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Proteostasis (Protein homeostasis), CHOP a transcription factor. and when…

- - - - Hsp70 (heat shock protein
        
        Major Chaperone family, located in cytosol, ER and mitochondria
      - Hsp90 (signalling) functions as a homodimer
      - Hsp 60 (a large chaperonin of the mitochondria)
      - TRic (chaperonon of the cytosol)
    - - Hsp40 ( regulates function of HSp70 fam
      - TPRs
    - - small Hsp's
  - - - Hsp40 is bound to a non native protien. (un/misfolded protien) and binds to binds to Hsp70 due to high association constant= stimulates ATP hydrolysis ( Pi and Hsp40 are released from complex and leaves = Hsp70 + misfolded protien + ADP). Hsp70 has undergone a conformational change and is 'closed' The kon is low; it will not readily accept or dissociated misfolded protiens. (TLDR: ADP bound state= HSP70 closed)
        
        Hip binds to the ADP bound Hsp70 + misfolded protien complex and stabilise so that Hsp70 is bound to the protein for longer so that it can longer to fold up correctly/ or it can be assisting in the folding mechanism.
        
        NEF( nuclar exchange factor) swaps the ADP for ATP and we are at the beginning of the cycle.
  - - - 1.during PS the full primary sequence is unavailable.
      - 2.Protein is exposed to the cytosol
      - 3.The C-terminus remains anchored to the ribosome= until that protien is fully removed from the ribosome, the protein will be unable to go into its native state. the Hydrophobic regions of the protein are also exposed to the aqueous environment= they will start to aggerate with other regions
      - (4) mRNA is translated on polysomes (ribosomes bound to the mRNA making the protein. the ribosomes can come into close proximity to each other which can causes aggregates) Neo-proteins cominto close proximity to one another = causing hydropgoci regions of adjacent proteins aggregate together
      - Solving the problem with chaperons
        
        Ribosomes have long exit channels that restricts the protein from misfolding, as the neo-protein is emerging however if the protein is large then this problem can still occur
        
        NAC(nascent-chain assocaited complex Chaperon)
        
        located at the end of the exit channel so that the protien has another oppertunity to correctly fold. theey are both ribosome binding chaperons to prevent misfolding/assit in correct folding
        
        RAC Ribsome assciated complex
- - - - HSF in a nutshell: HSF1( heat shock factor 1 is a transcription factor that translocate to the nucleus stimulates by cytosolic stress due to change in the environment. Role at the nucleus: increase the transcription of chaperone proteins= Increase in protection for the proteins that are being stressed due to environmental change.
      - 1. unstressed cell: HSF1 bound to Hsp70 + Hsp90.
        
        2. the cell is stressed through some stimuli: e.g. cell gets hot leads to the accumulation of misfolded proteins= Hsp70+90 bound to HSF1 dissociated and look after the misfolded protein= the liberation of HSF1 in the cell
        
        3. HSF1 activating post post-translation modifications'
        HSF1 trimerises after phosphorylation (3 HSF come together/phospohylation) and translocates to the nuclecus + binds to Heat shock shock elements (promoter regions on Hsp genes) this = increases trnascription of chaperon proteins= increases concentration of chaperones that bind to HSF1 to shut down the reposens and also bind to the misfolded protiens to refolded them or degreadeed them thus restoring balance.
      - The Heat shock response with age
        
        there is reduced HSF1 binding to DNA containing HSE (seen in young vs old mice) even if there stess is preseent this could be due to phosphorylation
        
        Reduced Phosphorylation of HSF1 at residues which promote DNA binding with age. if there is lesss phosphoylation at promoting sites then there will be less HSF1 to bind to the DNA containing HSE. so there is less HSF1 and also reduced function
        
        Increased HSF1 acetylation which prevents HSF1-DNA binding with age
        
        might also be more HSP70, 90 + 40 in aged tissues couples with decreased HSF1 which provides more of a blockade to HSF-1 movement to the nucleaus
        
        HSF1 with the same stress stimuli stays bound to the heat shock protien and the inhibatory post translation modification dominate so HSF1 cannot go to the nucluse and you get a comprosied heat shock resposne.
        
        HSF1 and lifespan: study showed that a mutation in HSF1 can alter lifespan. Reduced HSF1 reduces lifespan. increased HSF1 increase lifespan. and when it is overexpressed the lifespan was seen to increase.
      - HSF1+ Exercise
        
        Exercise increases HSF1 protein levels in the heart.
        
        Overexpressing HsF1 during pressure overload partially prevents fibrosis and cell death and improves function (pressure overload stimulates pathological hypertrophy that results in heart failure.
        
        Chaperone expression and exercise
        
        exerices= form of cellular stress increase= triggers a heat shock response.
        
