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BMS1. Proteins Folding and Degradation (MADURA) > Part 2: Protein Degradation > Flashcards

Part 2: Protein Degradation Flashcards

1
Q

The two separable aspects of the protein degradation system in eukaryotic cells:

A
  1. recognition of a substrate and its attachment to ubiquitin
  2. degradation of the ubiquitinated protein by a multicatalytic protease called the proteasome
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2
Q

Reasons for degrading intracellular proteins:

A
  1. Errors during synthesis - could lead misfolding of a protein, which is recognized by the cell.
  2. Environmental agents (such as heat and oxidation) can damage proteins.
  3. Proteins are no longer required, and the elimination of certain regulatory proteins is essential for normal cellular function.
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3
Q

Is the ubiquitin/proteasome pathway energy dependent?

A

Yes

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4
Q

Proteins are marked for degradation by attachment to the protein:

A

ubiquitin

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5
Q

Degradation of an ubiquitinated protein occurs in a compartmentalized protease called the:

A

proteasome

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6
Q

The breakdown of proteins is known as:

A

proteolysis

  • generates amino acids for use in new protein synthesis
  • generates intermediates for the synthesis of other metabolites
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7
Q

Zymogen:

A
  • inactive enzyme precursor
  • requires a biochemical change for it to become an active enzyme.

NON-SPECIFIC PROTEASES ARE EXPRESSED AS ZYMOGENS WHEN FIRST SYNTHESIZED.

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8
Q

Proteasome structure:

A
  • chambered barrel
    • a stack of four seven-subunit protein rings
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9
Q

The hydrolytic (degradation) activities of proteasomes occur where?

A
  • On the interior of the proteasome, in the chambered barrel.
  • Entry into the proteasome is restricted (gated).
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10
Q

What happens to substrates as they are about to enter the inner chamber of a proteasome for degradation?

A
  • they are unfolded by an ATP-dependent unfolding machine so that they can be threaded into the catalytic chamber
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11
Q

Combinatorial Diversity refers to:

A
  • the distinct targeting complexes that can be assembled from Ub-protein ligases (E3) and Ubconjugating enzymes (E2)
  • Each unique E2/E3 complex is likely to have only one or a few substrates to which they attach Ub.
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12
Q

Substrate-specificity in the Ub-proteasome degradation system is accomplished by forming unique combinations of targeting factors.

They are:

A
  1. Ub-protein ligases (E3)
  2. Ub-conjugating enzymes (E2)
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13
Q

Can a single Ub bind to a proteasome?

A
  • No
  • hydrophobic patch on a single Ub surface is not sufficient to form a high-affinity interaction
    • need Ub chain to form hydrophobic stripe
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14
Q

What is the precursor form of mono-Ub?

A
  • a fusion protein with other Ubs (polyubiquitin)
    • Reason: to block the reactive carboxy terminus of Ub.
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15
Q

What part of Ub functions in all covalent interactions Ub makes with proteins during the marking process?

A
  • carboxy terminus
    • highly reactive
    • reason for fusion precursor
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16
Q

What kind of bonds link Ubs together on a Ub chain coming off a marked protein?

A
  • isopeptide bond
    • lysine residue in the first Ub becomes covalently linked to the carboxy-terminus of the second Ub
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17
Q

The formation of a Ub chain coming off a substrate condenses to form:

A
  • a hydrophobic stripe
    • requires a minimum of 4 Ubs
    • forms strong interaction with proteasome
18
Q

Each Ub contains how many lysine residues?

A

7

19
Q

Ub-fusion proteins (precursors) are linked via:

A
  • peptide bonds
    • cleavage requires no energy
20
Q

What protein is the Ub-activating enzyme?

A
  • E1
    • cleaves single Ubs off of a Ub fusion protein in an ATP-dependent process
    • binds 2 Ub’s as a ubiquitin adenylate (Ub-AMP) and via a thioester bond ATP dependent step
21
Q

What type of bond forms between Ub coming off a Ub fusion protein and E1?

A
  • thioester linkage
    • Ub then transferred to E2 proteins
    • E2/E3 complex confers substrate specificity and delivers Ub to the target substrate
22
Q

Series of events in Ub-proteasome degradation pathway:

A
  1. Mono-Ub cleaved from a Ub fusion protein by E1 in an ATP-dependent manner.
  2. Ub transferred to an E2 enzyme.
  3. Ub-E2 complex interacts with E3 enzymes.
  4. Ub-E2-E3 complex recognizes and ubiquitinates substrate proteins.

THIOESTER CASCADE

23
Q

What cascade mobilizes Ub?

A

Thioester Cascade

  • between glycine-76 and a catalytic cysteine on E1, E2, E3
24
Q

E1:

A
  • Ubiquitin-activating enzyme.
  • A single E1 enzyme activates the carboxy terminus of Ub in an ATP-dependent reaction.
    • cleaves mono-Ub from Ub fusion protein
25
Q

What lysine residue on Ub is responsible for the formation of multi-Ub chains that target proteins for degradation?

