = OH
What are purines?
2 ring nitrogenous bases
What are pyrimidines?
Single ring nitrogenous bases
Which bases are purines?
A and G
Which bases are pyrimidines?
C, T and U
What types of duplex structure can form by complementary antiparallel arrangement?
DNA-DNA
RNA-RNA
DNA-RNA
What is the convention for writing polynucleotide sequences?
5’ –> 3’ left to right (complementary strand underneath 3’ –> 5’ for duplex structures)
Describe mitosis.
Forms 2 genetically identical diploid daughter cells
Somatic
One round of replication and one round of division
Homologous pairs
Describe meiosis.
4 haploid daughter cells - gametes One round of replication, 2 rounds of division Crossing over essential Homologous pairs form tetrads Generates genetic diversity
Where are the checkpoints in the cell cycle located?
End of G1 and G2
What is G0?
Resting phase where cell carries out its normal function
What method is used for DNA replication?
Semi-conservative
What is the function of DNA polymerase?
Control ribose-phosphate bind formation in semi-conservative replication of DNA
What happens when two facing DNA replication forks meet in semi-conservative replication?
DNA lipase joins fragments
Describe the process of DNA replication in prokaryotes.
Circular ‘naked’ chromosome –> initiation: strands separate –> elongation –> termination
Which enzyme gives DNA polymerase a kick start?
Primate
Why are Okazaki fragments formed in DNA replication?
Helical arrangement of DNA
What joins Okazaki fragments?
Lipase
Which ends of the DNA strand does DNA polymerase extend?
3’ only
How does the definition of a chromosome differ before and after replication?
Before 1 chromosome = 1 DNA molecule
After 1 chromosome = 2 DNA molecules = 2 identical sister chromatids
What is the function of DNA helicase?
Unwind double helix
How are chromosomes classified?
Position of centromeres
Describe the passage of neurones in the cell cycle.
Enter G0 and do not undergo mitosis after specialisation
What happens in mitotic prophase?
23 pairs of highly condensed chromosomes present
Kinetochore present for spindle connection
Nuclear membrane broken down
What happens in mitotic prometaphase?
Spindle fibres attach to chromosomes
Centrioles move to poles
What happens in mitotic metaphase?
Chromosomes randomly align along metaphase plate
What happens in mitotic anaphase?
Spindle moves to equator of cells
Sister chromatids pulled to opposite poles
Centromeres divide
What happens in mitotic telophase?
Nuclear envelope forms around both sets of separated chromatids
Nucleolus forms in both
Chromosomes decondense
Spindle fibres disappear
What happens in mitotic cytokinesis?
Cytoplasm divides
Parent cell becomes two daughter cells w/identical genetic information
What happens during metaphase I in meiosis?
Maternal and paternal chromosomes find each other to arrange in a tetrad
Crossing over incl. sister chromatids
Does crossing over of sister chromatids have a consequence?
No as they are identical
What happens in anaphase I during meiosis?
Homologous pairs separate
What happens in prophase II during meiosis?
Random arrangement of chromosomes
Retention of swapped over parts of chromatid
Why is there random arrangement of chromosomes in prophase II?
No homologous pairs
What happens in metaphase II of meiosis?
Chromosomes align along equator at 90 degrees to metaphase plate and spindle attaches
What happens in anaphase II of meiosis?
Chromosomes pulled to opposite poles
Give an overview of meiosis I and meiosis II.
I: homologous chromosomes of each chromosome pair divided
II: chromatids of each chromosome divided
What happens in spermatogenesis?
Spermatogonium (2n) –> primary soermatocyte (2n) –> spermatids (n) –> mature sperm
How long does spermatogenesis take?
~48 days
What happens in oogenesis?
Oogonium –> primary oocyte –> 3 polar bodies + 1 ovum –> mature ovum
How does the immature ovum compare to the three polar bodies formed during its genesis?
It has all of the energy stored within it
How long does oogenesis take?
12-50 years
Why is crossing over essential?
Ensures correct number of chromosomes in each cell by preventing chromosomes from sticking to each other
What does faulty meiosis lead to?
1/3 of all identified miscarriages
Infertility
Mental retardation
What is the frequency of faulty meiosis?
30 in 100
What gives 2^n possible arrangement of chromosomes where n is the number of chromosomes?
