QUESTION DYSFUNCTIONAL PROTEIN DEGRADATION NEURODEGENERATION ?

Amyotrophic Lateral Sclerosis NEURODEGENERATION associated with Alzheimer’s Disease Parkinson’s Disease Huntington’s Disease Amyotrophic Lateral Sclerosis

Neurodegenerative Disorders accumulation of misfolded proteins Disease Parkinson’s Disease Alzheimer’s Disease accumulation of misfolded proteins Huntington’s Disease Amyotrophic lateral sclerosis Spinocerebellar Ataxia Cell Death

Ubiquitin-Protein Aggregates HUNTINGTON’S ALZHEIMER’S f PD: ubiquitin c AD: tau d AD: ubiquitin PARKINSON’S LOU GEHRIG’S

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 factots Long-lived proteins Special cases (dentin, crystallins)

Proteins are not degraded at the same rate Protein Degradation Proteins are not degraded at the same rate ENZYME half-life Ornithine decarboxylase 11 minutes -Aminolevulinate synthetase 70 minutes Catalase 1.4 days Tyrosine aminotransferase 1.5 hours Tryptophan oxygenase 2 hours Glucokinase 1.2 days Lactic dehydrogenase 16 days HMG CoA reductase 3 hour

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

Two Sites for Protein Degradation Proteasomes Large (26S) multiprotein complex (28 subunits) Degrades ubiquitinated proteins Lysosomes Basal degradation – non-selective Degradation under starvation – selective for “KFERQ” proteins

The Ubiquitin/Proteasome PATHWAY

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 Three genes in humans: Two are stress genes (B and C) One, UbA as a fusion protein

Tetra-Ubiquitin Cook, W.J. et al. (1994) J. Mol. Biol. 236, 601-609

UBIQUITIN GENES

Ubiquitin/Proteasome Pathway Degradation by the 26S PROTEASOME Ubiquitination Ubiquitination

The Ubiquitin/Proteasome Pathway Four Main Steps: UBIQUITINATION RECOGNITION DEGRADATION DEUBIQUITINATION

UBIQUITINATED PROTEINS

UBIQUITIN CHAINS 6 11 27 29 33 MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG 48 63

Functions of Ubiquitination Mono-ubiquitination Receptor internalization Endocytosis – lysosome Transcription regulation Poly-ubiquitination Targets proteins from Cytoplasm, Nuclear & ER for degradation by the PROTEASOME DNA repair

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

UBIQUITIN ACTIVATION E1 ADENYLATE THIOL ESTER

UBIQUITIN CONJUGATION -----> E2-s-co-Ub + E1 E1-s-co-Ub + E2-SH -----> -----> E2-s-co-Ub + E1 UBC domain N C CLASS 1 – UBC domains only; require E3s for Ub; target substrates for degradation CLASS 2 – UBC domains & C-terminal extensions; UBC2 = RAD6 – DNA repair not degradation; no E3s CLASS 3 – UBC domains & N-terminal extensions; function not known

“recognins” = recognize a motif (DEGRON) on a protein substrate E2-s-co-Ub + Protein-NH2 -------> E2-SH + Protein-NH-CO-Ub (ubiquitin = polyubiquitin chains) UBIQUITIN LIGATION E3 “recognins” = recognize a motif (DEGRON) on a protein substrate

Three Major Classes of E3 1) HECT-domain E3s 3) multi-subunit cullin based E3s 2) RING finger-domain E3s

Ubiquitin Ligases (E3) 1) HECT-domain containing a conserved Cys 2) RING finger-domain Cys & His residues are ligands to two Zn++ ions stabilizes a molecular scaffold

Ubiquitin Ligases (E3) (cont.) 3) Complex E3s: Multiple subunits Ex: SCF-type E3, VBC-Cul2 E3 and other cullin based E3s, Anaphase promoting complex (APC) -they provide a Scaffold for Ub transfer -F-box – substrate recognition

ELONGATION = E4 U box = CHIP (+parkin) Non-U box = p300 (p53) E3-E4 complex = C. elegans

ACTIVATION OF A UBIQUITIN-LIGASE

RECOGNITION DEGRADATION SIGNALS substrates

N-end RULE

N-end RULE N-degron - signal N-recognin - E3

DEGRADATION

PROTEASOME COMPONENTS 19-3 20S Proteasome 19S Particle ATP 26S Proteasome

The 26S proteasome

Ubiquitinated proteins are degraded by the proteasome Ubiquitinated proteins are degraded in the cytoplasm and nucleus by the proteasome. Proteasomal protein degradation consumes ATP. The proteasome degrades the proteins to ~8 amino-acid peptides. Access of proteins into the proteasome is tightly regulated. The peptides resulting from the proteasome activity diffuse out of the proteasome freely.

