Protein decay Flashcards
What do we need ubiquitination for in proteasomal degradation?
- control pathway activation/ termination
- response to signalling
- cell cycle control
- recycle amino acids for new protein synthesis
What is the proteasome?
What is it important for?
- The proteasome is one of the major degradation machineries in eukaryotic cells.
- It terminates the existence of thousands of short-lived, damaged, misfolded or otherwise obsolete proteins and plays pivotal roles in protein quality control and other vital processes in the cell.
Whats the 26S proteasome?
- The 26S proteasome is a complex proteolytic machine of around 2.5 MDa.
- It consists of a barrel-shaped protein complex (core particle, CP) that can carry a regulatory lid (the regulatory particle, RP) on one or both ends.
- To be degraded in the proteasome proteins have to be tagged with Ubiquitin (Ub), in particular with chains of Ub molecules linked through lysine 48 (K48) of Ub.
What are ubiquitinated substrates recognised by when entering the proteasome?
- Ubiquitinated substrates are recognized by Rpn1, Rpn10, and Rpn13, three subunits of the RP that possess Ub-binding domains.
- Alternatively, substrates are delivered by Ub-binding shuttling proteins (p62, Rad23/HR23, Dsk2/PLIC/Ubiquilin, and Ddi1) that dock at the proteasome via interaction of their Ub-like domain with Rpn1, Rpn10, or Rpn13.
After the proteasome captures the ubiquitinated substrate what happens?
- After capturing the substrate, the Ub tag is released by an RP-associated deubiquitinating enzyme whereas the substrate is unfolded and threaded through a narrow gate into the interior of the CP, where it is degraded by chymotrypsin-, trypsin-, and caspase-like proteolytic activities.
Tell me why the ubiquitination pathway is so highly controlled?
This highly controlled process not only eliminates unwanted proteins, terminates or activates signaling pathways, and participates in cell cycle regulation but also provides an important source of amino acids for de novo protein synthesis
What is a decline in proteasomal activity associated with?
- Correspondingly, a decline in proteasomal activity is associated with aging, cancer, neurodegenerative diseases, and other late-onset diseases.
- On the other hand, the strong dependency of highly proliferating cells, such as cancer cells, on an active proteasome is exploited in anticancer therapies by using proteasome inhibitors for inducing cancer cell death.
Tell me the steps to proteasomal degradation
- The 26S proteasome is a large highly conserved protein complex ~2.5 Mda
- Proteins are tagged for degradation with a small protein called ubiquitin – ubiquitous – highly conserved between species.
- Ubiquitin tagging is catalysed by E1, E2, E3 ubiquitin ligases.
- Monoubiquitination signals polyubiquitination.
- The polyubiquitin chain is bound by the proteasome.
- Unfolded protein is proteolyzed into small peptides for recycling.
Tell me the structure and properties of the ubiquitin protein?
Structure of ubiquitin (Ub)
- Small 8.6 kDa protein (76 amino acids)
- Heat stable
- Highly conserved (95% sequence identity between human and yeast)
- Widespread distribution – ubiquitous
- Glycine at the C-terminus
- 7 lysine’s with an important Lys at position 48
Whats the mechanism for protein ubiquitination?
What is ubiquitylation a common signal for and what cascade does it involve?
What are the steps to this cascade?
- Ubiquitylation is a common signal for eukaryotic 26S proteasomes and involves a cascade of E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin ligase enzymes.
- In this cascade, E1 (plus ATP) first adenylates the carboxy-terminal carboxylate of ubiquitin (Ub), forming Ub–AMP, and then forms a Ub thioester intermediate (E1–Ub).
- Ubiquitin is transferred from E1 to E2, and then to the protein target with assistance from E3 (although ubiquitylation without E3 can occur118).
- Typically, an isopeptide bond is formed between the ubiquitin C-terminal carboxylate and the ɛ-amino group of a Lys side chain of the substrate protein or the growing ubiquitin chain (Lys48-linked ubiquitin chains are common signals for 26S proteasomes).
- Deubiquitylating enzymes within 26S proteasomes release and recycle ubiquitin during substrate protein degradation. PPi, inorganic pyrophosphate.
What happens in the first stage of protein ubiquitination?
- E1 ubiquitin activation: the formation of a reactive thioester between E1 and the C-terminal glycine
- requires ATP
What happens in the second stage of protein ubiquitination?
E2 ubiquitin conjugation: An E2 ubiquitin conjugation enzyme is ubiquitinated by a transacylation involving cysteine
What happens in the third stage of protein ubiquitination?
- E3 ubiquitin ligation: Transfer of ubiquitin to E3 ligase to which the protein to be degraded is already bound.
- The ligation involves transfer of ubiquitin to an ε-NH2 of a lysine of the protein to be degraded
Tell me about the mechanism of attachment between the ubiquitin protein and the target protein
- For a degradation signal, lysine 48 of one ubiquitin is bonded to the carboxyl group of the C-terminal glycine of the next ubiquitin
- This is repeated until a chain of typically 4 Ub’s is attached to the target protein
Mechanism of attachment of ubiquitin to target protein more images
What is the 26S proteasome structure and the subunits it is comprised of?
What are each of these subunits and how are they arranged?
The 26S proteasome
- catalytic 20S core particle (CP)
* (barrel of four stacked heptameric rings: two α- outer -rings and two β- inner -rings) - 19S regulatory particle (RP)
* base and the lid subcomplexes
Whats the role of the ubiquitin-proteasome system?
