UPS

Question 1

define proteostasis

Answer 1

regulation of the level, folding, interactions and localisation of proteins

Question 2

what are the 3 degradation pathways

Answer 2

cytosolic proteasome (UPS)
lysosomes
ERAD pathway which culminates in UPS

Question 3

how are the majority of cytosolic proteins degraded

Answer 3

by the UPS pathway

Question 4

how are proteins targeted to the UPS

Answer 4

ubiquitination

Question 5

what is ubiquitin

Answer 5

it is a 76aa protein which can be linked to other proteins via it’s Gly76 residue onto lysine residues

Question 6

describe the process of ubiquitination

Answer 6

e1 formas a thiolesther with the carboxyl group of Gly76 of Ub. E2 transiently carries Ub.
E3 promotes the transfer of Ub onto the substrate protein

Question 7

which protein in the ubiquitination process is responsible for substrate specificity

Answer 7

E3

Question 8

describe the structure and function of the proteasome

Answer 8

26S complex formed by a 20S core and 19S cap. it is a compartmentalised protease in which the inner core can hydrolyse bonds. the cap recognises the protein and removes the Ub as well as unfolding the protein to feed it into the core where hydrolysis occurs

Question 9

what are the 3 proteolytic activities of the 20S proteasome core

Answer 9

tryptic activity cleaves after basic residues
chymotryptic activity cleaves after hydrophobic residues
peptidylglutamylpeptidase activity cleaves after acidic residues

Question 10

what length are the peptides produced by the proteasome

Answer 10

3-22 aa

Question 11

how is unfolding of the substrate protein for the proteasome mediated

Answer 11

AAA+ ATPase activity in the 19S cap

Question 12

what is the role of cytosolic chaperones

Answer 12

recognise misfolded proteins, promote protein refolding, stabalise misfolded proteins

Question 13

how are chaperones and the UPS linked

Answer 13

cytosolic E3 ubiquitin ligase consitutive Hsc70-interacting protein (CHIP) which binds chaperones Hsc70, Hsp70 and Hsp90 and hence associated with misfiled proteins. CHIP mediated ubiquitination promotes the interaction of CHIP with the 26S proteasome via the co-chaperone BAG-1

Question 14

what is autophagy

Answer 14

it is the process by which cytoplasmic components are delivered to the lysosome for degradadtion

Question 15

what are the 3 classes of autophagy

Answer 15

macroautophagy, chaperone-mediated, microautophagy

Question 16

what are the 3 main enzymes find in lysosomes

Answer 16

cathepsin D cathpsin L and cathepsin B

Question 17

what is the role of cathepsin B

Answer 17

a cysteine protease whith both endopeptidase and carbocypeptidase activity

Question 18

what is the role of cathepsin L

Answer 18

a cysteine endopeptidase

Question 19

what is the role of cathepsin D

Answer 19

an aspartate endopeptidase

Question 20

what happens to the amino acids produced by proteolysis in the lysosome

Answer 20

they’re transported to the cytosol by membrane transporters

Question 21

what is the function of macroautophagy

Answer 21

removal of cytoplasmic components e.g. long lived cytosolic proteins, protein aggregates and cellular organelles

Question 22

what enhances macroautophagy

Answer 22

cell starvation

Question 23

describe the process of autophagosome formation

Answer 23

cytoplasmic components are surrounded by a double membrane (potentially from ER, golgi, mit or PM). formation requires the action of a number of protein complexes incl. 30AGT proteins in yeast

Question 24

describe lysosome fusion in macroautophagy

Answer 24

autophagosomes are transported along microtubules to the microtubule organising centre (MTOC). fusion then takes place involving SNARE proteins, VAMP8 and Vti1b

Question 25

which form of autophagy delivers protein directly to the lysosome

Answer 25

chaperone mediated autophagy

Question 26

what is the consensus sequence for delivery to the lysosome for chaperone mediated autophagy

Answer 26

KFERQ-like sequences which is normally buried but exposed on misfiled protein

Question 27

in what conformation can chaperone mediated autophagy degrade proteins

Answer 27

only as single subunits

Question 28

describe the pathway of chaperone mediate autophagy

Answer 28

Hsc70 recognises signal sequences. proteins are bound by the cytosolic portion of the lysosome membrane protein LAP-2A via the KFERQ motif. this promotes multimerisation of LAMP-2A and then unfolding and translocation of the substrate protein into the lysosome for degradation

Question 29

what is the hallmark of parkinson disease

Answer 29

levy bodies (mostly made up of a-synuclein) in the cytoplasm of affected neurones

Question 30

how is aggregation of a-syneuclin spread

Answer 30

it can be transmitted between cells

Question 31

what is the result of an additional coy of the a synuclein gene

Answer 31

increased risk of parkinsons

Question 32

how is a-synuclein degraded

Answer 32

both UPS and autophagy although CMA is dominant pathway

Question 33

what features do neurones of patients with parkinsons disease often have

Answer 33

decreased levels of protein degradation machinery e.g. cathepsin D, LAMP2a and Hsc70 resulting in enhanced transmission of aggregates

Question 34

how are do the A53T and A30P mutants of a synuclein appear to block the CMA pathway

Answer 34

they contain a KFERQ-like domain which binds Hsc70 for delivery to the lysosome but are not transported into the lumen and impair degradation of other substrates. increased levels of WT protein can have similar effects

Question 35

why is it thought macroautophagy is also linked to parkinson

Answer 35

in parkinson many cells accumulate autophagosomes demonstrating a failure in clearance

Question 36

why is mTOR a therapeutic target in parkinsons

Answer 36

activation supresses autophagy. Rapamycin inhibits mTor to promote macropautophay, promoting clearance of a synuclein and reduces neuronal death in animal models

Question 37

why is TFEB a therapeutic target in parkinsons

Answer 37

it is a transcription factor that regulates macroautophagy and lysosome biogenesis. up regulation promotes clearance of a synuclein and prevents neuronal death