Protein Degradation Flashcards
Structural proteins tend to have ____ lifetimes, while regulatory proteins tend to have _____ lifetimes
Structural proteins - long (low turnover rates)
Regulatory proteins - short (degraded after serving its function, and to prevent over regulation of activities)
What are the roles of protein degradation and why are they important?
- Ensure proper regulation of cell signalling pathways (e.g., signal transduction) through maintaining normal protein turnovers => no under/over regulation
- Remove misfolded and damaged proteins that can lead to abnormal cellular activities
- these non-functional/abnormal misfolded proteins can occur due to multiple cellular activities and enzymatic activities causing wear and tear
An example of a short-lived protein is the hypoxia inducible factor 1a (HIF-1a) which is a transcription factor responsible for oxygen homeostasis during hypoxic conditions.
Explain its function in hypoxic conditions.
Tissues sense change in environment during hypoxic conditions, and increase HIF-1a levels. HIF-1a induces expression of a panel of genes that can be involved in angiogenesis, cell migration, and glycolytic pathway
Explain what happens to HIF-1a in normoxia conditions
HIF-1a maintained at low levels in normoxia condition (usually undetectable levels)
It has short half-life of 5-8min
It is hydroxylated at Pro402 and Pro564 by oxygen-dependent prolylhydroxylases. Hydroxylated HIF1a is recognized and targeted for ubiquitination. Ubiquitinated HIF1a degraded by 26S proteasome.
In Von Hippel-Lindau (VHL) disease, mutation of the VHL gene causes it to encode for a mutated protein pVHL which causes ____________________
Failure to recognize and degrade HIF-1a by the ubiquitin proteasome system, accumulation of HIF1a
(because pVHL is a substrate-recognization component of multimeric E3 ligase of the ubiquitin-proteasome system)
What happens when HIF-1a is not degraded and accumulates?
Increased HIF1a transcriptional activity, increased expression of target genes
- Increase MMPs - chemokine processing, encourage cell migration and cell invasion
- Increase VEGF - increase invasion, metastasis, angiogenesis
=> Higher risk of tumor development
=> Tumors developed in VHL disease have high HIF1a transcriptional activity and high degree of vascularization
What are the two pathways of protein degradation in mammalian cells?
- Lysosomal degradation (minor)
- Proteasomal degradation (major)
[LYSOSOMAL DEGRADATION]
Explain lysosomal degradation
Proteolysis occurs in lysosomes
It is a non-specific process (any proteins in lysosomes will be degraded)
In higher eukaryotes, only membrane-associated/receptor-mediated proteins and alien proteins internalized by endocytosis will be degraded in lysosomes
[LYSOSOMAL DEGRADATION]
Contrast the 3 types of endocytosis
Phagocytosis ‘cell-eating’
- Large solid particles get phagocytosed into cells as phagosomes
- E.g., cell debris, bacterial cells
Pinocytosis ‘cell-drinking’
- Extracellular fluids and solutes are ingested by budding of small vesicles from the cell membrane
- It is a non-specific process (meaning any fluid can get ingested)
Receptor-mediated endocytosis
- Specific molecules (e.g., hormones, metabolites, virus) recognized by specific receptors
- Receptor and molecule get internalized into coated vesicles, which in turn fuse with endosomes
- Content in endosome sent to lysosomes for degradation or recycled to plasma membrane
[PROTEASOMAL DEGRADATION]
Explain proteasomal degradation
Intracellular proteins get degraded by 26S proteasome
It is a specific process, that takes place mostly for ubiquitinated proteins, and some non-ubiquitinated proteins
[PROTEASOMAL DEGRADATION]
What is the structure of the proteasome?
Large cyclindrical particle, consisting of at least 33 subunits
Huge protein with total MW 2.5MDa
[PROTEASOMAL DEGRADATION]
What is the role of the 26S proteasome?
*26S proteasome is the major proteasome found in all mammalian cells
Responsible for specific degradation of regulatory proteins and removal of damaged proteins
[PROTEASOMAL DEGRADATION]
What are the structures/components in the 26S proteasome?
