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SFM EXAM 2a/b 2020 > Functions and Dysfunctions of Protein Processing > Flashcards

Functions and Dysfunctions of Protein Processing Flashcards

1
Q

LOs #1-4 Functions and Dysfunctions of Protein Processing

A
  1. Illustrate the key components of protein synthesis (p. 350-353) including:
  • Ribosomes
  • mRNA
  • tRNAs and Aminoacyl tRNAs
  • Aminoacyl tRNA synthetases
  • Activation of amino acids

This LO is described in video and supplementary material and will not be covered in class.

  1. Describe the mechanism of translation (p. 353-357, Fig. 19.4):
  • Initiation
  • Elongation
  • Termination

This LO is described in video and supplementary material and will not be covered in class.

  1. Summarize the effects of various antibiotics and toxins on prokaryotic and eukaryotic protein synthesis (p. 357, Table 19.1)
  2. Evaluate the consequences of gene mutations and how they cause disease (p. 350)

Understand from the correlation boxes:

  • Sickle cell anemia (blue, p. 351)
  • Duchenne muscular dystrophy (blue, p. 352)
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2
Q

LOs 5-6 Functions and Dysfunctions of Protein Processing

A
  1. Summarize the methods of protein sorting and classify the two major pathways (p. 359-361, Figure 19.6, Table 19.2)

Cytoplasmic pathway

  • Mitochondrial signals and import into mitochondria (Figure 19.7)
  • Nuclear localization signals

Secretory pathway

•Translation on the ER (Figure 19.8)

  1. Describe the various cellular events involved in post-translational processing of proteins, including:

–Protein folding and the role of chaperones

–Proteolytic cleavage

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3
Q

Functions and Dysfunctions of Protein Processing

A
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4
Q

Table- Colinearity of nucleotide and amino acid sequences

A
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5
Q

Describe genetic code

A

§Genetic code: A ‘set of rules’ that convert the nucleotide sequence of a gene into the aa sequence of a protein using mRNA as an intermediary.

§Sequence of nucleotides in mRNA read consecutively in groups of three.

§Each group of 3 consecutive nucleotides in RNA is called a codon

§Each codon specifies either one amino acid or a stop to the translation process.

§61 triplet codons code for the 20 known amino acids and 3 stop codons

§The code is degenerate (some aa can be coded by more than 1 codon, and 3 codons do not code for any aa)

§Standard but not universal

§Not punctuated and is without commas

§Non-overlapping (with some exceptions)

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6
Q

Describe The General mechanism of translation (in prokaryotes)

A
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7
Q

Why is it important to study the difference in protein synthesis between prokaryotes and eukaryotes??

A
  1. To be able to selectively inhibit prokaryotic protein synthesis
    (Clinical use - molecular basis for the development of antibiotics)
  2. To be able to understand the mechanism of human diseases
    (Research use - allow for the development of treatment and/or prevention)
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8
Q

Table- components involved in prokaryotic and eukaryotic translation

A
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9
Q

Describe The Prokaryotic Translation Inhibitors

(Antibiotics)

A
  • Streptomycin: binds to 30S subunit and interferes with the binding of fmet-tRNA and impairs initiation. Interferes with 30S subunit association with 50S subunit.
  • Clindamycin and erythromycin: binds to large 50S subunit, blocking translocation of the ribosome.
  • Tetracycline: binds to small 30S subunit, blocks entry of aminoacyl-tRNA to ribosomal complex and impairs elongation.
  • Chloramphenicol: inhibits peptidyl transferase activity and impairs peptide bond formation.
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10
Q

Describe the Eukaryotic Translation Inhibitors

A
  • Shiga toxin and Ricin: binds to large 60S subunit (euk.), blocking entry of aminoacyl-tRNA to ribosomal complex.
  • Diphtheria toxin: inactivates GTP-bound EF-2, interfering with ribosomal translocation (euk.)
  • Cycloheximide: inhibits peptidyl transferase (euk.) and impairs peptide bond formation.
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11
Q

Describe Elongation Inhibitors

A

•Puromycin: causes premature chain termination (prok/euk).

–Resembles the 3’ end of the aminoacylated-tRNA.

–Enters the A site and adds to the growing chain.

–Forms a puromycylated chain, leading to premature chain release.

–More resistant to hydrolysis.

–Stops the ribosome from functioning.

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12
Q

Describe point mutations

A
  • Point mutations that affect a single base pair in the protein coding region or the open reading frame of a gene may result in a different amino acid being incorporated into protein.
  • Four different categories:

–Silent Mutation: does not change the amino acid.

