Ubiquitin proteasome System

1. Ubiquitinproteasome System JacareCardoza and Julia Bryarly

2. Maintaining Homeostasis in the Cell • Protein turnover: a balance between synthesis and degradation • Synthesis via translation • Degradation via lysosomes and proteosomes • Proteosomes account for 80-90% of protein breakdown

3. Degradation • Degradation is complex and temporally controlled • The highly regulated process plays a role in: • Cell life and Death • Cell cycle • Signal transduction • Gene expression • Development • Maintenance of proper protein folding

4. Degradation of a Protein via UPS Involves 2 Discrete Steps • 1. Protein substrate is tagged with ubiquitin • Protein + substrate= destruction marker • 2. Tagged protein degraded by 26S proteasome complex Step One Step Two

5. Step One: Tagging Substrate with Ubiquitin UBIQUITINPrimary Function: mark proteins for degradation

6. Step One: Tagging Substrate with Ubiquitin • Conjugation of ubiquitin to a protein substrate proceeds via a 3-step cascade mechanism. • Enzymes involved: • E1: Binds and activates ubiquitin, then transfers Ub to E2 • E2: Binds to E3 • E3: Binds to specific protein substrate and works in concert with E2 to transfer ubiquitin to the substrate

7. Step One: Tagging Substrate with Ubiquitin- A 3 Part Mechanism E2 E1 ATPAMP+ PPi Degradation Signal Second: Ubiquitin Conjugating Enzyme, E2 receives activated ubiquitin and escorts it to E3, the platform for the protein substrate Third:Ubiquitin is transferred to the substrate via the E2-E3 complex First: Ubiquitin-activating enzyme, E1, activates Ubiquitin Substrate E3

8. Mechanism of Step One

9. Step 2: Tagged protein degraded by 26S proteasome complex • 19S Caps • Contains a lid and base • Function: Lids: polyUb recognition/binding/ removalBase: substrate unfolding • 20S core • Contains 4 heptameric rings • Function: processive proteolysis

10. 19S Caps: Unfolding of Target Protein and Deubiquitination ATP-dependent AAA proteins unfold protein substrates Deubiquitination enzymes have hydrolase activity

11. 20S Core: Degradation of Target Proteins β subunits (1, 2, 5) have proteolytic activities β1: post-glutamyl peptide hydrolase-like β2: trypsin-like β5: chymotrypsin-like α7: Control the passage of substrates into and degradation products out of the proteasome

12. Step 2: Tagged protein degraded by 26S proteasome complex

13. Summary: Substrate Degradation by the Proteasome

14. Overview: Function of UPS • Maintains the right proteins, in the right amount, at the right time • Seeks and destroys damaged or faulty proteins, or those that exist in excess • Failure of the system can result in disease • Become over zealous= destruction of essential proteins • restrained= build-up of harmful proteins

15. Ubiquitin Proteasome System and its role in Parkinson’s Disease

16. Ubiquitin Proteasome System and its role in Parkinson’s Disease • Pathology of UPS involves two broad classifications: • Loss of function- mutations in a ubiquitin system enzyme or target substrate/protein  results in stabilization of proteins • Gain of function- abnormal or accelerated degradation of the protein target *In PD- aggregations of disease specific proteins inhibits activity of the UPS

17. Parkinson’s Disease (PD) • Neurodegenerative, motor disorder • Involves preferential degeneration of dopamine neurons of the substantia nigra pars compacta (SNc) of the midbrain Lost DA neurons from the SNc

18. Lewy Bodies In PD • Lewy bodies are a histological finding in post-mortem brains with Sporadic PD- proteinaceous, intracytoplasmic inclusions TEM of a Lewy Body • It is unclear if LBs are toxic to the cell or neuroprotective • Recent evidence suggests LBs are an attempt to sequester toxic aggregates when not properly degraded by the proteasome Lewy Body in the Soma

19. Implicated Defects In PD

20. UPS: Dysfunctional in Parkinson’s Disease

21. Mutation in the Coding Gene for Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1)

22. Mutation in the Coding Gene for Ubiquitin carboxy-terminal hydrolase (UCH-L1) Decreased hydrolytic activity of the monomeric form of UCH-L1 Increased ligase activity as a dimer Shortage of free ubiquitin Accumulation of toxic proteins Impairment of the UPS

23. Mutations in Alpha-Synuclein (PARK1)

24. Mutations in Alpha-Synuclein • Small protein  believed to regulate vesicle storage and DA neurotransmission • WT α-syn is monomeric • High concentrations oligomerizes to β-sheets, protofibrils

25. Protofibrils further aggregate, precipitate as insoluble amyloid fibrils (as in LBs) • Mutant forms (in N-terminal domain) form βsheets more easily  Gain of Function mutation • α-syn overexpression can induce apoptosis and increased sensitivity to toxic agents (ie proteasome inhibitors) • Failed attempts by the proteasome to degrade fibrils may lead to decreased functionality of the proteasome

