Part 2: Protein Degradation Flashcards
The two separable aspects of the protein degradation system in eukaryotic cells:
- recognition of a substrate and its attachment to ubiquitin
- degradation of the ubiquitinated protein by a multicatalytic protease called the proteasome
Reasons for degrading intracellular proteins:
- Errors during synthesis - could lead misfolding of a protein, which is recognized by the cell.
- Environmental agents (such as heat and oxidation) can damage proteins.
- Proteins are no longer required, and the elimination of certain regulatory proteins is essential for normal cellular function.
Is the ubiquitin/proteasome pathway energy dependent?
Yes
Proteins are marked for degradation by attachment to the protein:
ubiquitin
Degradation of an ubiquitinated protein occurs in a compartmentalized protease called the:
proteasome
The breakdown of proteins is known as:
proteolysis
- generates amino acids for use in new protein synthesis
- generates intermediates for the synthesis of other metabolites
Zymogen:
- inactive enzyme precursor
- requires a biochemical change for it to become an active enzyme.
NON-SPECIFIC PROTEASES ARE EXPRESSED AS ZYMOGENS WHEN FIRST SYNTHESIZED.
Proteasome structure:
- chambered barrel
- a stack of four seven-subunit protein rings
The hydrolytic (degradation) activities of proteasomes occur where?
- On the interior of the proteasome, in the chambered barrel.
- Entry into the proteasome is restricted (gated).
What happens to substrates as they are about to enter the inner chamber of a proteasome for degradation?
- they are unfolded by an ATP-dependent unfolding machine so that they can be threaded into the catalytic chamber
Combinatorial Diversity refers to:
- 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.
Substrate-specificity in the Ub-proteasome degradation system is accomplished by forming unique combinations of targeting factors.
They are:
- Ub-protein ligases (E3)
- Ub-conjugating enzymes (E2)
Can a single Ub bind to a proteasome?
- No
- hydrophobic patch on a single Ub surface is not sufficient to form a high-affinity interaction
- need Ub chain to form hydrophobic stripe
What is the precursor form of mono-Ub?
- a fusion protein with other Ubs (polyubiquitin)
- Reason: to block the reactive carboxy terminus of Ub.
What part of Ub functions in all covalent interactions Ub makes with proteins during the marking process?
- carboxy terminus
- highly reactive
- reason for fusion precursor
What kind of bonds link Ubs together on a Ub chain coming off a marked protein?
- isopeptide bond
- lysine residue in the first Ub becomes covalently linked to the carboxy-terminus of the second Ub
The formation of a Ub chain coming off a substrate condenses to form:
- a hydrophobic stripe
- requires a minimum of 4 Ubs
- forms strong interaction with proteasome
Each Ub contains how many lysine residues?
7
Ub-fusion proteins (precursors) are linked via:
- peptide bonds
- cleavage requires no energy
What protein is the Ub-activating enzyme?
- 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
What type of bond forms between Ub coming off a Ub fusion protein and E1?
- thioester linkage
- Ub then transferred to E2 proteins
- E2/E3 complex confers substrate specificity and delivers Ub to the target substrate
Series of events in Ub-proteasome degradation pathway:
- Mono-Ub cleaved from a Ub fusion protein by E1 in an ATP-dependent manner.
- Ub transferred to an E2 enzyme.
- Ub-E2 complex interacts with E3 enzymes.
- Ub-E2-E3 complex recognizes and ubiquitinates substrate proteins.
THIOESTER CASCADE
What cascade mobilizes Ub?
Thioester Cascade
- between glycine-76 and a catalytic cysteine on E1, E2, E3
E1:
- 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
What lysine residue on Ub is responsible for the formation of multi-Ub chains that target proteins for degradation?
lysine K48
E2:
- 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
E3:
- Ub-protein ligases.
- Associate with Ub-E2 complex to recognize and deliver Ub to target substrate.
- attaches Ub to substrate via an isopeptide bond
Ub carboxy-terminal hydrolase:
- cleaves peptide bond between Ubs on the Ub fusion protein, catalyzing the transfer of a mono-Ub to E1.
What are the 4 steps in the Ub-proteasome degradation pathway that requires ATP?
-
Ub activation
- Cleaving of mono-Ub from Ub fusion protein during transfer to E1
- Proteasome assembly
- Protein unfolding before entering the proteasome
- Recycling and peptide release
What are the two subunits of the proteasome?
- substrate binding (19S)
- there are two of these subunits
- substrate degradation (20S)
Cellular proteins are not degraded by a proteasome unless they are first recognized by:
- 19S subunit of the proteasome, unfolded and translocated.
- ATP is required for translocation of substrate into the 20S subunit.
Structure of the 20S subunit of the proteasome:
- 4 stacked rings
- 2 beta inner rings (degradate)
- 2 alpha out rings (prevent entry)
- these rings can self assemble
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:
- chaperone Ump1
- Ump1 is degraded upon assembly of the 20s subunit
Ump1:
- chaperone that catalyzes formation of the proteasome 20s subunit
- degraded by 20s subunit upon 20s subunit formation
What are the 5 key functions of the 19s subunit of the proteasome?
-
Multi-Ub chain binding.
- substrate recognition
-
Multi-Ub chain disassembly.
- removing Ub-chain from substrate
- Ub is then recycled
- Unfolds substrate via ATPases.
- Regulates entry into 20s channel.
- Binds regulatory proteins.
26s proteasome:
- Two 19S particles attach to each end of a 20S complex to form a dumbbell-shaped 26s complex.
- assembly requires ATP
Immune cells express a specialized proteasome.
Why is this proteasome special?
- 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.
The immunoproteasome has altered cleavage properties. How so?
- 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.
Mechanism of allosteric control of substrate entry into the proteasome chamber:
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.
Mechanism of recycling and peptide release from proteasome:
CHEW AND SPEW
- 26S complex dissociates into 20S and 19S particles following each cycle of protein degradation.
- peptides and amino acids released.
- REQUIRES ATP
The inflammatory response mechanism:
- NFκB transcription factor translated.
- NFκB processed into p65 by proteasome.
- p50/p65 dimer forms (active transcription factor).
- p50/p65 inhibited by IκBα in cytoplasm.
- Cell injury activates a kinase.
- Kinase phosphorylates IκBα.
- Specific E2/E3 factors recognize phosphorylated IκBα.
- IκBα degraded.
- p50/p65 enter the nucleus and activate transcription of stress-responsive genes.
- p50/p65 in the nucleus activates IκBα transcription.
- Pathway shut off.
Two types of E3 (Ub Ligase):
- Ring E3—contain 1 or more ring motifs (bind metal atoms), binds E2
- Hect E3 ligase—able to form a thioester bond with Ub
