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Intracellular Protein Degradation
Chris Weihl MD/PhD Department of Neurology
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How is trash handled?
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Protein Degradation in the Cell
Ub Nucleus Autophagy Ub Aggresome Ub UPS Ub Endocytosis
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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)
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Protein Degradation Turnover of protein is NOT constant
Half lives of proteins vary from minutes to infinity “Normal” proteins – hrs Short-lived proteins regulatory proteins enzymes that catalyze committed steps transcription factors Long-lived proteins Special cases (structural proteins, crystallins)
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Protein Degradation May depend on tissue distribution
Example: Lactic Acid Dehydrogenase Tissue Half-life Heart days Muscle 31 days Liver 16 days May depend on tissue distribution Protein degradation is a regulated process Example: Acetyl CoA carboxylase Nutritional state Half-life Fed 48 hours Fasted 18 hours
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Protein Degradation Ubiquitin/Proteasome Pathway 80-90%
Most intracellular proteins Lysosomal processes 10-20% Extracellular proteins Cell organelles Some intracellular proteins
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How are proteins selected for degradation?
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G K 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
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Ubiquitination of proteins is a FOUR-step process
First, Ubiquitin is activated by forming a link to “enzyme 1” (E1). AMP 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
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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 conjugating enzymes E3’s 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 conjugating enzymes E3’s ubiquitin ligase- drives specificity DUBs- 100 ubiquitin specific proteases- regulators of pathway
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PROTEASOME COMPONENTS
20S Proteasome 19S Particle ATP 26S Proteasome
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Hydrolysis peptide bonds after:
hydrophobic a.a. = CHYMOTRYPSIN-LIKE - 5 acidic a.a. = (-) CASPASE-LIKE -1 basic a.a. = (+) TRYPSIN-LIKE -2
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DEUBIQUITINATION De-ubiquitinating
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Pathways controlled by regulated proteolysis
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Mechanism of muscle atrophy
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MURF/Atrogin
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Knockout of Atrogin Rescues atrophy
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Proteasome inhibitors
ub-ub-ub-ub ub-ub-ub-ub ub-ub-ub-ub ub-ub-ub-ub proteasome ub-ub-ub-ub
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Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity
Uch37 Borodovsky, A et al EMBO J. 20: 2001
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The proteasomal DUB Usp14 impairs protein degradation
Lee, BH et al Nature 467:179-84 2010
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Decrease steady-state levels of aggregate prone proteins in the absence of Usp14
Lee, BH et al Nature 467:179-84 2010
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Lyosomal degradation Autophagy
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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)
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Yeast Genetics meets Human Genetics
Identification of >50 autophagy essential proteins with mammalian homologs
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Macroautophagy “Autophagic Flux” Fusion Sequestration Degradation
Lysosome FOXO3 Fusion Sequestration Phagophore Autolysosome Degradation Beclin ATG7 mTOR ATG5-ATG12-ATG16L1 Autophagosome Induction Nucleation Trafficking & Cargo loading “Autophagic Flux”
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Genetic knockout of autophagy initiating proteins
Complete loss of ATG5 leads to lethality
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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
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Basal Autophagy Autophagy has a “housekeeping” role in the maintenance of cellular homeostasis Autophagy is responsible for the clearance of ubiquitinated proteins
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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
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p62 as an autophagic tool p62 associates with ubiquitinated proteins and LC3 p62 is an autophagic substrate
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LC3 as an autophagic tool
LC3-I (18kD) LC3-II (16kD) GFP-LC3 starved
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IBMPFD myopathy
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Autophagosome proteins are elevated in IBMPFD
p62 protein levels (A.U) 1 2 Con WT RH9 RH12 1 2 LC3II protein levels (A.U) Con WT RH9 RH12 Ju et al, JCB 2009
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Autophagosome accumulate in IBMPFD cells
Ju et al, JCB 2009
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Why do autophagosomes accumulate?
Upregulation of functional autophagosomes Decrease in autophagosome degradation or “autophagic flux” Phagophore closure Autophagosome-lysosome fusion Absence of functional lysosomes
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Functional VCP is required for “autophagic flux”
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IBMPFD mutant VCP impairs “autophagic flux”
Ju et al, JCB 2009
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Autophagosomes and lysosomes coalesce in IBMPFD
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IBMPFD has “blocked” autophagy
Ub Nucleus
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Therapeutic interventions to treat autophagic disorders
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Rapamycin as an inducer of autophagy
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
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Rapamycin enhances autophagy in skeletal muscle
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Rapamycin treatment worsens IBMPFD
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IBMPFD has “blocked” autophagy
Ub Nucleus Ub Increase autophagic stimulus
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Future autophagic therapies
Depending upon the disease, stimulating or inhibiting autophagy may be appropriate. Identifying drugs that “facilitate” autophagy.
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