Transcript Ubiquitin-proteosome protein degradation ppt

Intracellular Protein
Degradation
Chris Weihl MD/PhD
[email protected]
Department of Neurology
How is trash handled?
Protein Degradation in the Cell
Ub
Autophagy
Nucleus
Aggresome
Ub
UPS
Ub
Ub
Endocytosis
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 Degradation
Turnover of protein is NOT constant
Half lives of proteins vary from minutes to infinity
“Normal” proteins – 100-200 hrs
Short-lived proteins
regulatory proteins
enzymes that catalyze committed steps
transcription factors
Long-lived proteins
Special cases (structural proteins, crystallins)
Protein Degradation
• May depend on tissue distribution
Example: Lactic Acid Dehydrogenase
Tissue
Half-life
Heart
1.6 days
Muscle
31 days
Liver
16 days
• Protein degradation is a regulated process
Example: Acetyl CoA carboxylase
Nutritional state Half-life
Fed
48 hours
Fasted
18 hours
Protein Degradation
 Ubiquitin/Proteasome Pathway
80-90%
Most intracellular proteins
• Lysosomal processes
10-20%
Extracellular proteins
Cell organelles
Some intracellular proteins
How are proteins selected for
degradation?
UBIQUITIN
 Small peptide that is a “TAG”
 76 amino acids
 C-terminal glycine - isopeptide
bond with the e-amino group of
lysine residues on the substrate
 Attached as monoubiquitin or
polyubiquitin chains
G
K
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
e-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 UPS is enormous!
The genes of the UPS constitutes ~5% of the
The genes of the UPS constitutes ~5% of the genome
genome
 E1’s- 1-2 activating enzymes
• E1’s1-210-20
activating
enzymesenzymes
E2’sconjugating
• E2’s10-20
conjugating
enzymes
E3’s500-800
ubiquitin
ligase- drives specificity
• E3’s500-800
specificity
DUBs100ubiquitin
ubiquitin ligasespecificdrives
proteasesregulators of pathway
• DUBs- 100 ubiquitin specific proteases- regulators of pathway
PROTEASOME COMPONENTS
20S
Proteasome
19S
Particle
ATP
26S
Proteasome
Hydrolysis peptide bonds after:
hydrophobic a.a. =
CHYMOTRYPSINLIKE - 5
acidic a.a. = (-)
CASPASE-LIKE -1
basic a.a. = (+)
TRYPSIN-LIKE -2
DEUBIQUITINATION
De-ubiquitinating
Pathways controlled by regulated proteolysis
Mechanism of muscle atrophy
MURF/Atrogin
Knockout of Atrogin Rescues
atrophy
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Proteasome inhibition increases Usp14
ubiquitin-hydrolase activity
Usp14
Uch37
Borodovsky, A et al
EMBO J. 20:5187-96
2001
The proteasomal DUB Usp14
impairs protein degradation
Lee, BH et al
Nature 467:179-84
2010
Decrease steady-state levels of aggregate
prone proteins in the absence of Usp14
Lee, BH et al
Nature 467:179-84
2010
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
Lysosome
FOXO3
Beclin
ATG7
mTOR
ATG5-ATG12-ATG16L1
Induction
Nucleation
Phagophore
Autophagosome
Sequestration
Trafficking
& Cargo loading
“Autophagic Flux”
Autolysosome
Fusion
Degradation
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
IBMPFD myopathy
LC3II protein levels (A.U)
p62 protein levels (A.U)
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WT
RH9
RH12
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
Ub
Nucleus
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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
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Inactive mTOR (or rapamycin treatment)
 Inhibition of protein synthesis and increased autophagic
degradation of protein
Ub
Ub
Increase autophagic stimulus
Nucleus
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Depending upon the disease, stimulating or
inhibiting autophagy may be appropriate.
Identifying drugs that “facilitate” autophagy.