Intracellular Protein Degradation Chris Weihl MD/PhD Department of Neurology
How is trash handled?
Protein Degradation in the Cell UPS Aggresome Autophagy Endocytosis Nucleus 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 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)
Protein Degradation Example: Lactic Acid Dehydrogenase TissueHalf-life Heart1.6 days Muscle31 days Liver16 days May depend on tissue distribution Protein degradation is a regulated process Example: Acetyl CoA carboxylase Nutritional stateHalf-life Fed48 hours Fasted18 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?
UBIQUITINK G 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 conjugating enzymes E3’s ubiquitin ligase- drives specificity DUBs- 100 ubiquitin specific proteases- regulators of pathway 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
PROTEASOME COMPONENTS 20S Proteasome 19S Particle 26S Proteasome ATP
Hydrolysis peptide bonds after: hydrophobic a.a. = CHYMOTRYPSIN- LIKE - 5 acidic a.a. = (-) CASPASE-LIKE - 1 basic a.a. = (+) TRYPSIN-LIKE - 2
DEUBIQUITINATIONDe-ubiquitinating
Pathways controlled by regulated proteolysis
Mechanism of muscle atrophy
MURF/Atrogin
Knockout of Atrogin Rescues atrophy
proteasome ub-ub-ub-ub
Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity Usp14 Uch37 Borodovsky, A et al EMBO J. 20:
The proteasomal DUB Usp14 impairs protein degradation Lee, BH et al Nature 467:
Decrease steady-state levels of aggregate prone proteins in the absence of Usp14 Lee, BH et al Nature 467:
Lyosomal degradation 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 Induction mTOR Beclin ATG7 Sequestration Phagophore ATG5-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
IBMPFD myopathy
0 1 2 LC3II protein levels (A.U) Con WT RH9 RH12 p62 protein levels (A.U) Con WT RH9 RH12 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.