A

lysine K48

26
Q

E2:

A
  • Ubiquitin-conjugating enzyme.
  • A large family of E2 enzymes (~50) can receive activated Ub via a trans-esterification reaction from E1.
  • Associate with E3 factors to recognize substrates.
  • mediates the transfer of Ub from E1 to E3 or substrate
27
Q

E3:

A
  • Ub-protein ligases.
  • Associate with Ub-E2 complex to recognize and deliver Ub to target substrate.
  • attaches Ub to substrate via an isopeptide bond
28
Q

Ub carboxy-terminal hydrolase:

A
  • cleaves peptide bond between Ubs on the Ub fusion protein, catalyzing the transfer of a mono-Ub to E1.
29
Q

What are the 4 steps in the Ub-proteasome degradation pathway that requires ATP?

A
  1. Ub activation
    • Cleaving of mono-Ub from Ub fusion protein during transfer to E1
  2. Proteasome assembly
  3. Protein unfolding before entering the proteasome
  4. Recycling and peptide release
30
Q

What are the two subunits of the proteasome?

A
  1. substrate binding (19S)
    • there are two of these subunits
  2. substrate degradation (20S)
31
Q

Cellular proteins are not degraded by a proteasome unless they are first recognized by:

A
  • 19S subunit of the proteasome, unfolded and translocated.
  • ATP is required for translocation of substrate into the 20S subunit.
32
Q

Structure of the 20S subunit of the proteasome:

A
  • 4 stacked rings
    • 2 beta inner rings (degradate)
    • 2 alpha out rings (prevent entry)
    • these rings can self assemble
33
Q

The 2 alpha rings and 2 beta rings that compose the 20s subunit of the proteasome come together to form the 20s subunit in a reaction mediated by:

A
  • chaperone Ump1
    • Ump1 is degraded upon assembly of the 20s subunit
34
Q

Ump1:

A
  • chaperone that catalyzes formation of the proteasome 20s subunit
  • degraded by 20s subunit upon 20s subunit formation
35
Q

What are the 5 key functions of the 19s subunit of the proteasome?

A
  1. Multi-Ub chain binding.
    • substrate recognition
  2. Multi-Ub chain disassembly.
    • removing Ub-chain from substrate
    • Ub is then recycled
  3. Unfolds substrate via ATPases.
  4. Regulates entry into 20s channel.
  5. Binds regulatory proteins.
36
Q

26s proteasome:

A
  • Two 19S particles attach to each end of a 20S complex to form a dumbbell-shaped 26s complex.
  • assembly requires ATP
37
Q

Immune cells express a specialized proteasome.

Why is this proteasome special?

A
  • degrades foreign proteins, and forms fragments of them that fit into the cleft of MHC molecules.
  • MHC molecules deliver protein fragment to the cell surface, where they are presented on antigen-presenting cells (APCs).
  • immune response triggered.
38
Q

The immunoproteasome has altered cleavage properties. How so?

A
  • they generate peptides of the correct length and hydrophobicity to be placed in a cleft present in the extracellular domain of MHC molecules.
  • MHC molecules appear on APCs to trigger immune response.
39
Q

Mechanism of allosteric control of substrate entry into the proteasome chamber:

A

BITE AND CHEW

  • polypeptide chains enter chamber, cut into large fragments.
  • high concentration of large fragments blocks further entry of polypeptide chain into the chamber.
  • as concentration is reduced into small peptides and amino acids, more polypeptide chain can enter.
40
Q

Mechanism of recycling and peptide release from proteasome:

A

CHEW AND SPEW

  • 26S complex dissociates into 20S and 19S particles following each cycle of protein degradation.
  • peptides and amino acids released.
  • REQUIRES ATP
41
Q

The inflammatory response mechanism:

A
  1. NFκB transcription factor translated.
  2. NFκB processed into p65 by proteasome.
  3. p50/p65 dimer forms (active transcription factor).
  4. p50/p65 inhibited by IκBα in cytoplasm.
  5. Cell injury activates a kinase.
  6. Kinase phosphorylates IκBα.
  7. Specific E2/E3 factors recognize phosphorylated IκBα.
  8. IκBα degraded.
  9. p50/p65 enter the nucleus and activate transcription of stress-responsive genes.
  10. p50/p65 in the nucleus activates IκBα transcription.
  11. Pathway shut off.
42
Q

Two types of E3 (Ub Ligase):

A
  • Ring E3—contain 1 or more ring motifs (bind metal atoms), binds E2
  • Hect E3 ligase—able to form a thioester bond with Ub

BMS1. Proteins Folding and Degradation (MADURA) (3 decks)

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