–Missense Mutation: changes amino acid in the protein with either no effect on protein function or a protein with vastly different function.

–Nonsense Mutation: codon changes into a stop codon causing premature chain termination. Also called null mutation. Protein either degraded or formed as a truncated version.

–Frameshift Mutation: one or more nucleotides are deleted or inserted into ORF. Out of frame causes change in the codon sequence and consequently alteration in the amino acid sequence of the protein (E.g., Duchenne Muscular Dystrophy, beta thalassemia)

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13
Q

What is Sickle Cell Anemia?

(example of missense mutation)

A
  • Arises from a missense mutation of 6th codon in the allele of the gene for human β-globin (HBB), a subunit of adult hemoglobin.
  • Mutation changes GAG to GTG which changes Glutamic acid (negatively charged, hydrophillic) to Valine (hydrophobic).
  • Change in the amino acid alters conformation of HbA which causes it to aggregate and form rigid, rod-like structures.
  • This deforms the RBCs into the sickle-like shape.
  • Deformed erythrocytes have poor oxygen capacity and tend to clog capillaries, further restricting blood supply to tissues.
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14
Q

What is Duchenne Muscular Dystrophy?

(example of frameshift mutation)

A
  • Large in-frame and out-of-frame (OOF) deletions to the dystrophin gene leads to partially or non-functioning dystrophin protein.
  • OOF deletions result in little/no expression of dystrophin protein, give rise to a severe form Duchenne Muscular Dystrophy (DMD).
  • Presented in 1:3,500 males
  • Leads to muscle wasting (confinement to wheelchair by the age of 12 and death by respiratory failure within 10 years, symptoms onset typically by years 3-5).
  • In-frame deletions result in expression of truncated forms of dystrophin, giving rise to a milder form of the disease called Becker muscular dystrophy.
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15
Q

Diagram- Eukaryotic

A
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16
Q

Steps After Protein Synthesis

A

•Protein sorting

–Cytoplasmic pathway

–Secretory pathway

•Post-translational modifications

–Glycosylation

–Phosphorylation

–Disulfide bonds

–Acetylation

  • Protein Folding
  • Proteolysis
  • Degradation
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17
Q

Describe Protein Sorting.

A
  • Two major pathways for protein sorting
  • Cytoplasmic pathway:

–for proteins destined for cytosol, mitochondria, nucleus, and peroxisomes.

–Protein synthesis begins and ends on free ribosomes in cytoplasm.

–Absence or presence of certain translocation signals play role in final targeting.

•Secretory pathway:

–for proteins destined for ER, lysosomes, plasma membranes, or for secretion.

–Translation begins on free ribosomes but terminates on ribosomes sent to ER.

–These proteins have ER targeting signal sequences present on the first 20 amino acid residues of the polypeptide.

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18
Q

Table- sorting of newly synthesized proteins

A
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19
Q

Describe mitochondrial protein import

A
  • Translocation sequences recognized by transporters present in the mitochondrial membrane (transporter in inner membrane, TIM and transporter in outer membrane, TOM)
  • Proteins are passed across TOM and TIM.
  • Fig. 19.7. From: Biochemistry: An Illustrated Review
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20
Q

Describe nuclear import

A
  • Imported via nuclear pores
  • Small proteins able to pass through specific pores
  • Large proteins (> 40 kDa) require nuclear localization signals
  • Four continuous basic residues (Lys and Arg)
21
Q

Describe the secretory pathway

A
  • Each protein in this pathway has an ER-targeting signal peptide
  • 15-60 amino acids at N terminus of protein
  • Two properties:

–1 or 2 basic amino acids (Lys or Arg) near N terminus

– An extremely hydrophobic sequence (10-15 residues) on C terminus of the basic residues

  • A signal recognition particle (SRP) binds to the ER-targeting signal and the ribosome during translation.
  • SRP wraps itself around ribosome-mRNA-peptide complex, tethering it to ER membrane and halting translation temporarily
  • Translation resumes when protein directed into the ER lumen
  • Enzymes on luminal side cleave the signal to release the protein
  • Protein undergoes post-translational modifications in ER and/or Golgi apparatus
  • Additional signal sequences serves to guide each protein to final destination
22
Q

Describe Signal Sequences in Secretory Pathway

A

•For ER Lumen proteins:

–KDEL K—Lysine, D—Aspartic acid, E—Glutamic acid, L—Leucine

•For lysosomal proteins:

–Mannose-6-phosphate (M6P)

•For membrane proteins:

–N terminal apolar sequences

•For secretory proteins:

–Tryptophan rich domain

23
Q

Translation on the endoplasmic reticulum

A
24
Q

Interaction of Ribosome with ER Membrane

A
25
Q

What is Inclusion cell disease?

