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Foundations 1 > Protein Homeostasis > Flashcards

Protein Homeostasis Flashcards

1
Q

What does it mean that the genetic code is specific, universal, and degenerate?

A

Specific: 1 codon => 1 amino acid

Universal: Approximately the same in all organisms

Degenerate: Codons are redundant–multiple codons can encode the same AA via 3rd base wobble. These different codons are used with different frequencies and regulate translation rate as the minor use codons are present in lower abundance/concentration

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2
Q

What is a reading frame?

A

The way in which an mRNA is divided into triplets for translation

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3
Q

Describe the structure of an aminoacyl-tRNA.

A

Stem loop cloverleaf structure forms due to base pairing between complementary antiparallel strands.

  • Anticodon loop
  • 2 other loops important for recognition

The amino acid is attached to the 3’ end of the tRNA via an acyl linkage connected to CCA.

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4
Q

How are aminoacyl tRNA’s synthesized?

A

Each amino acid has a specific amino acid tRNA sythetase that links the amino acid to the tRNA, requiring 2 ATP equivalents.

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5
Q

Describe the structure of the ribosome.

A

70 structural proteins + catalytic rRNA

Large and small subunits

3 tRNA binding sites: E, P, A

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6
Q

How do the ribosome, mRNA, and tRNAs assemble?

A
  1. eIF2 ternary complex formation
    - Initiator tRNA binds GTP-eIF2 (eukaryotic initiation factor 2)
  2. 43S complex formation
    - tRNA-eIF2 complex binds to the P site of the 40S (small) ribosomal subunit
  3. mRNA activation
    - mRNA 5’ cap binds cap binding protein (CBP)
    - CBP has many 20 subunits including eIF4E
    - binding recruits more initiation factors
    - poly-A tail binds the cap forming a circle
  4. 48S complex formation
    - mRNA-CBP associates with tRNA-40S complex
  5. Scanning for start codon
    - RNA helicase unwinds secondary structure from 5’ -> 3’ and scans for a start codon
    - requires ATP
  6. Start codon recognition
    - eIF2 conformation change and GTP hydrolysis
    - eIF2 dissociates
  7. 60S subunit binds to tRNA in P site with GTP hydrolysis and remaining initiation factors displaced
  8. 80S initiation complex is assembled.
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7
Q

How do eukaryotic and prokaryotic translation differ?

A

Prokaryotes: translation is co-transcriptional

Eukaryotes: transcription and translation are spatially and temporally separated

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8
Q

How is cap-independent translation initiated?

A

Found often in viruses (decapping enzymes) but also sometimes in vertebrates

IRES = internal ribosome entry site for internal start codons
- stem loop structure; all initiation machinery assembles here

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9
Q

What are the steps of elongation?

A
  1. Random association of tRNAs with mRNA in the A site
  2. Proofreading by EF1a-GTP
    - If proper pairing, tRNA lingers
    - GTP hydrolysis by EF1a
  3. Hydrolysis of AA-tRNA bond
    - AA on P site tRNA transfered to A site AA
    - peptidyl transferase
  4. Translocation
    - Ribosome moves along mRNA, moving new chain to P site
    - Old tRNA released
    - Requires EF2-GTP

Growing chain emerges via channel in large subunit and is folded

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10
Q

What are the energy requirements for elongation?

A

2 ATP to charge each tRNA
2 GTP to add each AA

50 kDa protein (~450 AA) = 1800 ATP

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11
Q

How is translation terminated?

A
  1. Release factor binds a stop codon in the A site
  2. Termination via hydrolysis:
    - polypeptide chain released from E site
  3. Ribosomal dissociation
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12
Q

What are polyribosomes?

A

During translation in the cytosol, after one ribosome moves from the initiation region another ribosome can bind. mRNAs usually have multiple ribosomes translating, > 80 nt apart.

In the ER, only 1 ribosome per mRNA because an SRP binds to the 5’ cap for translocation

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13
Q

How does heme regulate globin mRNA translation?

A

Hemoglobin = 2 a-globin + 2 B-globin + heme. Syntheses must be coupled to avoid energy waste and ROS production.

Heme regulated inhibitor kinase:

  • activated by low heme => phosphorylates eIF2 => inhibits protein synthesis
  • inactivated by high heme
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14
Q

What are ferritin and transferrin receptor and how do iron levels regulate their translation?

A

Transferrin receptor

  • transports extracellular Fe to cytosol
  • 3’ stem loop; if exposed -> endonucelolytic cleavage and mRNA degradation

Ferritin

  • binds cytoplasmic Fe
  • 5’ stem loop; if blocked translation is blocked

Cytosolic aconitase = cytosolic enzyme that binds stem loops in the absence of Fe. In the presence of Fe, binds Fe.

Iron starvation: ferritin translation blocked, transferrin receptor mRNA protected

Excess iron: ferritin translation ok, transferrin receptor mRNA degraded

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15
Q

How is mRNA stability determined?

