Presentation is loading. Please wait.

Presentation is loading. Please wait.

QUESTION DYSFUNCTIONAL PROTEIN DEGRADATION NEURODEGENERATION ?

Similar presentations


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

1 QUESTION DYSFUNCTIONAL PROTEIN DEGRADATION NEURODEGENERATION ?

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

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

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

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 Protein Degradation ENZYMEhalf-life Ornithine decarboxylase11 minutes  -Aminolevulinate synthetase70 minutes Catalase1.4 days Tyrosine aminotransferase1.5 hours Tryptophan oxygenase2 hours Glucokinase1.2 days Lactic dehydrogenase16 days HMG CoA reductase3 hour Proteins are not degraded at the same rate

7 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

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

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

16 UBIQUITINATED PROTEINS

17 UBIQUITIN CHAINS MQIFV K TLTG K TITLEVESSDTIDNV K A K IQD K EGIPPDQ QRLIFAG K QLEDGRTLADYNIQ K ESTLHLVLRLRGG

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

19 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

20

21 UBIQUITIN ACTIVATION E1 UBIQUITINADENYLATE THIOLESTER

22 E1-s-co-Ub + E2-SH -----> -----> E2-s-co-Ub + E1 UBIQUITIN CONJUGATION 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 UBC domain NC

23 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 3) multi-subunit cullin based E3s 1) HECT-domain 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 1)U box = CHIP (+parkin) 2)Non-U box = p300 (p53) 3)E3-E4 complex = C. elegans

28

29 ACTIVATION OF A UBIQUITIN-LIGASE

30 DEGRADATION SIGNALS substrates RECOGNITION

31 N-end RULE

32

33 N-degron - signal N-recognin - E3

34

35 DEGRADATION

36 PROTEASOME COMPONENTS S Proteasome 19S Particle 26S Proteasome ATP

37 The 26S proteasome

38 Ubiquitinated proteins are degraded 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.  Ubiquitinated proteins are degraded in the cytoplasm and nucleus by the proteasome.

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

40 DEUBIQUITINATIONDe-ubiquitinating

41

42

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

44 Ubiquitin – like modifiers

45 LYSOSOMES

46 Digestive System of the Cell DigestsDigests –ingested materials –obsolete cell components DegradesDegrades macromolecules of all types –Proteins –Nucleic acids –Carbohydrates –Lipids HeterogeneousHeterogeneous

47 Lysosomal Enzymes 50 different degradative enzymes Acid hydrolasesAcid hydrolases –Active at pH 5 (inside lysosome) –Inactive if released into cytosol (pH 7.2) Acidic pHAcidic 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). KFERQ  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 AUTOPHAGY - Macroautophagy – inducible (mTOR) (autophagy) - Microautophagy - constitutive - Chaperone-mediated autophagy (CMA) – KFERQ motif (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 Stress TOR target of Rapamycin Tight association (negative regulation) 1) INDUCTION

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

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

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

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

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

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

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

62 The End

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

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

65

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


Download ppt "QUESTION DYSFUNCTIONAL PROTEIN DEGRADATION NEURODEGENERATION ?"

Similar presentations


Ads by Google