Independent assortment of chromosomes
What pattern of inheritance does Cystic Fibrosis show?
Autosomal recessive
How does an autosomal recessive inheritance pattern appear on a pedigree chart?
Appears to ‘come out of nowhere’
Can skip generations
Makes and females equally affected
What pattern of inheritance does Huntingdon’s disease show?
Autosomal dominant
What is the probability of heterozygotes with an autosomal recessive having affected offspring?
25%
What is the probability of heterozygotes with an autosomal dominant disease having affected offspring?
50%
Why are autosomal dominant diseases rare in the homozygous state?
Likely to result in termination
How do autosomal dominant inheritance patterns appear on a pedigree diagram?
Present in ever generation unless de novo
Males and females equally affected
What pattern of inheritance does Haemophilia A show?
X-linked recessive
Who is affected by an X-linked recessive inheritance pattern disease?
Hemizygous males and homozygous females
How does an X-linked recessive inheritance pattern appear on pedigree diagram?
More common in males
Every affected female has carrier mother and affected father
Daughters of affected males always at least carriers
What is the probability of a female carrier of an X-linked recessive genetic disease having an affected son?
50%
What is co-dominance?
New phenotype is formed when neither allele is dominant over another
Use blood groups to describe co-dominance.
A and B both dominant over O but neither dominant over the other –> AB blood type
What codes for glycoproteins on RBC surface which determine blood group?
Human isoglutamin gene
Which antigens are present on the RBCs of blood group O?
None
Which blood those can be given to AB blood group patients?
All
How can receive blood transfusion of type O?
All blood groups
What is a gene?
Stretch of DNA at a specific chromosomal locus
What are genes condensed into?
Chromatin
What are the types of RNA?
Messenger Ribosomal Transfer Micro Noncoding
What is microRNA used for?
Control of gene expression in eukaryotes
What enzymes are needed for DNA replication, transcription and translation?
Replication: DNA polymerase
Transcription: RNA polymerase
Translation: ribosome
Describe the process of initiation in transcription.
Initiation: sequence dependent promoter recognition, transcription initiation factors, RNA polymerase
Describe the process of elongation in transcription.
5’ to 3’ chain growth
What does the termination of transcription depend on?
Sequence
What happens in the initiation stage of transcription?
TATA box recognised in 5’ –> 3’ direction
General transcription factors recruit polymerase II
RNA polymerase II phosphorylated
How does initiation of transcription vary between eukaryotes and prokaryotes?
Eukaryotes: TATA box at -30, variety of upstream sequences, complex regulation
Prokaryotes: Pribnow box at -10, upstream sequences at -35, simple regulation
How is transcription regulated?
Protein-DNA and protein-protein interactions
Promoters have numerous sequence activators
What happens to the DNA molecule during the elongation stage of transcription?
Opens to allow for gene expression the closes when DNA polymerase is finished
In which direction does mRNA synthesis take place?
5’ –> 3’
What is capping?
Methylated guanine creates a 5’-5’ triphosphate bridge immediately after transcription to prevent degradation
What is polyadenylation?
Addition of large numbers of adenine residues to 3’ end of mRNA
What is the function of polyadenylation?
Protect against degradation
Signal export from nucleus
What does the length of the polyadenine tail on a mature mRNA molecule indicate?
Half-life
What is splicing?
Removal of introns and some exons depending on sequence
What is likely to happen to a protein if splicing goes wrong?
Likely to be ineffective
Give an example of a condition where there is an error with splicing.
PKU
Describe the structure of a mature mRNA molecule.
5’ cap –> 5’ UTR –> ORF –> 3’ UTR –> 3’ poly A tail
Where in the cell does translation take place?
Cytoplasm
How many kinds and copies of rRNA are present in a eukaryotic cell?
6-8 kinds
Many copies of each
Which type of RNA polymerase is associated with rRNA?
I
How many kinds and copies of mRNA are there in a cell?
100,000s of kinds
Few copies of each only present when proteins need to be made
Which type of RNA polymerase is associated with mRNA?
II
How many kinds and copies of tRNA are there in a cell?
~100 kinds
Lots of copies of each
Which type of RNA polymerase is associated with tRNA?
III
How many rRNAs are present in a eukaryotes?
4
How many proteins are there in eukaryotes?
82
Describe the structure of a eukaryotic ribosome.
