1. The Ubiquitin Proteosome Pathway The Ubiquitin Proteosome Pathway Swati Pradhan Mayura Dange Vidyadhar Daithankar

2. Overview  Background  Protein misfolding & degradation  Ubiquitin & proteosome structure  Ubiquitin Proteosome Pathway  Mechanism  Structures of enzymes involved in pathway  Pathogenic implication of defective pathway  Biological functions of pathway  Diseases & drug development

3. The Central Dogma

4. Translational Folding of a Protein

5. Chaperone Mediated Protein Folding & Misfolding

6. Post-Translational Modification Acetylation Acetylation Glycosylation Glycosylation Phosphorylation Phosphorylation Ubiquitination Ubiquitination http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmdbooks&doptcmdl/Figure+6-79

7. Degradation of Misfolded Proteins Lysosomal (extracellular) protein degradation Lysosomal (extracellular) protein degradation –Protein degraded by lysosomal enzymes Cytosolic (intracellular) protein degradation Cytosolic (intracellular) protein degradation –The Ubiquitin Proteosome pathway

8. Lysosomal degradation Proteins delivered via endocytosis Proteins delivered via endocytosis Lysosomes Lysosomes –The cellular dust- bins –Contain many hydrolytic enzymes Proteases Proteases Lipases Lipases Glycosidases Glycosidases

9. Cytosolic protein degradation The Ubiquitin Proteosome Pathway The Ubiquitin Proteosome Pathway www.ihf.de/forschung/ popup/ubiquitin.html

10. 2004 Nobel Prize in Chemistry The discovery of ubiquitin-mediated protein degradation The discovery of ubiquitin-mediated protein degradation –Aaron Ciechanover –Avram Hershko –Irwin Rose Cells give a chemical "kiss of death" to proteins that need to be destroyed. Cells give a chemical "kiss of death" to proteins that need to be destroyed.

11. Targeting by Ubiquitin Despite help from chaperones, more than 80% fold incorrectly Despite help from chaperones, more than 80% fold incorrectly Proteins are dislocated back into the cytosol Proteins are dislocated back into the cytosol –Oligosaccharides are removed –Deglycosylation is catalyzed by N-glycanase One third of the newly made polypeptide chains are selected for degradation One third of the newly made polypeptide chains are selected for degradation

12. The Export of Misfolded Proteins

13. Ubiquitin 76 amino acids, 8.5 kDa protein 76 amino acids, 8.5 kDa protein Heat stable Heat stable Folds into a compact globular structure Folds into a compact globular structure Found throughout the cell Found throughout the cell Found in all eukaryotic cells Found in all eukaryotic cells Human and yeast ubiquitin share 96% sequence identity Human and yeast ubiquitin share 96% sequence identity Involved in many cellular processes Involved in many cellular processes http://www.sanger.ac.uk/Users/sgj/thesis/html/node93.html

14. The Proteosome Professional protein degrading organelles Professional protein degrading organelles An abundant ATP- dependent protease An abundant ATP- dependent protease Constitutes nearly 1% of cellular protein Constitutes nearly 1% of cellular protein Present in many copies throughout the cytosol and the nucleus Present in many copies throughout the cytosol and the nucleus Consists of a central hollow cylinder (20S) Consists of a central hollow cylinder (20S) Ends of the cylinder are associated with the 19S cap Ends of the cylinder are associated with the 19S cap http://walz.med.harvard.edu/Proteasome_Complexes/

15. The Structure of 20S Proteasome http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.3206

16. Types of Ubiquitination Mono-ubiquitination Mono-ubiquitination –Transcription, histone function, endocytosis and membrane trafficking Lys48, Lys11 or Lys29 linked poly ubiquitination Lys48, Lys11 or Lys29 linked poly ubiquitination –Target proteins to the proteasome Lys63 linked poly ubiquitination Lys63 linked poly ubiquitination –Signaling, DNA repair, stress response, endocytosis and signal transduction

17. UBIQUITIN PATHWAY

18. Covalent Attachment of multiple ubiquitin molecules Covalent Attachment of multiple ubiquitin molecules Degradation of the tagged protein Degradation of the tagged protein 3 Enzymes : Ub – Activating enzyme E1 3 Enzymes : Ub – Activating enzyme E1 Ub – Conjugating enzyme E2 Ub – Conjugating enzyme E2 Ub – Ligases E3 Ub – Ligases E3 UBIQUITIN PATHWAY

