SPOONING Flashcards
(1) What favours aggregation of proteins
Macromolecular crowding
(1) Issue with nascent proteins
They are in a non-native, aggregation-prone conformation
(1) Process of protein folding with chaperone
1) Hydrophobic patches on unfolded proteins found by heat shock protein 40 family (Hsp40 co-chaperone)
2) Deliver substrate to ATP-bound (open comf), bind Hsc 70 and stim ATPase activity of Hsc70
3) Results in ADP-bound (Closed conf), Hsc70 shielding hydrophobic patches, preventing aggregation (allows folding)
4) ADP exchanged with ATP (by NEF), Hsc70 opens and releases protein to snap into final conf
(1) Roles of cystosolic molecular chaperones
1) Prevent aggregation of unfolded proteins
2) Provide a controlled environment for folding
3) Permit assembly of multimeric complexes
4) Direct proteins with folding problems for destruction
(1) Process of targeting protein to proteasome
1) Ub activated by E1 ubiquitin-activating
2) Activation Ub is transferred to an E2 ubiquitin-conjugating enzyme
3) E2-Ub conjugate associates with E3 ubiquitin liagse
4) E3-E2-Ub conjugate binds the target protein
5) Transfers Ub to the target
(1) The ubiquitin proteasome system (UPS)
1) Ub is activated by an E1 ub-activating enzyme
2) Ub is transferred to an E2 ub conjugating enzyme
3) Ub conjugated E2 selects E3 Ub liagse
4) E3 transfers Ub to a lysyl side chain of target protein
5) Process repeated until tetra-ubi
6) Ub-target protein binds proteasome RP and degraded in core DUBs recycle ub
(1) List the 9 cytosolic post-translational modifications
1) Protein folding (Mediated by chaperones/co-chaperones and chaperonins)
2) Assembly of multimeric complexes (Mediated by chaperones/co-chaperones)
3) Polyubiquitylation (Results in proteasomal targeting)
4) Proteasome (mediated proteolysis)
5) Proteolytic cleavage to activate proteins when required
6) Addition of lipids (Permit membrane targeting)
7) Phosphorlyation (on/off switches)
8) ADP ribosylation
9) Methylation
(2) Modification of mRNAs for secreted proteins
Addition of 5’ signal sequence, encodes a N-terminal signal peptide –> targets into ER lumen
-SPs are free to evolve rapidly as long as they keep: +ve charge towards the N terminus and hydrophobic stretch
(2) The SRP (signal recognition particle) cycle
1) ER signal peptide captured by SRP
2) Directs SRP/SP complex (targeting) to A subunit of SRP receptors on ER membrane
3) Recruitment of closed translocon
4) Translocon opens to allow entry of SP (while SRP/SRP receptor complex is dismantled and SRP recycled)
5) Signal peptidase removes SP ( then released into ER membrane)
6) signal peptide peptidase (SPP) that cuts released SP in place of membrane, allowing easy removal of 2 hald SPs.
7) As translation finishes, protein freed into ER lumen and folds.
(2) Enzyme that N-glycosylates proteins
Oligosaccharide transferase (OST)
(2) Role of N-glycans
1) Increase protein stability and reduce aggregation problems in folding in ER
2) N-glycans bulky so constrain A-carbon backbone, influencing folding rates and find protein conformation
3) Influence activity and interactions with other molecules
4) Flags for folding and ER quality control. Allows interactions with ER chaperones (e.g. calreticulin)
(2) Enzyme that catalyses disulphide bond formation
Protein disulphide isomerase (PDI) and oxidising conditions
(2) 3 other secretory system post-translational mods
1) Attachment of sugars to O atoms of aa (O-glycosylation)
2) Proteolytic cleavage
3) Addition of lipids to permit/maintain membrane targeting (e.g. GPCRs have myristic acid lipid anchor)
(2) Process of matrix-targeting sequence and N-terminal leader peptides
1) LP recognised by specific import receptors of TOM complexes (Translocase outer membrane)
2) Pass through transport channel of TOM40
3) Rare contact sites, protein passed to a channel of TIM23 and TIM17 (Translocase inner membrane)
4) Protein enters the matrix
5) TIM-mediated transport requires proton-motive force
6) ATP used by matrix Hsp70 mortalin (bound to TIM44)
7) mt Hsp70 prevents aggragation mt proteins.
8) Protein released from mortalin and matrix targeting signal removed by matrix protease