40S and 60S subunits
80S ribosome
Describe a prokaryotic ribosome.
30S and 50S subunits
70S ribosome
How many rRNAs are there in prokaryotes?
3
How many proteins are there in prokaryotes?
56
In which direction is the template read during translation?
5’ to 3’
In what direction does the polypeptide extend during translation?
Amino to carboxyl
What initiates translation?
AUG
Which codons terminate translation?
UAA
UAG
UGA
Why is the genetic code said to be degenerate?
More than one codon codes for the same amino acid
What differences in bacterial translation give opportunity for attack?
Simple promoter Different transcription factors 1 RNA polymerase Coupled transcription and translation No post transcriptional processing Short lived mRNAs Simpler ribosomes Distinctive translation initiation Different translation factors
What is the wobble position on a tRNA molecule?
5’ base of anticodon and 3’ base of codon which allows single tRNA to recognise more than one codon
What is inosine?
Purine derived aspecific nucleotide
What is the function of inosine?
Give extra degeneracy
Less susceptibility to mutation
What happens during initiation of translation?
5’ cap recognised –> (40S subunit) methionyl recognises AUG –> phosphorylation (60S subunit) –> fully functional subunit
What happens during the elongation stage of translation?
tRNA occupies P site –> tRNA w/correct codon binds to A site –> peptidyl transferase forms peptide bond –> ribosome moves –> empty A site
What happens during termination in translation?
P site full –> stop codon recognised by uncharged tRNA which physically moves ribosome along –> water, peptide and tRNA formed
When do ribosomes attach to the ER membrane?
If protein is destined for membrane or secretory pathway
How do ribosomes attach to the ER?
Co-translational insertion
When do ribosomes remain in the cytosol during protein synthesise?
If the protein is destined for the cytosol or posttranslational import into organelles
How are proteins directed to the correct destinations?
Intrinsic signal
Receptor recognises signal and directs to correct membrane
Translocation machinery transports across membrane
Energy available to transport protein to new place
Where can proteins exist in mitochondria?
All areas
How are mitochondrial matrix proteins directed?
Chaperone keeps protein folded –> signal binds to receptor –> protein through TOM –> protein through TIM –> targeting signal cleaved
Where is the signal sequence on a mitochondrial matrix protein located?
N-terminus
What are TOM and TIM?
Channels which allow passage of a mitochondrial matrix protein mRNA across the outer and inner mitochondrial membranes
What causes pyruvate dehydrogenase deficiency?
Mutation at codon 10 in precursor protein
What does the mutation in pyruvate dehydrogenase deficiency cause in the precursor protein?
Pro replaces Arg
Lose basic residue on hydrophilic face of amphipathic helix
Reduce mitochondrial uptake
What supplies the energy for nuclear import of proteins destined for the nucleus?
GTP hydrolysis
What happens in the cytosol before nuclear import?
Importin binds cargo containing a nuclear localisation signal
After migrating the rough the nuclear pores, what happens to importin?
Ran-GTP binds –> conformational change –> cargo released –> importin w/bound Ran-GTP recycled to cytoplasm
What binds with cargo that has a peroxisomal targeting signal?
Peroxisomal import receptor
What happens to the peroxisomal protein as the receptor integrated into the translocon?
Remains folded
What happens when the receptor integrates with the translocon in peroxisomal protein synthesis?
Translocon opens
PTS dissociated from receptor
Receptor uses ATP to move to cytosol
Where can a mutation occur in peroxisomal protein synthesis?
In receptor for subset of peroxisomal proteins
How are proteins for the ER or secretory pathway directed?
Co translational transport
Describe how proteins destined for the secretory pathway are synthesised.
Produced by ER ribosomes w/ co translational transport in/across ER membrane –> bud off in vesicles –> fuse w/Golgi –> cisternae progression –> trans-Golgi network –> lysosome or secretion
What is another name for cisternae progression in the secretory pathway?
Retrograde transport
What is constitutive cellular secretion?
Proteins always secreted e.g. extracellular matrix enzymes
What kind of cellular secretion do specialist cells carry out?
Regulated
What are secretory cells polarised?
So secretory granules at apical side and nucleus basal
What prevents damage caused by secretion of enzymes into tissue?
Polarisation of secretory cells
Signal sequences at which terminus of secretory proteins are hydrophobic?
N
What does the pre- prefix of a protein indicate?
