intracellular protein degradation chris weihl md/phd department of neurology
TRANSCRIPT
Intracellular Protein Degradation
Chris Weihl MD/PhDDepartment of Neurology
How is trash handled?
Protein Degradation in the Cell
UPS
Aggresome
Autophagy
Endocytosis
Nucleus
Ub
Ub
Ub
Ub
Consequence of impaired protein degradation• Protein aggregates• Ubiquitinated inclusions• Vacuolation• Damaged organelles• Secondary impairment in other cellular processes• Cell Death
• Underlying pathogenesis of degenerative disorders (neurodegeneration, muscle degeneration, liver degeneration, lung disease, aging)
Protein DegradationTurnover of protein is NOT constantTurnover of protein is NOT constant
Half lives of proteins vary from minutes to infinityHalf lives of proteins vary from minutes to infinity
““Normal” proteins – 100-200 hrsNormal” proteins – 100-200 hrs
Short-lived proteinsShort-lived proteinsregulatory proteinsregulatory proteins
enzymes that catalyze committed stepsenzymes that catalyze committed stepstranscription factorstranscription factors
Long-lived proteinsLong-lived proteinsSpecial cases (structural proteins, crystallins)Special cases (structural proteins, crystallins)
Protein Degradation
Example: Lactic Acid DehydrogenaseTissue Half-lifeHeart 1.6 daysMuscle 31 daysLiver 16 days
• May depend on tissue distribution
• Protein degradation is a regulated processExample: Acetyl CoA carboxylase
Nutritional state Half-lifeFed 48 hoursFasted 18 hours
Protein Degradation Ubiquitin/Proteasome Pathway
80-90%Most intracellular proteins
• Lysosomal processes10-20%
Extracellular proteinsCell organellesSome intracellular proteins
How are proteins selected for degradation?
UBIQUITIN
KK
GG
Small peptide that is a “TAG” 76 amino acids C-terminal glycine - isopeptide
bond with the -amino group of lysine residues on the substrate
Attached as monoubiquitin or polyubiquitin chains
Ubiquitination of proteins is a FOUR-step process
First, Ubiquitin is activated by forming a link to “enzyme 1” (E1).
Then, ubiquitin is transferred to one of several types of “enzyme 2” (E2).
Then, “enzyme 3” (E3) catalizes the transfer of ubiquitin from E2 to a Lys -amino group of the “condemned” protein.
Lastly, molecules of Ubiquitin are commonly conjugated to the protein to be degraded by E3s & E4s
AMP
The UPS is enormous!
The genes of the UPS constitutes ~5% of the genome
• E1’s- 1-2 activating enzymes
• E2’s- 10-20 conjugating enzymes
• E3’s- 500-800 ubiquitin ligase- drives specificity
• DUBs- 100 ubiquitin specific proteases- regulators of pathway
The UPS is enormous!The UPS is enormous!