A
  • Lysosomal proteins not tagged with M6P
  • Defective or missing GlcNAc phosphotransferase, an enzyme that adds M6P to lysosomal hydrolases. The enzymes are not phosphorylated and hence not sorted into vesicles and not delivered into lysosomes.
  • They are instead carried to cell surface and secreted (found in blood).
  • High plasma levels of lysosomal enzymes.
  • By 6 months: failure to thrive and developmental delays and physical manifestations.
  • Development delays of motor skills more pronounced than cognitive delays.
  • Hepatomegaly, splenomegaly, defective heart valves.
  • Death frequently occurs by age 7, usually due to congestive heart failure or recurrent respiratory tract infections.
26
Q

Describe Post-translational Processing

A
  • Protein Folding: Small proteins can fold into native conformations spontaneously.
  • Large proteins cannot and are at risk for (a) aggregation and (b) proteolysis.
  • Large proteins need auxiliary proteins called chaperones.

–Protect the protein and help fold into proper tertiary structure

•Other proteins called chaperonins have barrel shaped compartments that admit unfolded proteins and catalyze their folding in an ATP-dependent manner.

27
Q

Chaperones and chaperonins in protein folding

A
28
Q

What is proteolytic cleavage?

A

•Proteolytic Cleavage:

–converts inactive forms to active enzymes by unmasking active site (e.g., trypsinogen and chymotrypsinogen to trypsin and chymotrypsin, respectively)

–Converts nascent precursor proteins to mature ones (e.g., proinsulin to insulin)

29
Q

What are the Post-translational Modifications?

A

•Covalent Modifications

– Glycosylation

– Phosphorylation

– Disulfide bond formation

–Acetylation

30
Q

Describe glycosylation

A
  • Extracellular proteins (cell surface proteins and plasma proteins) glycosylated.
  • Covalently linked to sugar residues in the ER lumen
  • As protein trafficking through ER and Golgi, residues processed to form mature protein.
  • Glycoproteins: either O-glycosidic or N-glycosidic linkage.
  • O-links are formed with the hydroxyl groups of Ser or Thr residues.
  • N-linked are always with Asparagine.
31
Q

What is acetylation?

A
  • Proteins are typically acetylated on lysine residues.
  • Use Acetyl CoA as the acetyl group donor.
  • Histones are acetylated and deacetylated on their N terminal lysines – critical for gene regulation.
  • Acetylation reaction catalyzed by Histone acetyltransferase (HAT) and deacetylation by histone deacetylase (HDAC) enzymes.
  • Acetylation and deacetylation regulate protein function - important post-translational mechanism.
  • Also facilitate crosstalk with other types of modifications such as phosphorylation, methylation, ubiquitination, etc for dynamic control of cellular signaling.
  • Pattern of Histone modifications are heritable – Epigenetics (study of potentially heritable changes in gene expression that does not involve changes to the underlying DNA sequence.
32
Q

Summary of Key Concepts- Functions and Dysfunctions of Protein Processing

A
  • Translation – 3 stages (Initiation, Elongation, Termination).
  • Sorting mechanism by which proteins “shipped” to various destinations (organelles) within cell.
  • Two major pathways of protein sorting (based on their final destination)

–Cytoplasmic pathway (cytoplasm, mitochondria, nucleus, peroxisomes)

–Secretory pathway (ER, Golgi, Lysosome, Plasma Membrane, Secretory)

•Post-translational processing

–Folding

–Proteolytic cleavage

  • Post-translational modifications
  • Glycosylation
  • Acetylation
  • Defects in protein folding – lead to neurodegenerative disorders (covered in the last SFM lecture/board discussion/small group activity)
33
Q

O-linked Glycosylation Form

A
34
Q

N-linked (mannose rich type) glycosylation form

A
35
Q

N-linked (complex type) glycosylation form

A
36
Q

What is mRNA?

A

After transcription, pre-mRNA edited to form mRNA

Then exported to the cytoplasm for translation

Eukaryotic mRNA contains:

  • Codons (present in coding region)
  • 7-methylguanosine cap at the 5′ end
  • Poly(A) tail at the 3′ end.
37
Q

What is tRNA?

A

tRNA serve as adaptors

Have binding site for both codons (in mRNA) and amino acid

Match Amino Acids to Codons in mRNA

38
Q

Describe the general structure of tRNA.

A
  • Cloverleaf secondary structure.
  • Two regions of unpaired nucleotides are crucial to its functions.
  • Anticodon loop, a set of 3 consecutive nucleotides that pair with a complementary codon in mRNA.
  • 3’ CCA terminal region which binds the amino acid that matches the corresponding codon.
39
Q

What are Aminoacyl tRNAs?

