macromolecules Flashcards
Why do proteins undergo degradation?
- Different lifespan across proteins
- Structural tend to have longer lifespans (low turnover)
- (most) Regulatory tend to have shorter lifespans
- To ensure proper regulation of cell signalling pathways through maintaining normal protein turnovers
- To remove misfolded (no native conformation) and damaged (eg. oxidised) proteins that can lead to abnormal cellular activities
What if proteins cannot undergo degradation?
If no degradation —> accumulation of toxicity —> body cannot function properly —> disease
what is an example of the consequences of a protein not being able to undergo degradation?
Short-lived protein: Hypoxia inducible factor 1 alpha (HIF-1 alpha)
what normally happens to HIF-1 alpha in normoxia conditions?
- A transcription factor that is produced during hypoxia (insuff O2 in tissues for adequate homeostasis) to maintain oxygen homeostasis
- MOA: Induces expression of genes involved in angiogenesis (form new blood vessels), cell migration, glycolytic (glucose is broken down to form pyruvate) pathway
- In normoxia (normal O2 levels), HIF-1 alpha maintained at low levels (negligible) as not needed —> so half life is only 5-8 mins
- Short half-life due to prolyhydroxylases, which is only active when there is O2 (normoxia) —> hydroxylated HIF-1 alpha —> recognised and targeted for ubiquitination —>
- Ubiquitinated HIF-1 alpha degraded by 26S proteasome (aka degraded by ubiquitin-proteasome system)
- pVHL: Part of the ubiquitin-proteasome system that recognises HIF-1 alpha (which is a substrate of pVHL) —> HIF-1 alpha gets ubiquitinated —> Proteasomal degradation
what if HIF-1 alpha cannot be degraded?
- If HIF-1 alpha cannot be degraded —> Von Hipel-Lindau (VHL) disease
- Mutated pVHL —> Cannot degrade HIF-1 alpha —> HIF-1 alpha accumulates (so this happens despite being in normoxia) —> increase in HIF-1 alpha transcriptional activity —> increase expression of target genes: Increase matrix metalloproteinases (MMPs: enzymes that encourage cell migration and invasion), increase vascular endothelial growth factor (VEGF: promote angiogenesis) —> increase invasion, metastasis (spread of cancer cells), angiogenesis
what are the clinical outcomes if HIF-1 alpha cannot be degraded?
- Clinical outcomes: Tumour like pheochromocytomas, hemangioblastomas of CNS, clear-cell renal carcinomas, retinal capillary angiomas ] all have high degrees of vascularisation due to angiogenesis and high HIF-1 alpha transcriptional activity 😖
why does healthy HIF-1 alpha have a short half life?
Short half-life due to prolyhydroxylases, which is only active when there is O2 (normoxia) —> hydroxylate two proline residues (Pro402 and Pro564) in HIF-1 alpha —> hydroxylated HIF-1 alpha —> recognised and targeted for ubiquitination
what is VHL?
- VHL: Heriditary disease, caused by autosomal dominant mutation in one of the alleles of gene VHL which encodes for VHL protein (aka pVHL)
what are the Types of protein degradation in mammalian cells?
- Proteasomal degradation (80-90%)
- Lysosomal degradation
note: Therapeutic proteins (from drugs) will also get degraded by either pathways —> Either identified as alien (since exogenous and taken up by lysosomes) or recombinant protein (eg. recombinant insulin and taken up by proteasome)
who is proteasomal degradation for?
- Normally for ubiquitinated proteins (exceptions for very small minority)
- By 26S proteasome
who is lysosomal degradation for?
Only for membrane-associated proteins and alien proteins (extracellular proteins) —> internalised into a vesicle in lysosomes by endocytosis
which degradation type is a specific process?
proteasomal degradation!
Specific process: Unique for each protein
lysosomal degradation: Not specific: Will be degraded regardless of identity
how does lysosomal degradation occur?
When proteins enter lysosomes that has acidic interior of pH 4.5 —> proteolysis occurs (cleavage of peptide bond)
what is the Structure of Proteasome?
- Very huge cylindrical (core is hollow) protein in cells
- Consist of min 33 subunits, with total molecular weight ~2.5MDa
- Major proteasome: 26S proteasome
- Found in all cells
- Degrades and removes regulatory proteins
- Composed of a 20S core particle: Capped by a 19S regulatory particle at lid and base
- 20S core particle is made up of 4 heptameric rings assembled to form cylindrical structure —> 2 outer rings = 2 alpha subunits AND 2 inner rings = 2 beta subunits
- Inner rings contain proteolytic active sites
how is proteasomal degradation a Selective process?
- Narrow entry into 20S core particle for selectivity
- Partially unfolded proteins need to assume a primary polypeptide chain to fit through —> Upon entry, will totally unfold to stretch along channel to reach/ be translocated to inner rings —> Gets hydrolysed to short peptides of 3-25AA —> released from opposite end of channel
- 19S regulatory particles contain ATPase subunits to gate entrance, remove Ub, unfold and transfer unfolded protein to chamber
- Gate normally closed
- Only allows ubiquitinated proteins into channel
- But ubiquitin is a very small molecule, so need multiple of it to act as a tag on protein to be recognised by proteasome —> Polyubiquitin chain (min 4 ubiquitin molecules that is linked through Lys48)
- Prior to entry, deubiquitinating enzymes (DUBs) will cleave tag into monomers
- Monomers escape proteasome into cytoplasm and are recycled to tag other protein substrate
- Proteasome engages protein substrate —> Polypeptide unfolds to enter and gets translocated into 20S core to be degraded
What is ubiquitin (Ub)?
- Peptide, only has 76 residues with high lysine content of 7Lys
- Amino group of Lys gets utilised to form polyubiquitin chain to be attached to protein substrate
what is Monoubiquitination?
- Attachment of one Ub to protein
- Not targeted for degradation
- Post-translational modification event: Activated or not to carry out cellular function
what is an example of monoubiquitination?
- Monoubiquitination of histones and transcription factors to allow activation of transcription
- Monoubiquitination of surface cell receptors to activate them to participate in endocytosis and degradation in lysosomes
what are the Types of ways that protein substrates reach proteasome?
- Close proximity: Substrates bind directly to proteasome by interacting with 19S regulatory particle subunits (which can recognise ubiquitin tag)
- Substrates brought to proteasome by adaptor proteins that bind to both proteasome and polyubiquitin chain
- Minority: Substrates gets degraded without being ubiquitinated
what are the Types of endocytosis?
- Phagocytosis (cell eating)
- Pinocytosis (cell drinking)
- Receptor-mediated endocytosis
what is endocytosis: Phagocytosis (cell eating) under lysosomal degradation?
- By cytotoxic T cells, innate immunity (eg. macrophages)
- Substance is quite big: Cell debris, dead cells, protein aggregates, pathogenic microorganisms, dust particles, particulate non-living matter
- Membrane bound upon being eaten up