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

Ubiquitin – like proteins “UBP” Small Ubiquitin-like Modifier

Ubiquitin – like modifiers

LYSOSOMES

Digestive System of the Cell Digests ingested materials obsolete cell components Degrades macromolecules of all types Proteins Nucleic acids Carbohydrates Lipids Heterogeneous

Lysosomal Enzymes 50 different degradative enzymes Acid hydrolases Active at pH 5 (inside lysosome) Inactive if released into cytosol (pH 7.2) Acidic pH of lysosomes maintained by a proton pump in the lysosomal membrane Requires ATP, thus mitochondria

Different pathways lead to the lysosome 1) Phagocytosis Cell “eating” of material > 250nm 2) Pinocytosis Cell “drinking” < 150nm 3) Receptor Mediated Endocytosis -clathrin-coated pits 4) Autophagy “self eat” of old worn out organelles, important in cell degradation during apoptosis

Protein degradation in the lysosomes Lysosomes degrade extracellular proteins that the cell incorporates by endocytosis. Lysosomes can also degrade intracellular proteins that are enclosed in other membrane-limited organellas. In well-nourished cells, lysosomal protein degradation is non-selective (non-regulated). In starved cells, lysosomes degrade preferentially proteins containing a KFERQ “signal” peptide. The regression of the uterus after childbirth is mediated largely by lysosomal protein degradation

AUTOPHAGY - Macroautophagy – inducible (mTOR) (autophagy) - Microautophagy - constitutive - Chaperone-mediated autophagy (CMA) – KFERQ motif

AUTOPHAGY

AUTOPHAGY (MACRO) PATHWAY Oxidative stress Infection Protein aggregates

AUTOPHAGY (MACRO) PATHWAY Lysosome

AUTOPHAGY PATHWAY 17 genes = Atg 1) INDUCTION TOR target of Rapamycin Stress (negative regulation) Tight association

2) AUTOPHAGOSOME FORMATION AUTOPHAGY PATHWAY 2) AUTOPHAGOSOME FORMATION LIPID KINASES (PHOSPHATIDYL INOSITOL) SIGNALING COMPLEX NEW MEMBRANE = ER

AUTOPHAGY PATHWAY 3) DOCKING & FUSION MEMBRANE ASSOCIATION DIMERIZATION MEMBRANE ASSOCIATION phosphatidylethanolamine PROTEIN CONJUGATION SYSTEM ~ TO THE UBIQUITIN SYSTEM

remain associated with the AUTOPHAGY PATHWAY 4) BREAKDOWN RECYCLING & RETRIEVAL Only Atg19 & Atg8-PE remain associated with the Autophagosome; Others are re-cycled

AUTOPHAGY PATHWAY - PD, AD, HD and TSE - aggregate removal Neurodegenerative diseases - PD, AD, HD and TSE - aggregate removal Infectious diseases - remove pathogens Cancer - sequester damaged organelles - promote autophagic death

Degradation of Proteins 80-90% 10-20% AUTOPHAGY/LYSOSOME PATHWAY 26S PROTEASOME

Ubiquitin-Protein Aggregates Why?  26S Proteasome Environmental & Genetic Insults Inflammation Aging Ub AGGREGATES

? SUBSTRATE AUTOPHAGY INDUCTION CROSS-TALK Lysosome 26S Proteasome Ub MODEL SUBSTRATE Ub Ub Ub Ub AUTOPHAGY INDUCTION CROSS-TALK ? Lysosome 26S Proteasome

The End

Human neuroblastoma SK-N-SH cells Rat Spinal Cord Organotypic Cultures AUTOPHAGY & UPP Human neuroblastoma SK-N-SH cells & Rat Spinal Cord Organotypic Cultures Model for ALS

ENVIRONMENTAL & GENETIC INSULTS CELL RECOVERY  AUTOPHAGY UPP  CELL DEATH ENVIRONMENTAL & GENETIC INSULTS  Protein Aggregates Other ? ?  Protein Degradation p62/SQSTM1 HDAC6

Amyotrophic Lateral Sclerosis Neurodegeneration = ubiquitin inclusions HUNTINGTON’S Disease ALZHEIMER’S Disease PARKINSON’S Disease Amyotrophic Lateral Sclerosis f PD: ubiquitin c AD: tau d AD: ubiquitin