Composed of 20S core particle capped by a 19S regulatory particle (at either one or both ends)
20S core particle (aka degradation chamber) is made up of 4 heptameric rings assembled to form cylindrical structure
- 2 outer rings = 2a subunits
- 2 inner rings = 2B subunits
The inner rings house a hollow central cavity, and the walls of the inner ring contain proteolytic active sites
[PROTEASOMAL DEGRADATION]
Explain the role of 19S regulatory particle in protein degradation through the 26S proteasome
19S regulatory particle (arranged into lid and base)
- Contains ATPase subunits (energy-dependent) to gate entrance into the degradation channel/chamber (entry is 13A)
- Plays a role in substrate recognition, unfolding, translocation into degradation chamber
=> 19S cap hydrolyzes ATP to provide energy to drive removal of Ub, protein unfolding, and transfer/translocation of unfolded protein into the inner degradation chamber of the proteasome
[PROTEASOMAL DEGRADATION]
Explain the role of 20S core particle in protein degradation through the 26S proteasome
20S core particle (degradation chamber)
- Protein arrives in the degradation chamber through a channel running along the long axis of the core particle
- There is a narrow entrance into the channel (13A), gated by 19S regulatory particle
- Folded particles must be partially unfolded in the 19S regulatory particle before they can be translocated into the 20S core particle
- Upon entering the channel, protein unfolds further, and stretches along the channel
- The proteins are hydrolyzed to short peptides of 3-25AAs at the proteolytic active sites (in the 2B inner rings)
- The AAs are eventually released from the opposite 19S cap
[PROTEASOMAL DEGRADATION]
Why is proteasomal degradation also known as ubiquitin-dependent protein degradation?
Proteolysis of substrate in 26S proteasome is selective as 19S regulatory particle must recognize the polyubiquitin chain
- Proteins must be marked by >1 ubiquitin for recognition
=> Upon recognition by 19S cap, 19S cap hydrolyzes ATP to provide energy to drive removal of Ub, protein unfolding, and transfer/translocation of unfolded protein into the inner degradation chamber of the proteasome
[PROTEASOMAL DEGRADATION]
How are proteins marked by ubiquitin?
- Where do ubiquitin monomers come from?
- How do they get attached to the protein?
Ubiquitin tag is cleaved by deubiquitinating enzymes (DUB) into monomers
The monomers escape from the proteasome and are recycled to label other protein substrates to be degraded
Each monomer is a very small protein, 76-residue polypeptide containing 7 lysines
The monomer is attached to substrate protein through an isopeptide bond between C-terminal Gly of Ub and the amino group of Lys in the substrate (via biochemical reaction catalyzed by enzymes)
A minimal signal of a chain of 4 ubiquitin monomers linked through Lys48 are needed for proteasome targeting
[PROTEASOMAL DEGRADATION]
Monoubiquitination is a __________
Monoubiquitination is a predominant regulatory modification (PTM) - to activate/inactivate proteins to carry out different cellular functions: E.g.,
- Monoubiquitnination of histones and transcription factors regulate transcription
- Monoubiquitination of surface cell receptors signal for their endocytosis and degradation in lysosomes
However, some monoubiquitinated protein have been found to be targeted for proteosomal degradation
[PROTEASOMAL DEGRADATION]
What are the various routes of delivery of substrates (intracellular proteins) to the proteasome?
- Substrates bind directly to the proteasome by interacting with 19S regulatory particle that recognizes the polyubiquitin tag
- Substrates are brought to the proteasome by adaptor proteins that bind both the proteasome and the polyubiquitin chain on the substrate (bring them close in proximity)
- Uncommon: some protein substrates are degraded by proteasomes without being ubiquitinated
[PROTEASOMAL DEGRADATION]
The proteasome itself is a large protein that can be recognized for degradation, explain how it might get degraded?
Proteasome has long half-life, and exists intracellularly.
It may leak out of the cell if the cell is dying. If proteasome is found extracellularly, it means that the cell is reaching the end of its lifespan. Large proteasome can be recognized and targeted for degradation.
*Extracellularly, can be degraded by proteases, immune cells etc.