        : increases levels of chaperones across several tissues affors tissue additional protections agaisnt misfolded proteins
        
        Exercise and heat are factors that will induce a heat shock response and in turn increases level of chaperons. they also increases Hsp70 expression in the brain
  - - - most proteins are important to the ER during protein synthesis (co-translational translocation). Protein syntheses and modified in the ER are either memebrane bound or to be secreted from the cell ER has its own set of specialises chaperones to assist correct folding.
    - - 1. ATF6 is bound to a Bip in an unstressed ER. (a HSp70 for the ER).
        
        2.With ER stress, Bip dissociates from HSp70 and binds to the misfolded proteins
        
        ATF6 is liberated is transported to the golgi (via COPII vesicle
        
        At the Golgi ATF6 proteases cleave ATF6 into 2 parts. ATF6(n-terminus) behaves a functional transcription factor
        
        ATF6 leaves the golgi goes to the nucleus and increases the transcription of chaperons
      - 1.PERK is bound to Bip (a HSP70) in an unstressed ER (the perk arm is the arm that is responsible for shutting down protein synthesis)
        
        ER stress= Bip dissociates and binds to misfolded proteins
        
        PERK( normally a monimor when bound to BIP). two perks come together to form a dimer, upon forming a dimer they autophosphorylates= this switches on it's kinase activty= PERK can now act as a kinase
        
        Perk then phosphorylates EIF2alpha(upon phosphorylation it inhibits protein synthesis
        
        ATF4 moves to the nucleus and acts like a transcription factor which produces factors than can resolve ER stress.
      - IRE1 (is a free entiey) it binds directly to misfolded proteins in the ER
        
        IRE1- then forms a Dimer and the dimer then trans-autophosphorylates = each dimer phosphyolates each other
        
        Phosphorylation stimulates IRE-1 endoribonuclease activty (the ability of a protein to degrade RNA).
        
        when IRE-1 is acting as an endoribonuclease it will splice EBP1 into XBP1s( a functional transcription factor) which then translocated to the nucleus and increases factors that resolve ER stress
        
        IRE-1 as a endoribonuclease also degrades MRNA that about to move into the ER via co translocation. But IRE1 inhbits this process as it doesnt want to add to the burdan within the ER.(this process is called regulated IRE1 dependent decay (RIDD)
- - - - The inability of peripheral tissues to uptake glucose in response to insulin (insulin resistance) which puts excessive demand on the pancreatic Beta cells to produce more insulin. which ultimately drives Beta cell death and Chronic hyperglycaemia= long term damage cardiovascular system, nervous system, kidneys and eyes .
    - - Manipulation of CHOP: Beta cells die in diabetes due to chronic ER stress that leads to apoptosis when chop is present. When chop is knocked out the beta cells dont undergo apoptosis.
        
        In diabetic mice: the deletcion of CHOP prevents cell death and restores glycaemic control as there is more beta cells to secrete insulin thus reducing the stress on the beta cells. there is a link to diabetes and ER stress that leads to Chop induced apoptosis which leads to stress on beta cells to produce insulin and a reduced beta cell capacity.
        
        Insulin resistnace results in increased proinsulin secretory load in the ER. subsequent chronic ER stress (the demand exceeds the capacity) and UPR (er) results in Beta cell death and type 2 diabetes via induction of CHOP.
  - - - study in worms/cells found that with age, inducing the upr(er) the response is damped. i.e. the UPR(er) loses the ability t omount an adaptive response to cellular stress
    - - there are also tissue specfic effects: in neurons: you can see that the consitutive expression of EXB1s extendend life span, but looking at muscle tissue there was an negative effect on life span. it depends on the tissue, depends on the time of life (similar to heterochronic parabiosis)
  - - - E1 UB activating enzyme: the cysteien from the E1 enzymes froms a thioester bond with the COOH of Ub terminal gylcine= activated E1 enzyme. through the hydrolysis of ATP. the Ub molecule has been activated
        
        carrying the E2 to the substrate: the Ub is transferred from the E1 to E2 carrier enzyme.
        
        There are 20-30 types of E2 enzymes which introduces specificty to the system
        
        E3 ubiquitin ligases: E3s transfer the Ub from an E2 to the substrate protein. E3s recognise the target substrate (i.e. misfolded protein) via exposed hydrophobic regions
        
        Two types of E3:
        
        HECt domains: accept the Ub from E2 and transfer it to the substrate
        
        RING fingers: most abundant E3, act as a scaffold to bring E2 and substrate close enough for direct transfer of Ub
        
        up to 10000 different E3s
        
        each cell has one E1 isoform(type)
        
        Activation stage of Ubiquitin
    - - the 19s subunit recognises the polyubiqutinted protien. the deubiquitnates is to be recycled
        
        the protein is being held in the proteasome, and the protien is unfolded by ATPases opens the 20s core
        
        in the 20s core, numouers proteases degrade the protein into amino acids to be used by the cell.