Activator sequence still attached
What are hydrophobic signal sequences rich in?
Basic side groups
What is signal recognition protein?
Multi domain riboprotein that mediates 3-way association w/SRP-receptor in the ER, ribosome, and signal peptide
What is the structure of the SRP receptor in the ER membrane?
Alpha-beta tetradimer
What happens after SRP and GTP bind to SRP receptor in synthesis of secretory proteins?
Translocon is opened, sequence enters –> signal peptidase cleaves signal –> protein synthesis –> released into cell
Where must proteins destined for plasma membrane or internal membrane of secretory pathway insert?
ER membrane
What exists in a membrane protein which anchors it during protein synthesis and prevents further transfer into the ER lumen?
Second hydrophobic sequence
What sequence in a membrane protein remains in the cytosol during its synthesis?
Stop transfer anchor sequence
What are the functions of ER in protein synthesis?
Insertion of proteins into membrane Glycosylation Proper protein folding Hydroylation of selected Lys and Pro residues Specific proteolytic cleavage Formation of disulphide bonds Assembly of multi subunit protein
Why is N-linked glycosylation of proteins important?
Correct protein folding
Protein stability
Deficiency –> sever congenital disorder
What is the oligosaccharide preassembled on in N-linked glycosylation?
Lipid carrier - dolichol
What is the oligosaccharide in N-linked glycosylation transferred to after preassembly?
Asparagine
Is the same oligosaccharide always used in N-linked glycosylation?
Yes
How is the oligosaccharide in N-linked glycosylation extensively modified in the ER and Golgi?
Trimming and addition of further sugars
What is the function of peptidyl-prolyl isomerases?
Accelerate inter conversion of cis/trans isomers of proline residues
What is the role of protein disulphide isomerase in the ER lumen?
Form disulphide bonds
How does protein disulphide isomerase carry out its role?
It is itself oxidised and can then be reduced by oxidising the substrate protein
What prevents secretion of ER lumen proteins from the ER during their synthesis?
Specific signal
What recognises Lys-Asp-Glu-Leu sequence in soluble ER resident proteins to return them back to ER from cis-Golgi cisternae?
KDEL receptor
What is anterograde transport?
KDEL receptor points to vesicle formation –> pH prevents direct KDEL binding –> secreted protein w/out KDEL
What is retrograde transport?
Decrease in Golgi pH causes KDEL binding –> higher ER pH causes dissociation of ER resident protein
What folding problems can arise during protein secretion?
Trapped in mis-folded conformation
Protein contains mutation resulting in misfolding
Protein incorrectly assoc. w/other subunits
How does the ER respond to misfolded proteins?
Chaperone attempts to correct protein
Retained unfolded proteins in ER –> downregulates protein synthesis
What happens to proteins if misfolding cannot be corrected?
Protein –> cytosol for degradation
Protein accumulates to toxic levels in ER
What diseases arise as a result of protein misfolding?
Parkinson’s
Wilson disease
What happens to lysosomal enzymes as they pass through the Golgi?
Phosphate group added to hydroxyl group of C6 mannose sugar
Which enzymes are found in the Golgi body that act on lysosomal enzymes?
Glucosamine phosphotransferase
Phosphodiesterase
What mediates lysosomal enzyme delivery by determining whether vesicle buds off from trans-Golgi network?
Mannose-6-phosphate receptor
What is O-linked glycosylation?
Attachment of sugar to hydroxyl group of serine (mainly) or threonine
What type of molecule is O-linked glycosylation important in?
Proteoglycans
Where is O-linked glycosylation prevalent?
Extracellular matrix and mucus secretions
Where is O-linked glycosylation prominent?
ER and Golgi
Give an example of a protein released by the unregulated constitutive secretory pathway.
Collagen
Give an example of a protein released by the regulated secretory pathway.
Insulin
Which action in the secretory pathway determines whether a protein is released by regulated or constitutive pathway?
Whether membrane fusion is regulated or not
Which protein is the most abundant in the body?
Collagen
What secretes collagen in connective tissue?
Fibroblasts
What is the basic unit of collagen?
Tropocollagen
What is the repeating structure in collagen?
Gly-X-Y
How many polypeptide alpha chains are in one molecule of tropocollagen?
3
Why are the alpha chains of tropocollagen highly extended?
