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QUESTION DYSFUNCTIONAL PROTEIN DEGRADATION NEURODEGENERATION ?

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Presentation on theme: "QUESTION DYSFUNCTIONAL PROTEIN DEGRADATION NEURODEGENERATION ?"— Presentation transcript:

1 QUESTION DYSFUNCTIONAL PROTEIN DEGRADATION NEURODEGENERATION ?

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

3 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

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

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

6 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 days Tyrosine aminotransferase 1.5 hours Tryptophan oxygenase 2 hours Glucokinase days Lactic dehydrogenase 16 days HMG CoA reductase 3 hour

7 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

8 Protein Degradation Ubiquitin/Proteasome Pathway 80-90%
Most intracellular proteins Lysosomal processes 10-20% Extracellular proteins Cell organelles Some intracellular proteins

9 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

10 The Ubiquitin/Proteasome
PATHWAY

11 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

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

13 UBIQUITIN GENES

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

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

16 UBIQUITINATED PROTEINS

17 UBIQUITIN CHAINS 6 11 27 29 33 MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG 48 63

18 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

19 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

20

21 UBIQUITIN ACTIVATION E1
ADENYLATE THIOL ESTER

22 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

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

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

25 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

26 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

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

28

29 ACTIVATION OF A UBIQUITIN-LIGASE

30 RECOGNITION DEGRADATION SIGNALS substrates

31 N-end RULE

32

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

34

35 DEGRADATION

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

37 The 26S proteasome

38 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.

39 Hydrolysis peptide bonds after:
hydrophobic a.a. = CHYMOTRYPSIN-LIKE - 5 acidic a.a. = (-) CASPASE-LIKE -1 basic a.a. = (+) TRYPSIN-LIKE -2

40 DEUBIQUITINATION De-ubiquitinating

41

42

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

44 Ubiquitin – like modifiers

45 LYSOSOMES

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

47 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

48 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

49 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

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

51 AUTOPHAGY

52 AUTOPHAGY (MACRO) PATHWAY
Oxidative stress Infection Protein aggregates

53 AUTOPHAGY (MACRO) PATHWAY
Lysosome

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

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

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

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

58 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

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

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

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

62 The End

63 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

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

65

66 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

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