19. Hierarchical structure Several E2 transfer Ub from E1 to E3 to which substrate protein is bound Several E2 transfer Ub from E1 to E3 to which substrate protein is bound E3s catalyze covalent attachment to the substrate and recognize the substrate E3s catalyze covalent attachment to the substrate and recognize the substrate

20. Ubiquitin Pathway

21. Ubiquitin Activating Enzyme E1 Adenylation Adenylation Thio-ester bond formation Thio-ester bond formation E2 association E2 association

22. Mechanism E1 activates C-terminus of Ub by forming acyl -adenylate intermediate E1 activates C-terminus of Ub by forming acyl -adenylate intermediate Catalytic Cys residue forms thioester bond with Ub Catalytic Cys residue forms thioester bond with Ub Another Ub is adenylated Another Ub is adenylated Transfer of Ub to E2 forming a thioester bond Transfer of Ub to E2 forming a thioester bond

23. Ubiquitin Conjugating enzyme E2 Carries activated Ub from E1 to the substrate Carries activated Ub from E1 to the substrate Cys residue positioned in a shallow groove Cys residue positioned in a shallow groove Relatively inflexible structure Relatively inflexible structure Conserved Asn may be required for H- bond network OR plays a catalytic role in isopeptide bond formation Conserved Asn may be required for H- bond network OR plays a catalytic role in isopeptide bond formation

24. Ub Ligases E3 Final target selection and specificity Final target selection and specificity Place activated Ub near Lys of substrate Place activated Ub near Lys of substrate Isopeptide formation of Gly of Ub with the є –NH 2 Lys or to the N-terminal residue of the substrate Isopeptide formation of Gly of Ub with the є –NH 2 Lys or to the N-terminal residue of the substrate

25. Categories of E3 Ligases HECT domain: Homologous to E6-AP C terminus HECT domain: Homologous to E6-AP C terminus RING domain: Really Interesting New Gene RING domain: Really Interesting New Gene

26. Conserved 350 amino acids Conserved 350 amino acids Catalytic contribution Catalytic contribution Forms thiol ester bond with Ub before transferring it to the substrate Forms thiol ester bond with Ub before transferring it to the substrate N lobe and C- lobe form ‘L’ or ‘inverted T’ shape N lobe and C- lobe form ‘L’ or ‘inverted T’ shape Flexibility of hinge loop is required for catalytic activity Flexibility of hinge loop is required for catalytic activity C lobe accepts Ub form E2 and transfers it to the substrate C lobe accepts Ub form E2 and transfers it to the substrate Sequential addition / Indexation Sequential addition / Indexation HECT Ub Ligases E3

27. L – shaped E2/E3 complex

28. 15 th most common domain in Human genome 15 th most common domain in Human genome Conserved Cys and His Zn 2+ co-ordinating residues Conserved Cys and His Zn 2+ co-ordinating residues Interact directly with E2s Interact directly with E2s Allosterically activate E2 enzymes Acts as molecular scaffold Brings Ub-E2 and substrate closer Increase # Lys in the vicinity of E2 RING Ub Ligases E3

29. Polyubiquitination Poly Ub chain synthesized by adding Ub moieties to Lys of the previous Ub Poly Ub chain synthesized by adding Ub moieties to Lys of the previous Ub Another enzyme E4 may be catalyzing this step Another enzyme E4 may be catalyzing this step

30. Deubiquitination Thiol proteases Thiol proteases Ubiquitin processing (UBP) enzymes Ubiquitin processing (UBP) enzymes  Removes Ub from polyubiquinated proteins Ubiquitin carboxy terminal hydrolases (UBH) Ubiquitin carboxy terminal hydrolases (UBH)  Regenerates monomeric Ub

31. Pathological implication of defective ubiquitin-proteosome pathway

32.  Ubiquitin proteasome pathway is ubiquitous & targets many processes and substrates.  Several complex processes are mediated via degradation or processing of specific proteins.  Aberrations in these systems associates with pathogenic conditions either directly or indirectly.

