regulation of protein degradation

Question 1

pathways of protein degradation (3)

Answer 1

lysosomes
extracellular proteases
ubiquitin-proteasome pathway

Question 2

reasons why cells degrade their own proteins (4)

Answer 2

1: physiological removal of rate-limiting enzymes and regulatory proteins
2: quality control of misfolded proteins
3: usage of proteins as energy source (primarily in skeletal muscle during catabolic states)
4: antigen processing by immune system (extracellular antigens processed by endosome/lysosome pathway and intercellular by ubiquitin-proteasome pathway)

Question 3

ways proteases are classified (3)

Answer 3

by:
active site nucleophile
- serine
- cysteine (lysosmal proteins)
- threonine (proteasome proteases)
- water (metalloproteases and acidic proteases)
substrate specificity for:
- positively charged AA (eg trypsin)
- negatively charged AA
- hydrophobic AA (eg chymotrypsin)
cleavage location
- aminopeptidase - can only start at N terminus
- endopeptidases - can cleave protein anywhere
- carboxypeptidase - can only start at C terminus

Question 4

ways cells regulate protein degradation (4)

Answer 4

1: natural protease inhibitors (eg serpins)
2: inactive enzyme precursors (zymogens) - enzyme must be activated like prohormones, etc.
3: compartmentalization (lysosomes)
4: specific death signals (ubiquitin)

note: proteolytic degradation of peptide bonds is energetically favorable - need ATP to regulate the protein degradation process

Question 5

lysosomal pathway of protein degradation

Answer 5

1: primary lysosomes formed from Golgi apparatus
2: these fuse with endosomes (have membrane proteins or cells that bind to membrane proteins outside cell like LDL) or autophagosomes (bulk canabalism - takes up large amount of cytoplasm and proteins by engulfing it with membrane - stimulated in states of starvation) to make secondary lysosomes
3: lysosomal proteases break down contents

Question 6

regulation of lysosomal pathway

Answer 6

proteases (pepsins in lysosomes) regulated by:
1: compartmentalization - put membrane around pepsins
2: requirement for acidic conditions (provided by ATP-dependent lysosomal proton pump - pepsins only active at low pH)
3: specific cathepsin inhibitors (called cystatins) - inhibit any lysosomal proteins that leak out
pathway also regulated by hormonal signals during catabolic states

Question 7

cathepsin K deficiency - pycnodysostosis

Answer 7

cathepsin K involved in regulation of lysosomal degradation of proteins
deficiency results in genetic disorder of bone development

Question 8

extracellular protease pathway of protein degradation

- required in which processes?

Answer 8

synthesized by cells and secreted into bloodstream

1: tissue remodeling (matrix metalloproteases and elastase)
2: complement cascade (immune response)
3: clotting (thrombin cascade)
4: fibrinolysis (aka plasmin cascade; breaking down of the clot)
5: digestion (pancreatic proteases)

Question 9

serpins

Answer 9

natural serine protease inhibitors
regulate extracellular proteases
genetic diseases associated with serpin deficiency:
	1: alpha1-antitrypsin deficiency
	2: C1INH deficiency
	3: antithrombin deficiency

Question 10

ubiquitination steps

Answer 10

for selective protein degradation for proteins in cytoplasm, nucleus and ER

1: ubiquitin-activating enzyme (E1) activates carboxyl group of ubiquitin - E1’s active site is cysteine - results in reactive thioester - uses ATP, generates AMP + 2 Pi
2: activated ubiquitin is transferred to carrier proteins (E2)
3: E2 binds to E3 to make ubiquitin ligase complex (E1 there too)
4: degradation signal on target protein binds to E3 (E3 has binding pockets specific for certain proteins, or for an aspect of misfolded proteins such as a hydrophobic domain)
5: complex facilitates transfer of ubiquitin to e-amino groups on lysine residues in substrate proteins => isopeptide bond - energy neutral way so don’t need more ATP
6: the E1 that had the ubiquitin leaves and is replaced by another E1 that has an ubiquitin, which can then be attached to the target protein, allowing for polyubiquitination
7: proteasome then degrades ubiquinated peptide into small peptides and AA

note: cells have different combinations of E2 and E3 and different combinations recognize different protein substrates

