Intracellular Protein Degradation Chris Weihl MD/PhD weihlc@neuro.wustl.edu Department of Neurology

How is trash handled?

Protein Degradation in the Cell Ub Nucleus Autophagy Ub Aggresome Ub UPS 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 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

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?

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

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

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 ATP 26S Proteasome

Hydrolysis peptide bonds after: hydrophobic a.a. = CHYMOTRYPSIN-LIKE - 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

Proteasome inhibitors ub-ub-ub-ub ub-ub-ub-ub ub-ub-ub-ub ub-ub-ub-ub proteasome ub-ub-ub-ub

Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity 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 “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”

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

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

Autophagosome accumulate in IBMPFD cells Ju et al, JCB 2009

Why do autophagosomes accumulate? Upregulation of functional autophagosomes Decrease in autophagosome degradation or “autophagic flux” Phagophore closure Autophagosome-lysosome fusion Absence of functional lysosomes

Functional VCP is required for “autophagic flux”

IBMPFD mutant VCP impairs “autophagic flux” Ju et al, JCB 2009

Autophagosomes and lysosomes coalesce in IBMPFD

IBMPFD has “blocked” autophagy Ub Nucleus

Therapeutic interventions to treat autophagic disorders

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

Rapamycin enhances autophagy in skeletal muscle

Rapamycin treatment worsens IBMPFD

IBMPFD has “blocked” autophagy Ub Nucleus Ub Increase autophagic stimulus

Future autophagic therapies Depending upon the disease, stimulating or inhibiting autophagy may be appropriate. Identifying drugs that “facilitate” autophagy.