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