33. Biological function of Ubiquitin Proteosome pathway

34. Consequences of Defects in Ubiquitination

35. Pathological Conditions Associated with Ubiquitin Proteosome Pathway – –Malignancies – – Neurodegenerative disorders – – Genetic disease Cystic fibrosis, Angelman’s syndrome & Liddle’s syndrome – – Immune and inflammatory responses

36. Malignancies Oncoproteins like NMyc, c-Myc, c-Fos, are substrates of U-P pathway. Destabilization of tumor suppressor genes like p53 and p27. Extremely low levels of p53 in uterine cervical carcinoma. Prostate, Colorectal and breast cancer: – –Tumor suppressor protein p27 is CDK inhibitor of the cell cycle. – –Healthy individuals have high levels of p27. Mitogenic stimuli rapidly degrades the protein. – –Cancer patients has low levels of p27 in quiescent cells. – – Defects in ubiquitin system accelerates degradation of suppressor. – –Strong correlation of low levels of p27 and aggressiveness of cancer.

37. Cell Cycle Regulators and Cancer Defect in ubiquitin pathway ( Skp2) Degradation of P27 Skp2 Polyubiquitination

38. Neurodegenerative disorders  Alzheimer's disease  Parkinson's disease  Huntington’s disease  Spinocerebellar ataxias  Spinobulbar muscular dystrophy (Kennedy’s syndrome) Formation of inclusion bodies (Ref: http://w3.dbb.su.se/~oliveberg/images/bildstrat1.jpg)

39.  Accumulation of ubiquitin may be secondary reflecting unsuccessful attempts of ubiquitination.  Abnormal protein associate with each other forming aggregates.  Hypothesis: Aggregated proteins inhibit ubiquitin proteosome pathway. ( Ref: http://www.neurodegeneration.uni-goettingen.de/index.html?/en/p311.html) Parkinson’s disease and Lewy Bodies

40. Liddle’s Syndrome  Hereditary form of hypertension.  Caused due to deletion of proline rich (PY) region in the β and γ subunits of epithelial Na + channel (hENaC).  HECT domain of E3 binds to PY motif of hENaC.  Mutation in PY motif leads to stabilization of channel complex and E3 ligase cannot bind to PY motif.  Increased expression of hENaC channel causing excessive reabsorption of sodium and water. Stabilization of channel

41. Angleman syndrome  Ubiquitin system is considered to be involved in brain development.  Defective synthesis of gene coding for E3 ligase E6-AP  Characteristic symptoms involve mental retardation, seizures, out of context frequent smiling and laughter.  Brain proteins that could be stabilized by mutation have not been identified. Cystic fibrosis  Gene codes for a protein, CFTR, which is chloride ion channel.  Small fraction of protein matures to the cell surface.  Mutation in protein ΔF508, CFTR ΔF508 doesn't reach the cell surface.  Ubiquitination degrades mutant CFTR ΔF508, resulting in complete lack of cell surface expression.

42. Immune and inflammatory responses  Ubiqutin proteosome pathway is involved in processing of antigenic proteins.  Epitopes are presented on class I MHC molecule generating T cell immune response. Ubiquitin proteosome pathway Native protein Foreign protein CLASS I MHC molecule No immune response Immune response

43. Drug Development for Ubiquitin Dysfunction  Inhibition of enzymes common to entire pathway would target the process non- specifically.  Narrow window between benefits and toxicity needs to be identified.  Develop completely specific E3 ligase inhibitors that would affect the pathways of interests.  Better approach would be development of small molecules that would be specific for substrates.

44. Conclusions  Ubiquitylation plays a fundamental role of protein degradation at cellular level. (Levels of proteins in nucleus, cytoplasm, ER lumen and transmembrane protein are kept in check by ubiquitin proteosome pathway.)  Ubiquitylation is highly complex, temporally controlled and tightly regulated process.  Enzymologically Ubiquitination is more complex pathway compared to other post translational modification.  Mechanism of catalysis by E3 ligase still remains unclear.  Elucidation of complete catalytic mechanism of ubiquitylation will provide considerable insight on cellular functions.

45. Questions

46. extra

47. Extra www.mekentosj.com/ubiquitin/proteasome.html

48. Extra (The Central Dogma)