Protein Misfolding Flashcards
1
Q
in vivo folding
A
- uses molecular chaperones
- designed to prevent aggregation
- Hsp 60 and 70 (heat shock)
2
Q
Hsp70
A
- binds to proteins as they are synthesized from ribosome when they are in intermediate but not native folding conformations
- protect against aggregation by covering sticky hydrophobic patches
3
Q
Hsp60
A
- 14 Hsp60 monomers associate to form a large double donut structure thats hollow
- misfolded proteins enter the cavity
- groES cap closes over groEL with protein inside
- ATP hydrolysis is used to unfold misfolded protein
- can refold when protected inside the cavity
- if they don’t fold in the time the groES dissociates, they try again on their own
- cyclic process, may take several rounds to fold especially problematic proteins
4
Q
chaperones
A
- on;y used to fold a very small fraction of proteins synthesized in the cell
- large mutidomain proteins are their principle targets
- cell has mechanisms to prevent misfolding but sometimes the mechanisms get overwhelmed
5
Q
GroEl/GroES possible mechanisms
A
- true enzyme (lowers E of transition state for folding)
- Anfinsen box (isolation)
- iterative annealing
- fold faster in box than in solution-active, protective container (confinement)
-likely a combination of box, annealing, and confinement
6
Q
iterative annealing
A
- unfolded/misfolded protein binds in GroEL cavity
- GroES binds (due to affinity for ATP bound version) deforms cavity, changes interior residues from non-polar to polar, which mechanically unfolds the protein by dissociating the hydrophobic interactions
- ATP hydrolysis by GroEL ~13 sec timing mechanism
- GroES dissociates, protein is released to try and refold again
7
Q
Protein degredation
A
- turnover is necessary; many cellular processes are regulated by ubiquitin/ proteasome pathway
- proteasome inhibitors in clinical trials for cancer, HIV, cardiovascular disease
- many proteins are supposed to be only transiently active
- also plays a response in immunological response (MHC class 1)
8
Q
ubiquitin/proteasome pathway
A
- E1- binds to Ub and activates; ATP driven reaction, thioester bond
- E2 transfers the activated Ub to the target protein which is bound to a specific E3 molecule (via thioester E2-Ub intermediate)
- E3 catalyzes final transfer of Ub to the epsilon amino group of one or more specific lysine residues on the target protein- this is repeated until at least 4 for degredation
9
Q
proteasome
A
- 26S particle
- made of 20S core and 19S regulatory cap
- 6 AAA-ATPase (gold) subunits in the 19S cap and other non-ATPase domains
- 66 proteins and 1.5 megadaltons
- poly Ub proteins bind to the non ATPase subunits of the 19S cap
- AAA-ATPase subunits use ATP to unfold the bound protein
- unfolded protein is threaded into channel of 20S cylinder
- de Ub enzymes cleave Ub to be used again (in non-ATPase subunits)
- 20S particle cleaves protein into peptides from 3-30 AA (important but unresolved), context dependence on where, influenced by 19S
- peptides transported through the ER for antigen presentation by MHC class I or simply recycled to build new proteins (endo and exopeptidases in cell)
10
Q
diseases of Ub-proteasome pathway
A
- cancer- increased growth rates make cancer cells more dependent on proteasome (destroying protective cells), increased degradation of p53 and p27
- neurodegenerative-AD, parkinson, Huntington-accumulation of Ub proteins in plaques, Lewy bodies; cause or by-product?
CF- clears misfolded deltaF508 CFTR (no protein at all)
Autoimmune-improper processing of peptide antigens
11
Q
molecular basis of disease
A
- loss of protein function
- formation of alternate conformations-poison nearby cells, tissues, organs
- aggregates involved in above 2
- gain of function- increases activity or gains a new function- always on mutations
12
Q
function, structure, and balance
A
- native conformation is correct function
- direct knockout
- destabilization
- toxic conformation
13
Q
direct knockout
A
- mutation of a residue that is essential for function
- ex-involved in substrate binding
- structure and stability of the protein are essentially unchanged, can’t function because a critical side chain has been altered
14
Q
destabilization
A
- pushes equilibrium toward unfolded state
- can’t fold (can’t muster energy)
- change of side chain in a hydrophobic core to one that is substantially different
15
Q
toxic conformation
A
- mutations shifts the conformational equilibrium to an incorrectly folded state
- mutating a surface charged residue to a hydrophobic one
- causes aggregations
- can cause formation to change dramatically-amyloid diseases
16
Q
p53 cancer
A
- ~50% of all tumors have point mutations in p53
- most frequently mutated protein in cancer
- over 15,000 mutations cataloged to date
- TF
- activated by DNA damage or other insult
- triggers cell cycle arrest or apoptosis (by binding to DNA and activating transcription of certain genes)
- prevents accumulation of chromosomal mutations
17
Q
p53
A
- p53 is modular design
- three domains
- tetramerizes at short c terminal domain
- large central domain is where site specific DNA binding occurs
- n terminal domain facilitates transcription by binding to other proteins and recruiting them to approproate sites
- over 90% of tumorogenic mutations are in central DNA binding domain
18
Q
p53 mutations and stability
A
- DNA contact mutations alter side chains that bind to DNA
- reduce DNA binding without changing overall protein structure and stability
- stability mutants in Beta-sandwich don’t change DNA binding but reduce stability greatly, disrupt hydrophobic, electrostatic, H bonding or VdW forces
- less stable p53 leads to faster degradation by the UB/proteasome pathway-not enough p53 to do its job
- can cause aggregation due to incorrect folding of hydrophobic cores (intermediate is stable but native isn’t)- associates with itself
19
Q
drugging p53
A
- design a small molecule to bind to the protein and stabilize native state
- look for unique features on solvent-accessible surface area rendering
- find small cavity or pocket for the molecule to fit for shape/size and chemical compatibility
- should only stabilize this protein
- involves computer based docking, screening libraries for lead compounds, chemical synthesis and re-screening, structure activity relationships via X-ray or NMR, animal studies and clinical trials
20
Q
druggin p53 2
A
- block interaction between p53 and MDM2 (E3 that recognizes p53)
- allow mutant p53 to accumulate so even though specific activity is low, overall activity is restored by elevating total levels
-normal p53 has expiration code and if it’s mutated its tagged earlier-try and keep it around
21
Q
CF
A
- fatal, 30 yr average life span
- thick sticky mucus in lung, pancreas, intestine
- affects sweat, tear, and salivary glands
- inability to absorb nutrients-high infant mortality
- buildup of fluid in lungs-infection and lung degeneration
- 70% caused by deltaF508 in CFTR protein
- most lethal in caucasian people
- gated chloride channel of unknown structure.
22
Q
proposed structure of CFTR
A
- ABC transporter
- gated ion channel
- pump solutes in and out of cells
- nucleotide binding domain
- uses ATP to pump