The genes of the UPS constitutes ~5% of the genome
E1’s- 1-2 activating enzymes E2’s- 10-20 conjugating enzymes E3’s- 500-800 ubiquitin ligase- drives specificity DUBs- 100 ubiquitin specific proteases- regulators of pathway
The genes of the UPS constitutes ~5% of the genome
E1’s- 1-2 activating enzymes E2’s- 10-20 conjugating enzymes E3’s- 500-800 ubiquitin ligase- drives specificity DUBs- 100 ubiquitin specific proteases- regulators of pathway
PROTEASOME COMPONENTS
20S Proteasome
19S Particle
26S Proteasome
ATP
Hydrolysis peptide bonds after:Hydrolysis peptide bonds after:
hydrophobic a.a.hydrophobic a.a. = = CHYMOTRYPSIN-CHYMOTRYPSIN-LIKE - LIKE - 55
acidic a.a.acidic a.a. = (-) = (-)CASPASE-LIKE CASPASE-LIKE --11
basic a.a.basic a.a. = (+) = (+)TRYPSIN-LIKE TRYPSIN-LIKE --22
DEUBIQUITINATIONDEUBIQUITINATION
De-ubiquitinatingDe-ubiquitinating
Pathways controlled by regulated proteolysisPathways controlled by regulated proteolysis
Mechanism of muscle atrophy
MURF/Atrogin
Knockout of Atrogin Rescues atrophy
prot
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prot
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ub-ub-ub-ub
ub-ub-ub-ub
ub-ub-ub-ubub-ub-ub-ubub-ub-ub-ub0
100000
200000
300000
400000
500000
600000
700000
800000
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Rel
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Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity
Usp14
Uch37
Borodovsky, A et alEMBO J. 20:5187-962001
The proteasomal DUB Usp14 impairs protein degradation
Lee, BH et alNature 467:179-842010
Decrease steady-state levels of aggregate prone proteins in the absence of Usp14
Lee, BH et alNature 467:179-842010
Lyosomal degradation
• Autophagy
Autophagy
• Lysosomal degradation of proteins and organelles• Occurs via three routes
• Macroautophagy• Microautophagy (direct uptake of cellular debris via the
lysosome)• Chaperone mediated autophagy (selective import of
substrates via Hsc70 and Lamp2a)
Yeast Genetics meets Human Genetics
• Identification of >50 autophagy essential proteins with mammalian homologs
Macroautophagy
Autophagosome
InductionmTOR
BeclinATG7
SequestrationPhagophoreATG5-ATG12-ATG16L1
Nucleation
Lysosome
Autolysosome
Degradation
FOXO3
Trafficking Fusion
“Autophagic Flux”
& Cargo loading
Genetic knockout of autophagy initiating proteins
Complete loss of ATG5 leads to lethality
Tissue specific knockout of autophagy
• Degeneration of CNS tissue; Hara et al 2006
• Hepatomegaly in Liver; Komatsu et al 2005
• Atrophy and weakness of skeletal muscle; Masiero et al 2009
• Pathologic similarities • Ubiquitinated inclusions• Aberrant mitochondria• Oxidatively damaged protein
Basal Autophagy
• Autophagy has a “housekeeping” role in the maintenance of cellular homeostasis
• Autophagy is responsible for the clearance of ubiquitinated proteins
Selective Autophagy
• Aggregaphagy– p62/SQSTM1, Nbr1• Mitophagy – Parkin, Nix• Reticulophagy – endoplasmic reticulum• Ribophagy – translating ribosomes• Xenophagy – e.g. Salmonella via optineurin• Lipophagy – autophagy mediated lipolysis
• Performed by an expanding group of ubiquitin adaptors
p62 as an autophagic tool• p62 associates with ubiquitinated proteins and LC3• p62 is an autophagic substrate
LC3 as an autophagic tool
LC3-I (18kD)LC3-II (16kD)
GFP-LC3
starved
0
1
2
LC
3II
pro
tein
levels
(A
.U)
Con WT RH9 RH12
p62
pro
tein
levels
(A
.U)
0
1
2
Con WT RH9 RH12
Ju et al, JCB 2009
Ju et al, JCB 2009
Upregulation of functional autophagosomes
Decrease in autophagosome degradation or “autophagic flux” Phagophore closure Autophagosome-lysosome fusion Absence of functional lysosomes
VCP
Ju et al, JCB 2009
Nucleus
Ub
Immunosuppressant used to treat transplant rejection
Inhibits the mTOR pathway mTOR integrates extrinsic growth signals
and cellular nutrient status and energy state
Active mTOR Protein synthesis and cell growth
Inactive mTOR (or rapamycin treatment) Inhibition of protein synthesis and increased
autophagic degradation of protein
Nucleus
Ub
Increase autophagic stimulus
Ub
Depending upon the disease, stimulating or inhibiting autophagy may be appropriate.
Identifying drugs that “facilitate” autophagy.