Question 11

proteasome steps

Answer 11

multienzyme complex

1: entry lid has receptor proteins that bind to ubiquitin-tagged proteins - only binds polyubiquitinated ones
2: deubiquitinating enzymes (isopeptidases) cleave isopeptide bonds and ATP-dependent enzymes (both types of chaperone proteins) unfold the target proteins
3: proteases within cylindrical core have differing substrate specificities - hydrolyze proteins in central channel of proteasome

note: all proteases threonine proteases (no other threonine proteases known)

Question 12

diseases involving ubiquitin-proteasome pathway

Answer 12

cervical cancer
parkinson’s
multiple myeloma (plasma cell tumor) treated with proteasome inhibitors

Question 13

dynamic state of cellular proteins

Answer 13

always turning over
example: crystalin, life of 80 years, as age turnover slows, why need lens replaced
hemoglobin, life of 120 days
sperm cells - exzyme ornithinie decarboxylase enzyme has life of 11 minutes

Question 14

anabolic state

Answer 14

body builders on steroids

Question 15

catabolic state

Answer 15

diabetes, starvation

Question 16

physiological removal of rate-limiting enzymes and regulatory proteins

Answer 16

cyclin degraded to allow movement out of G2 into mitosis

inflammation regulated this way too

Question 17

protein quality control

Answer 17

when proteins not folded properly
if have hg point mutation, unstable so is degraded
can have normal DNA sequence too - post translational modifications occur incorrectly or damage to protein after its been folded - by reactive O2 species (esp. due to smoking)
can have transcription errors (30% of transcripts have errors)
or folded improperly (like if chaperones aren’t available)
clinical correlation: cystic fibrosis - chloride channel is mutated so that cell mistakes it for being folded wrong or a mistake and it gets degraded
another example: alcoholic liver damage - proteins in liver cells denatured due to long term exposure to alcohol

Question 18

use of proteins as energy source

Answer 18

if malnourished, use for energy to get AA for gluconeogenesis

Question 19

protein degradation as antigen processing by the immune system

Answer 19

necessary for both MHC1 and MCH2

Question 20

alpha1-antitrypsin deficiency

Answer 20

clinical correlation for extracellular protease protein degradation
smoker with emphysema and liver failure
many neutrophils in lungs and protein deposits in liver
genetic, recessive
1/1000 people of european descent
alpha1-antitrypsin inhibits elastase secreted by neutrophils at site of inflammation - allows itself to be target for elastase and cleaved by it - intermediate clings to elastase so inhibits it (suicide substrate)
if deficiency, less effective substrate for elastase - smoking makes it worse because more inflammation
get misfolded alpha1-antitrypsin protein aggregating in liver
curable with liver transplant

Question 21

anaphylactic shock

Answer 21

recurrent tongue swelling and shortness of breath
can be spontaneous - not due to allergy
genetic lack of C1 inhibitor - get massive compliment activation, angioedema

Question 22

antithrombin deficiency

Answer 22

clinical correlation for protease deficiency
pregnant woman develops blood clot in leg and lung after miscarriage
thrombosis in leg that breaks off and lodges in lung
predispose to it by antithrombin deficiency - thrombin converts fibrinogen to fibrin to create clot

Question 23

biochemistry of ubiquitin attachment

Answer 23

ubiquitin attaches to internal lysine group in protein

new form of chemistry - isopeptide bond

Question 24

cervical cancer

Answer 24

HPV that causes cervical cancer has E6 viral proteins - degrades p53

Question 25

velcade

Answer 25

new treatment for cancer

proteasome inhibitor that can kill multiple myeloma cells - more sensitive to drug than other cells