Presence of proline and hydroxyproline in X and some Y parts of the repeating structure
What are the structural features of the RH triple helix which forms collagen?
Non-extensible
Non-compressible
High tensile strength
What stabilises the structure of the RH triple helix in collagen?
H binds b/w alpha chains
Which type of collagen accounts for 90% of all body collagen?
I
Which unites are present in collagen type I?
2 x alpha-1
1 x alpha-2
Where is type II collagen found?
Cartilage
Intervertebral discs
Which units make up type II collagen?
3 x alpha-1 units
Where is type III collagen found?
Foetal skin
CVS
Which units make up collage type III?
3 x alpha-1
Where is type IV collagen found?
Basement membrane
Where is type V collagen found?
Placenta and skin
What is associated with protein disulphide isomerase in the ER and is used for collagen synthesis?
Prolly hydroxylase
What dos prolyl hydroxylase require to function?
Vitamin C
Iron(II) ions
How does prolyl hydroxylase increase the stability of collagen?
Creates hydrogen bonds
What happens to the tropocollagen triple helices in scurvy?
They are weak
Why is collagen not synthesised within the cell?
Molecules too long
Give the first four stages of the modification and synthesis of collagen in the ER.
Synthesis and entry of chain into ER lumen
Cleavage of signal peptide
Hydroxylation of selected proline and lysine residues
Addition of N-linked oligosaccharides
What causes the transition of prepro-alpha to pro-alpha in collagen synthesis?
Signal peptidase
What causes hydroxylation of selected proline and lysine residues in collagen synthesis?
Prolyl hydroxylase
Where are N-linked oligosaccharides added in collagen synthesis?
C-terminal propeptide
Give an example of a sugar that can be added to collagen when N-linked oligosaccharides are added to the C-terminus.
Galactose
What is added to hydroxylysine residues in collagen synthesis?
Galactose
What occurs in chain alignment of 3 alpha chains during collagen synthesis?
Formation of disulphide bonds
How is the triple helical procollagen assembled in collagen synthesis?
From C to N terminus
What is found at both the N and C termini of a collagen molecule?
Extra amino acids that do not form the triple helix
How is completion of O-linked oligosaccharide chains achieved?
Addition of glucose
What links the N and C terminal extensions in the transport vesicle?
Disulphide
What happens to the N and C terminal propeptides extracellularly?
Extensions removed
Alpha-chain formation stimulated
How is a collagen fibre formed once the N and C terminal propeptides have been cleaved?
Lateral association of molecules
Followed by covalent cross linking
How do collagen fibrils form a fibre?
Aggregation
What is formed by lateral association of individual collagen units?
Form 50 nm fibrils
Why does collagen have a striated appearance?
Formed of 50 nm fibrils
What happens when heavy metal staining is used on collagen?
Sit in gaps b/w molecules giving striated appearance
How is an aldol cross-link formed?
Lysine residues –> aldehyde derivatives –> potentially spontaneous aldol cross-links
Which enzyme converts lysine residues to aldehyde derivatives?
Lysyl oxidase
Which enzyme is affected in Ehlers Danlos syndrome?
Lysyl oxidase
What is needed for lysyl oxidase function?
Vitamin B6
Copper(II) ions
What is the action of lysyl oxidase?
Form covalent cross-links
What are the two steps in insulin processing that take place in the ER?
Hydrophobic sequence cleaved from proinsulin
Specific folding stabilised by 3 sulphide group bonds
What happens post-Golgi to leave a complete two-chain insulin molecule?
Connecting peptide removed
Which three enzymes are involved in the ER processing of insulin?
PC3 endoprotease
PC4 endoprotease
Carboxypeptidase
Where is the proinsulin antibody found?
Secretory glands waiting to mature
What is the third sulphide bond in an insulin molecule important for?
Structure
Why is proteolytic processing needed for small secretory products such as enkephalins in the secretory pathway?
They are too short to enter the ER via co-translational mechanism
What would happen if hydrolytic enzymes were activated in the cell instead of being proteolytically processed in the secretory pathway?
They would be destructive to the cell
How are multiple bio active products produced from the same polypeptide?
Proteolytic processing in the secretory pathway
How is activation of the insulin receptor avoided in the secretory pathway?
Proteolytic processing
How are bio active growth factor and insulin prevented from acting at the same time?
Proteolytic processing in the secretory pathway