- Ubiquitinated substrates are recognized by Rpn1, Rpn10, and Rpn13, subunits of the RP.
- OR substrates are delivered by Ub-binding shuttling proteins (p62, Rad23/HR23, Dsk2/PLIC/Ubiquilin, and Ddi1)
- Ub is released by RP-associated deubiquitinating enzyme
- Substrate is unfolded and threaded into the interior of the CP for degradation by chymotrypsin-, trypsin-, and caspase-like proteolytic activities.
Tell me the stages to the mechanism of attachment of ubiquitin to the proteasome
What are ubiquitinated proteins targeted to?
The proteasome
What is the proteasome? What does it consist of? How is it arranged? What activity does the core have?
- The proteasome is an ATP driven large multi-enzyme 26S complex
- Consists of a 20S core of 28 subunits arranged as 4 rings of 7 units
- Arranged as: α 1-7, β 1-7, β 1-7, α 1-7
- Crystal structure shows a cylinder with central core β units containing proteolytic activity
- A 19S cap binds at both ends of the barrel
- The cap binds the polyubiquitin and also has ATPase activity that is involved in unfolding the protein
- The core β subunits have 3 different proteases producing peptides of 7 – 9 amino acids
- The core has iso-peptidase activity, the ubiquitin is cleaved from the peptide and recycled
The ubiquitination pathway is used for the degradation of what?
- Incorrectly synthesized proteins
- Ageing proteins
- Regulatory proteins with short-half lives
- Regulatory proteins after phosphorylation
- ……….
What happens to incorrectly synthesised proteins in the proteasome?
- Normal protein synthesis is a rapid process prone to error
- About 30% of newly synthesized proteins contain errors
- These proteins are immediately degraded by the ubiquitin pathway
What happens to proteins which have aged and loss stability in the proteasome?
- The result of damage while functioning within the cell – normal ware and tare
- These proteins must show conformational changes for ubiquitination
What happens to proteins with short half lives in the proteasome?
- Many regulatory proteins have short half-lives and are rapidly degraded.
- (e.g., signal transduction pathways, cell-cycle control, transcription, apoptosis, antigen processing, biological clock control)
How is all the recognition in the proteasome achieved?
- N-terminal sequence affects half-life e.g., proteins with N-terminal
- Ala, Gly have half-lives of 20+ hours; Arg or Lys last a few minutes only
- PEST sequence (Pro-Glu-Ser-Thr) also mark a protein for rapid degradation
Whats the N-end rule of the proteasome and what does it do?
Dependence of half-lives of cytoplasmic yeast proteins on amino-terminal residues
Summary of lecture 20
- The life span of intracellular proteins is largely determined by their susceptibility to protein degradation
- Many proteins are marked for destruction by polyubiquitination
- Ubiquitin is ligated to proteins (or other ubiquitin molecules) in three key steps (E1: Activation, E2: Conjugation, E3: Ligation)
- The proteasome is the molecular machine used to degrade proteins. It consists of 50 subunits (20S core has 28 subunits arranged =α 1-7, β 1-7, β 1-7, α 1-7)
- Ubiquitination is reversible due to the activity of deubiquitinating enzymes
Tell me about protein quality control?
- Partially misfolded proteins can be rescued by chaperones
- Proteins that are more seriously damaged (incorrectly synthesized, badly misfolded, damaged) are degraded by the ubiquitin system
What do inflammatory signals (TNF) result in, in ubiquitination dependent control of inflammation?
Inflammatory signals (TNF) result in the phosphorylation of IκBα
Cell cycle
Tell me about the other phases of the cell cycle and ubiquitins role in this?
- The S-Phase cyclin-CDK complexes begin to accumulate in G1 but are inhibited by Sic1.
- This inhibition prevents initiation of DNA replication until the cells have completed all G1 events.
- G1/S phase CDKs assembled in late G1, phosphorylate Sic1, marking it for ubiquitination by the SCF ubiquitin ligase and subsequent proteasomal degradation.
- The active S phase CDKs then trigger DNA synthesis
In many cancers, proteins that normally kill cancer cells are rapidly degraded, how does Bortezomib’s effect this?
- Bortezomib’s mode of action is to inhibit the proteasome and allow those proteins to kill the cancer cells
- Currently used for multiple myeloma and mantle cell lymphoma
Tell me some other roles of ubiquitin modification?
- So far: ubiquitin modification of a protein results in degradation
- However, ubiquitin modification of a protein can has other functions
- The multiple lysine’s are involved in other cellular processes
- C-terminus is site of attachment to protein that is to be degraded
Tell me the multiple forms of ubiquitin and its roles?
After ubiquitinated Hrs directs cargo proteins into vesicle buds and recruits cytosolic ESCRT complexes to the membrane, what happens?
- After ESCRT/Hrs complexes mediate membrane fusion and pinching off the completed vesicle
- they are disassembled via AAA-ATPase Vps4 and returned to the cytosol
The HIV life cycle
Tell me similarities with the C-terminus of Gag?
Similarities in sorting and budding established by mutations of C-terminus of Gag, Tsg101 and VPS4 individually leading to accumulation of Gag at the plasma membrane but abrogating budding
How are ubiquitin patterns distinguished?
Ubiquitinated proteins are recognized by receptors that contain ubiquitin-binding domains (UBDs)
Mechanisms for specificity in ubiquitination?
Other protein modifications are being discovered
What does SUMO have a function in?
Function in nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, stress response, cell-cycle
The ‘ubiquitination code’