A

5’ cap structure:

  • prevents recognition by exonucleases
  • decapping (by virus or cleavage from thermal instability) => rapid 5’ -> 3’ degradation

poly-A tail length:

  • ~200bp bound with poly-A binding proteins
  • gradual shortening: exposed regions between proteins will be randomly cleaved in the cytosol
  • binding proteins cannot bind when tail < 30 => rapid 3’ -> 5’ degradation by exonucleases
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16
Q

What are micro RNAs?

A

20-22bp ssRNAs homologous to RNAs

Bind mRNAs and modify translation depending on binding site

  • initiation block: binding cap or start codon
  • elongation block: cause ribosome drop off or proteolysis
  • decapping
  • deadenylation of tail

1 microRNA can regulate 100s of mRNAs; multiple microRNAs may regulate 1 mRNA

17
Q

How does mTOR regulate cell growth and proliferation?

A

mTOR = “master regulator” kinase

  1. Growth factors and nutrient sensing activate mTOR
  2. mTOR upregulates ribosome synthesis and (de)phosporylates ribosomal subunit proteins and initiation factors
18
Q

What is proteostasis?

A

Maintaining proper protein composition by balancing synthesis and stability

Molecular chaperones help with correct folding.

Degradation systems deal with improperly folded proteins.

19
Q

How does the ubiquitin proteasome system degrade proteins?

A

Ubiquitin proteasome = collection of proteases surrounded by caps

  1. Tagging for degradation
    - E1 (only 1) activates the ubiquitin monomer, requiring ATP
    - Activated ubiquitin transferred to E2 (dozens), which binds E3.
    - E3 (100s–substrate specific) binds the substrate and transfers the ubiquitin from E2 to substrate
  2. Recognition of poly-ubiquitin chain by proteasome caps
  3. Cleavage of ubiquitin and polypeptide degraded to individual AAs
20
Q

What are the functions of lysosomes?

A

Digestion of macromolecules from phagocytosis, endocytosis, autophagy, or direct transfer using acid hydrolases

Destruction of microbes

Lysosomotropism of drugs

21
Q

How are misfolded ER proteins degraded?

A

ERAD = ER Associated Degradation

  1. Misfolded protein carried thru ER lumen by chaperone to ER protein translocator
  2. Sugars removed by N-glycanase
  3. Ubiquination and degradation
22
Q

What is the unfolded protein response and when does it occur?

A

Lots of ERAD suggests a mutation and triggers unfolded protein response

Sensors for misfolded proteins in the ER lumen are kinases and transcription factors with cytosolic effects

Activation of genes to increase protein folding capacity of ER like chaperones and glucosyl transferases

23
Q

What is consumed via macroautophagy?

A
  • virus
  • protein aggregates
  • organelles: degrade proteins to recycle AAs when there is a need for AAs
24
Q

What are the steps of macroautophagy?

A
  1. Autophagy induction by LC3
  2. Phagophore formation
  3. Elongation of phagophore and engulfment
  4. Autophagosome formation
  5. Fusion with lysosome or early endosome to add hydrolases
  6. Lysosomal degradation
25
Q

What is microautophagy?

A

Lysosomes engulf small amounts of cytoplasm: bulk or selectively (substrates bind hsc70, which binds to a receptor on the lysosome and is then engulfed)

26
Q

What is chaperone mediated autophagy?

A

Direct transfer of proteins into lysosomes:

  1. Chaperone complex (including hsc70) recognizes KFERQ motif
  2. Binding of LAMP2 receptor on lysosomal surgace
  3. Unfolding and translocation of protein into lysosome
27
Q

What are the paths of protein degradation?

A

Ubiquitin proteasome system

ERAD and URP

Lysosomal degradation:

  • macroautophagy
  • microautophagy
  • chaperone mediated autophagy
28
Q

What can cause misfolded proteins leading to aggregation?

A
  • Mutations
  • Defects in translation
  • Environmental stress
  • Aging
29
Q

What causes the formation of amyloid?

A

Nonphysiological proteolysis

Defective proteolysis

Mutations

30
Q

How is amyloid formed?

A
  1. Proteins rich in a-helical structures misfold, forming antiparallel beta sheets.
  2. Soluble oligomers of B-sheets aggregate forming protofibrils
  3. Protofibrils aggregate into fibrils
31
Q

What diseases are related to protein aggregation and amyloid deposits?

A

ALS, Alzheimers, CJD, mad cow, scrapie, Parkinson’s (Lewy bodies), Huntington’s (intranuclear exclusions)

32
Q

What hypotheses exist about how protein aggregates affect cellular functions?

A

Depletion of normal protein => loss of function

Misfolded/aggregated protein and protein deposits => inflammation

Misfolded/aggregated protein and protein deposits => gain of toxin

33
Q

What are prions?

A

Infectious amyloid with self-seeding growth

Host-to-host, molecule-to-molecule, and cell-to-cell transmission

CJD, kuru, mad cow, scrapie, fatal familial insomnia, Gerstmann-Straussler-Scheinker

Foundations 1 (26 decks)

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