W/S 6 - Ubiquitin Proteasome Pathway (UPP)

Question 1

proteasomes

Answer 1

protein complexes that degrade damaged/toxic proteins by proteolysis

Question 2

proteolysis

Answer 2

chemical reaction that breaks peptide bonds

Question 3

key components of proteasome pathway - ubiquitin

Answer 3

small protein

tags other proteins for degradation

Question 4

key components of proteasome pathway - proteasome

Answer 4

multi-subunit protease complex

degrades ubiquitin-tagged proteins

main proteasome = 26S (20S core + 19S regulatory particle)

Question 5

E1

Answer 5

activates ubiquitin in ATP dependent manner

Question 6

E2

Answer 6

transfers activated ubiquitin to target proteins

Question 7

E3

Answer 7

facilitates transfer of ubiquitin from E2 to substrate, providing specificity

Question 8

steps in UPP

Answer 8

E1 activates ubiquitin using ATP

activate ubiquitin transferred to E2 enzyme

E3 ligase binds both substrate protein + ubiquitin-loaded E2

ubiquitin attached to lysine residue on substrate protein, forming isopeptide bond

additional ubiquitin molecules linked to form polyubiquitin chain = signal for degradation by proteasome
-> recognised by 19S, which removes ubiquitin chain (recycled) and unfolds substrate
-> unfolded substrate translocated to 20S core; then degraded into small peptides

ubiquitin molecules cleaved from substrates + recycled by deubiquinating enzyme (DUBs)

Question 9

UPP in Alzheimer’s

Answer 9

[low activity]

accumulation of misfolded proteins
-> Aβ plaques
-> hyperphosphorylated tau tangles

Question 10

UPP in cancer

Answer 10

[high activity]

degradation of oncogenes, tumour-suppressors + regulatory proteins

mutations in E3 ligases = increase degradation of p53 - allows cancer cells to evade programmed cell death

Question 11

why does high proteasome turnover occur?

Answer 11

compensatory response to increased protein misfolding

Question 12

Aβ

Answer 12

= cleavage product of Amyloid Precursor Proteins (APP) when processed by B-secretase 1 (BACE 1) followed by γ-secretase

Question 13

effect of proteasome impairment on APP

Answer 13

altered cleavage

increases Aβ plaques and tau phosphorylation

Question 14

Key findings from (Chocron et al, 2022)

Answer 14

1. elevated insoluble Aβ and tau phosphorylation in hippocampus of Alzheimer’s patients
2. overexpression of 20S proteasome subunit core in drosophila
   -> delayed cognitive effects
   -> reduced cell death
   -> extended lifespan
3. mice inserted with additional copy of PSMB5 = increased 20S + 26S and crossed with AD mouse models
   -> transgenic mice = lower mortality and cognitive decline vs controls
4. development of TATI-8,9TOD and TATI-DEN
   -> proteasome-activating peptidomimetics
   -> able to cross BBB
   -> reduced cell death and cognitive deficits in fly model of AD

Question 15

drug from (Frisira et al, 2019) paper

Answer 15

NPI-0052 = novel proteasome inhibitor (3rd generation)

binds to 20S subunit

penetrates BBB (suitable for brain tumour)

Question 16

(Frisira et al, 2019) - medulloblastoma phenotype

Answer 16

substantial upregulation of all 19S/20S proteasome subunits in MB

Question 17

(Frisira et al, 2019) - synergistic effect with γ-radiation

Answer 17

sig. synergist effect in post-treatment

increased oxidative stress (eliminates damaged proteins) and stabilisation of p53-family proteins

= apoptosis in MB cells

Question 18

(Manasanch and Orlowski, 2017) - Bortezomib

Answer 18

first generation proteasome inhibitor

prevents degradation of ubiquitin tagged misfolded proteins

side effect = thrombocytopenia

Question 19

first generation proteasome inhibitor

Answer 19

binds to 20S core of 26S proteasome

Question 20

(Manasanch and Orlowski, 2017) - Carfilzomib

Answer 20

irreversible proteasome inhibitor (2nd generation)

irreversibly binds to β5 subunit of 20S subunit (targets chymotrypsin activity) - prevents degradation

disrupts cell-cycle regulation and tumour growth signalling pathways

stabilises tumour suppressor proteins and inhibits pro-survival pathways (e.g. NF-kB)

side effects = thrombocytopenia and cardiotoxicity

Question 21

(Manasanch and Orlowski, 2017) - UPR

Answer 21

[unfolded protein response]

enhances protein-folding capacity of ER, degrades misfolded proteins and decreases production of new proteins

maintains protein homeostasis

Question 22

(Manasanch and Orlowski, 2017) - Advantages of Carfilzomib over Bortezomib

Answer 22

1. irreversible binding = sustained proteasome suppression
2. greater specificity for chymotrypsin-like activity = less off-target effects
3. lower incidence of neuropathy

Question 23

(Manasanch and Orlowski, 2017) - resistance to proteasome inhibitors

Answer 23

1. enhanced proteasome activity
   -> upregulation of proteasome subunits
   -> switches to other proteasome forms
2. drug efflux
   -> overexpression of drug transporters pumps inhibitors out cells
3. mutations of ubiquitin ligases or deubiquinating enzymes
4. upregulation of autophagy
5. activation of survival pathways
   -> NF-kB signalling
   -> increased expression of heat shock proteins (HSP)
   = promotion of cancer cell survival
6. alterations in apoptotic pathway
   -> downregulation of pro-apoptotic proteins
   -> upregulation of anti-apoptotic proteins
7. cell cycle and DNA repair
   -> bypass cell-cycle checkpoints

Question 24

(Manasanch and Orlowski, 2017) - overcoming resistance

Answer 24

combination therapies
-> autophagy inhibitors
-> NF-kB inhibitors