A
  • Complex of tRNA with amino acid (AA)
  • AA esterified to CCA sequence at 3’-end of cognate tRNA
  • AA needs to be “Activated”
  • Catalyzed by enzymes called aminoacyl tRNA synthetases
  • Each AA has its own aminoacyl tRNA synthetase
  • Aminoacyl tRNA synthetases serve as a second genetic code
  • Each tRNA charged with the correct AA to maintain fidelity of protein synthesis
40
Q

Activation of Amino Acids
Two step process:

A
  1. Aminoacyl tRNA synthetase catalyzes addition of AMP to COOH end of AA
  2. AA transferred to cognate tRNA
41
Q

What are ribosomes?

A
  • Translational machinery assembled on ribosomes
  • Large complexes of proteins and rRNA
  • Have 2 subunits

–Large Subunit

–Small Subunit

  • Large and small SU assemble together into an active complex in the presence of mRNA
  • Structures differ in prokaryotes and eukaryotes

–How is this medically relevant?

–Use antibiotics to target prokaryotic translational machinery

42
Q

Describe the ribosomal complex.

A

•Three important sites on the complex

–Acceptor (A) site (where mRNA codon exposed to receive aminoacyl tRNA, except the met tRNA)

–Peptidyl (P) site (where aminoacyl tRNA is attached)

–Empty (E) or exit site (location occupied by empty tRNA before exiting ribosome)

Small and large subunits assemble around the mRNA, the small positioning the mRNA and the large removes each amino acid and add it to the growing peptide chain.

43
Q

Describe translation

A
  • Translation occurs in the 5’ à 3’ direction
  • Consists of 3 steps:
  1. Initiation: formation of mRNA, small ribosomal subunit and initiator tRNA pre-initiation complex
  2. Elongation: activated AA attached to initiating Met by forming a peptide bond
  3. Termination: peptide chain is released

From the ribosomal complex

44
Q

Describe the initation step of translation

A

§Site at which protein synthesis begins is crucial.

§Determines reading frame for the whole length of the mRNA

Eukaryotes: 5’ cap, 3’ poly-A tail, and an ATP-dependent mRNA scan

§All known mRNA molecules contain signals that define the beginning of each encoded polypeptide chain.

§Pre-initiator complex is first assembled. Large su ribosome then added to form initiation complex

§A special initiator tRNA to which a GTP is bound is attached to P site of small su.

§The inititator tRNA-methionine complex (N-formylmethioninyl tRNA in prokaryotes and methioninyl tRNA in eukaryotes) loaded onto the small su of ribosomes on the P- site.

§Other initiation factors (IF in prokaryotes) and eukaryotic initiation factors (eIFs) are added.

§Large su added

§Translation begins with the initiation codon AUG (codes for methionine)

45
Q

Describe the elongation step of translation

A

§Activated aa attached to initiating methionine via a peptide bond

§Polypeptide chain extended by the action of the ribosome and involves the following steps:

§Loading of an aminoacyl tRNA onto the ribosome such that its anticodon base pairs with codon positioned on A site

§Prior to loading, the aminoacyl tRNA is attached to a GTP-bound elongation factor.

§Loading accompanied by GTP hydrolysis and release of factor from aminoacyl tRNA

§Peptide bond formation between aa in A and P site catalyzed by peptidyl transferase

§Energy comes from high energy bond between aa and tRNA

46
Q

Describe the termination step of translation

A
  • Peptide chain released from the ribosomal complex and the latter dissociates into its components
  • Termination triggered by Stop codons (UAA, UAG and UGA).
  • Not recognized by any tRNA and do not code for any amino acid.
  • Signal ribosome to stop translation.
  • The stop codons are recognized by Release Factors (RFs), proteins that promote the release of completed protein from the tRNA.
  • RFs bind to A site of ribosome containing the stop codon and cleaves the ester bond between the C terminus of the polypeptide and the tRNA.
  • Catalyze the addition of a water molecule instead of an amino acid. Forms COOH end of polypeptide.
  • Completed protein released from ribosome into cytoplasm.
  • GTP hydrolysis dissociates ribosomal comple-
47
Q

What are polysomes?

A
  • Clusters of ribosomes simultaneously translating a single mRNA molecule
  • Each synthesizing a polypeptide
  • Makes protein synthesis more efficient
48
Q

What are the STOP codons?

A

Stop codon – UAA, UAG and UGA

SFM EXAM 2a/b 2020 (14 decks)

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