Lecture 2 chaperons and folding Flashcards
What are chaperones?
Proteins that bind to exposed hydrophobic regions of other misfolded proteins preventing their aggregation and assisting their correct native state.
What do they prevent?
The accumulation of toxic aggregates such as amyloid. These disrupt the function of the heart, kidney, liver etc causing damage.
Who discovered the shock response in Drosophilia and what did it show?
Feruccio Ritossa 1962. Drasophila have polytene chromosomes in salivary glands form puffs during active transcription. When larvae were exposed to heat stress, specific puffs appeared to certain chromosomal sites. The puffs indicated increased transcription of a small set of genes that encode heat shock proteins which act as molecular chaperones to refold the proteins and protect the cells from stress.
How were Arabidopsis used to look at protein chaperones?
Treated with mild heat shock. Allowed to recover and challenged with second heat shock. This was associated with expression of heat shock proteins.
What is thermotolerance?
The tolerance for a higher temperature. Can observe increase in expression of certain proteins. Chaperones are not just related to heat shock it was the first way they were found.
What was the radioactive experimental approach?
Cells were incubated with 3H-leucine a radioactive amino acid to track newly synthesized proteins. Heat shock. Extracted and run on SDS-PAGE. This confirmed the selective induction of HSPs in response to heat stress.
What are the stress conditions that lead to the activation of heat shock proteins?
Extreme temperatures, oxidative conditions, expression of foreign proteins e.g., viral, chemicals e.g., heavy metals.
How are HSP involved in other pathways?
Regulate transcription factors. Need to increase the transcription level of Hsps in a higher temperature to stabilise the proteins that have lost certain bonds.
What are the different heat shock chaperone families?
hsp100 (E. coli ClpA, ClpB), hsp90 (pro- and eukaryotes), hsp70 (E. Coli DNAK, yeast SS), hsp60 (E. Coli GroEL), small hsps hsp40 (E. Coli DNAJ, yeast YDJ), hsp25.
What are the general properties of chaperones?
- Assist folding. Confirmation of target still determined by sequence.
- Often recognise exposed hydrophobic residues. Can involve co chaperones like hsp40 aids hsp70.
- ATPase activity. ATP hydrolysis associated with confirmational change in chaperone so change in target. ATP binding releases polypeptide.
- Compartment specific. e.g., BiP (hsp70-related) in endoplasmic reticulum.
- Important element of quality control or proteastasis mechanism.
- Many cycles of polypeptide binding and release, until it folds or is passed on. Binding and release of ATP until native protein state is achieved.
What is the role of Hsp70?
Clamp. Diverse functions: transmembrane translocation, folding, disaggregation, endocytosis, exocytosis, protein degradation (via chaperones). They are abundant and ubiquitous.
What is the structure of Hsp70?
2 domains. ATP binding/hydrolysis (ATPase) and substrate binding/lid, joined by linker. Binds extended hydrophobic regions of polypeptides and holds them in that state. Non charged or non polar amino acids in the substrate binding domain.
What is the ATP binding and hydrolysis cycle of Hsp70?
With the ADP bound state the linker region extends, allowing the lid to close over the substrate. SBD holds onto the substrate tightly preventing aggregation. With the ATP bound state ATP binding causes a conformational change where the lid and SBD bind the ATPase domain. Opens the SBD leading to substrate release.
What are the domains of Hsp70?
Nucleotide binding domain binds ATP/ADP and controls activity. Substrate binding domain holds unfolded or misfolded proteins. Lipid domain covers and stabilizes the substrate in the ADP bound state.
What is the transition of ADP to ATP caused by?
Driven by ATP hydrolysis. ATP to ADP and Pi triggers a confirmational switch from open to close. Hsp70 cycles through substrate binding and release.
What is the mechanism of action of Hsp70 assisted by Hsp40?
Unfolded or partially folded substrate presented by co-chaperone e.g., hsp40. Hsp40 recruits Hsp70 in its ATP-bound state. The unfolded protein binds to Hsp70. Hsp40 promotes ATP hydrolysis in Hsp70 which closes the substrate binding clef stabilizing the bound protein. Hsp70-interacting protein Hip helps maintain this closed state. A nucleotide exchange factor NEF such as Bag (Bcl-2-associated athanogene) facilitates the exchange of ADP for ATP. Substrate protein is released and either folds properly or rebinds for additional folding assistance.
What is the structure of Hsp40?
Diverse family (40 in mammals) target Hsp70 to different sites/functions. Is an elongated V-shaped dimer. The helical J domain activates Hsp70 ATPase activity by binding near ATPase-SBD linker. The substrate binding domain has 2 beta-subdomains.
How are Hsc70 substrates identified?
Pulse-label cell 5 min with radioactive amino acid. Hsc70 associates with newly synthesised protein. The cells lysed and centrifuged. Immunoprecipitation with antibody to Hsp70 traps associated polypeptides.
What percentage of newly synthesized mammalian cytosolic polypeptides are precipiated by anti-hsc70?
15-20%
What percentage of bacterial soluble polypeptides are precipitated by anti-DNAK?
10%
How does protein misfolding cause cystic fibrosis?
Loss of function mutations in CFTR destabilize protein so misfolding in the ER leading to subsequent degradation.
How does protein misfolding lead to retinitis pigmentosa?
Mutations of highly abundant photoreceptor protein rhodopsin. Affects folding and transport leads to photoreceptor cell death and blindness.
How does protein misfolding lead to neurodegenerative conditions?
Aggregation of a diverse set of peptides/proteins associated with the conversion to amyloid-like fibrillar assemblies.
When do Hsp70 proteins act?
Before the protein leaves the ribosome.
