lf206 lecture 10 - protein modification (cytosolic events)

most protein synthesis starts in free ______ ribosomes

cytosolic

protein synthesis in the cytosol

1. cytosolic ribosomal ________ are used to assemble _______ on mRNA encoding ______ proteins

2. these proteins remain in the _______.

3. multiple ribosomes assemble and produce __________ (polysomes)

4. newly made protein are released and the ribosomes are _______ and recycled

1. subunits, ribosomes, cytosolic

2. cytosol

3. polyribosomes

4. dismantled

the cytosol is ______ crowded

so estimates may differ by _____ of magnitude

a single mRNA translated by multiple ribosomes (__________) the nascent proteins are prone to __________

very

orders

polysomes/polyribosomes

aggregation

newly synthesised proteins are often non-________

unfolded proteins are prone to ________

folded proteins are stable, functional and resistant to _______

_______ are proteins that ensure correct polypeptide folding

functional

aggregation

proteases

chaperones

how do chaperoned correctly fold proteins?

1. ________ patches on nascent/unfolded proteins are recognised and bound by ____ _____ _____ __ (Hsp 40 co-chaperone)

2. Hsp 40 delivers the substrate to an ____ conformation Hsc 70 chaperone to stimulate its ______ activity

   1. when ___ bound it has an open conformation

3. this hydrolysing activity results in ___-bound (closed conformation) Hsp70 which then _____ the hydrophobic _____

4. this prevents ______ and allows for time to ___ correctly

5. upon _______ exchange of ADP to ATP, Hsc70 adopts an open conformation, releases the partly folded substrate which will then __ into its final conformation.

1. hydrophobic, heat shock protein 40

2. open, ATPase

   1. ATP

3. ADP, shields, patches

4. aggregation (clumpi/cluster), fold

5. nucleotide, snap

why is studying in vivo chaperone interactions hard?

the cytoplasm is very crowded

studying chaperones in vitro

1. ________ of unfolded proteins leads to insolubility

2. heat target protein to separate aggregate (P, pellet) and ______ (S) fractions by ________

3. heating in presence of Hsp40 increases _ fraction

4. heating in presence of Hsc70 has a ______ effect

5. maximal solubilisation requires Hsp40, Hsc70 and ___

1. aggregation

2. soluble, centrifugation

3. S

4. greater

5. ATP

Hsc70 is an anti-aggregation device.

it doesn’t determine a protein’s ________

the protein can have a productive (folding, activation) or __ -______ (destruction, inactivation) fate

productive → released & finds stable conformation, passed onto other ______ for further folding

non-productive → transported to a ______ or passed on to proteasomes for _______

conformation

non-productive

chaperone

lysosome

degradation

how is Hsc70 released

requires _______ ____ _____ (NEF) to bind to Hsp70:____ complex and removes ADP _______-binding site

this promote nucleotide _____ allowing entry of ATP

nucleotide exchange factors

client

nucleotide

exchange

Hsp90 can provide a platform for the assembly of _________ complexes by bringing in other partially folded substrates.

chaperonins (for example _____) are composed of two rings of 7 or 8 or 9 subunits per ring depending on ______

they can interrupt/join after Hsc70: client complex to release a _____ folded protein

the chaperonins provide a cage that _______ small folding proteins like _______ and actin.

multimeric

Hsc60

species

fully

isolates

tubulin

__-chaperones determine the fate of protein/chaperone client complex by _______ with others.

provide an example.

co-chaperones

competing

HOP transfers clients from Hsc70 to Hsp90

BAG-1 releases Hsc70 clients to proteasome = destruction

BAG-2 releases clients away from proteasome = folding

HIP competes with NEFs to maintain Hsc70 client interaction

what does Hsc70 stand for?

heat shock cognate protein 70

atomic ____ microscopy shows Hsc70 in the presence of Hsp40 and NEF ____ can disaggregate

1. DNAJB1 (a ______) binds as dimers to aSyn fibres

2. this recruits Hsc70

3. ____ triggers more Hsc70 binding

4. the binding stretches the target causing _______ -

force

Apg2

1. Hsp40

2. Apg2

3. disaggregation

PROTEASOME STRUCTURE

has a central core of 2 ring of 7 ______ subunits

flanked by a ring of 7 ____ subunits above and below it

the central core is flanked by a 19S _________ ___ (RP) cap ontop to be described as a __S proteasome

or a 30S proteasome with an RP ______

the active sites of the proteasome are at/encoded by ____ subunits.

beta

alpha

regulatory particle

26

below it

beta

the 20S core particle of a __________ has 3 proteolytic activities.

what are they?

proteasome

1. chymotrypsin-like

2. trypsin-like

3. caspase-like (peptidylglutamyl-peptide hydrolysing).

proteins are targeted to the protein by _________ (Ub).

this is a conserved protein of __ amino acids found in all __karyotic cells

proteins marked with a chain of _____ Ub proteins means it is doomed for ____________

monoubiquitinated proteins are targeted to the _______ instead.

ubiquitin

76

eukaryotic

4

degradation

lysosomes

a protein that has failed Hsc70-mediated _______could be targeted to the proteasome.

1. Ub is activated by __ ubiquitin-activating enzyme

   1. there are roughly _ of these enzymes in mammalian cells

2. activated Ub is transferred to __

   1. there a roughly __ of these enzymes in mammalian cells

3. the E2-Ub conjugate associates with E3 ubiquitin _____

   1. there are roughly ___s of E3 ligases in mammalian cells

4. the __-__-__ conjugate binds to the target ________ and transfers Ub to the target

folding

1. E1

   1. 9

2. E2

   1. 30

3. ligase

   1. 100s

4. E2-E3-Ub, protein

features of a protein that E3 targets. [4]

• extended residence in a chaperone system

• their N-terminus

• misfolded regions

• exposure of a degradation signal

the chain of 4 Ub is detected by ________ _____ ___ of the proteasome

regulatory particle cap

PROTEASOME DESTRUCTION

1. polyubiquitylated proteins bind to the 19S _______ _____ of the proteasome

2. RP uses ___ for energy to unfold the protein

3. _________ (DUBs) remove Ub molecules and they are recycled

4. protein drawn through proteasome core and degraded - the _____ proteolytic activities are encoded by ____ subunits of 20S core.

5. protein degraded into small peptides. ~7-9 ____ acid residues long

1. regulatory particle

2. ATP

3. deubiquitylases

4. three, beta

5. amino

the proteasome is not just a destructive machine

in the RP subunit, there is a chaperone called _____

it directs some proteins to be __-_______

RPT5

re-folded

when proteasomes/E3 fails

• proteins ________

• can lead to formation of aggregates - driving _________ diseases

• if cell cycle proteins aren’t degraded properly it can lead to cell _________ (cancer)

overactive proteasomes

• have been implicated in ________ disease. e.g systemic lupus erythematosus and rheumatoid arthritis

accumulate

Alzheimer’s

proliferation

autoimmune

proteolytic cleavage to ______ a protein

inactive precursor procaspase 3 is cleaved by CASP8, 9 & __

they all cleave in the _____ place

________ into an active procaspase 3

activate

10

same

rearranged