26. Alpha-Synuclein: Toxic or Neuroprotective? • Lewy Bodies that contain insoluble amyloid fibrils may be neuroprotective • May allow cell to sequester toxic, undegradable proteins away from UPS machinery, and other sensitive cellular machinery (ie transcriptional) • Soluble protofibrils maybe toxic to UPS • Ataxins in Spinocerebellar Ataxis have been found to adhere to the proteasomal cap • Normal vs. Dysfunctional UPS

27. Mutations in Parkin (PARK2)

28. Normal Parkin (PARK2) • 465 AA residue, ~52kDa • Parkin is a ubiquitin-protein ligase (E3) • Acts with ubiquitin conjugating enzymes • 2 RING finger motifs at the C-terminus • RING finger domain most likely involved in recruiting E2 • UBL domain in the N-terminal region (UBiquitin-Like) • UBL proteins modify enzymes and substrates of the UPS • Affects activity (ie increases affinity of enzymes, or affect stability of substrates/availability of substrates to UPS machinery) • UBL can serve as proteasome binding motiffacilitates transfer of polyubiquitination on the substrate to proteosome • May enhance the efficiency of the proteolytic process through better binding of components of the system (KM)

29. Mutated Parkin • Deletion/point mutations found in ~50% of Autosomal Recessive-Juvenile Parkinson’s (AR-JP= early onset, no LBs) • Mutation in Parkin can prevent binding to proteasome (KM) • Mutations in Parkin reduce the activity of the enzyme • Reduce interaction with other enzymes • Ie CHIP, co-chaperone • Possibly a dominant mutant allele that can bind substrate but cannot ubiquitinate it  stabilization of the protein  • Alpha synuclein is believed to be a substrate of Parkin Defects in Parkin results in accumulation of substrates through disruptions in the UPS

30. Decreased Proteasomal Efficacy in Parkinson’s Disease

31. In Sporadic-PD: • αsubunits, but not βsubunits of 26/20S proteasome are lost • Losses are within dopaminergic neurons • In the SNc, proteasome enzymatic activities are inhibited

32. Inhibition of the Mitochondrial Complex i • Substantia nigra DA neurons produce ROS • Exposure to toxins (ie pesticides) can possibly then overload the cell (unable to cope with additional oxidative stress) • Leads to inhibition of the mitochondrial respiratory chain especially in pigmented neurons • Failure to produce adequate amounts of ATP • Significantly decreased activity of the UPS (ATP-dependent reactions) • Accumulation of α-synuclein

33. Summary of the Pathogenesis of Neurodegeneration in Parkinson’s

34. Burn, David J., et al. Chapter 62: Dementia with Lewy Bodies. “Neurobiology of Mental Illness”. 2011; 1010-1030. Ciechanover, Aaron. The ubiquitin proteasome system in neurodegenerative diseases: Sometimes the chicken, sometimes the egg. Neuron 2003. (40) 427-4446. Fornai, Francesco, et al. Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitin proteasome system and α-synuclein. PNAS 2005. (102) 3413-3418. McNaught, Kevin St. P., et al. Altered proteasomal function in sporadic Parkinson’s disease. Experimental Neurology 2003; (179) 28-46. McNaught, Kevin St. P., et al. Failure of the ubiquitin-proteasome system in Parkinson’s disease. Nature Reviews: Neuroscience 2001; (2) 589-594. Taylor, J. Paul, et al. Toxic Proteins in Neurodegenerative disease. Science 2002; (296) 1991-1995. Mani, A., Gelmann, Edward P., The Ubiquitin-Proteasome Pathway and Its Role in Cancer. J. of Clinical Oncology 2005; (23, 21). 4776-4789. Marques, Antonio J., et al. Cstslytic Mechanism and the Assembly of the Proteasome. Chem. Review 2009; (109) 1509-1536. Meyer, RJ., et al. Protein Degradation: The Ubiquitin Proteasome System. (2). Google Bookshttp://books.google.com/books?id=xyB_hIpnE2YC&pg=PA17&lpg=PA17&dq=Rpt5+recognition+of+ubiquitin&source=bl&ots=fW2SKPMr40&sig=q6H_hHWERU4Jc1sbBkWBXHmnhJk&hl=en&ei=-rLJTracPOS-2AWKuoDYDw&sa=X&oi=book_result&ct=result&resnum=6&ved=0CEQQ6AEwBQ#v=onepage&q=Rpt5%20recognition%20of%20ubiquitin&f=false Berg, JM., et al. Biochemistry, 5th Edition. New York; W H Freeman; 2002. “Ubiquitin”. http://www.search.com/reference/Ubiquitin. http://mol-biol4masters.masters.grkraj.org/html/Co_and_Post_Translational_Events6-Protein_Degradation.htm References

35. Images references Parkin: http://www.biomedicine.lu/news-display-pages/perspectives/perspectives-detail/the-shaking-palsy-and-the-hunt-for-a-cure/ Proteasome: http://udel.edu/~mayurad/page6.html UPS system on YouTube: http://www.youtube.com/watch?v=4DMqnfrzpKg&feature=youtu.be Alpha Synuclein Aggregation Pathway and Monomeric Form: http://en.wikipedia.org/wiki/Alpha-synuclein Implicated Defects in PD: Credit to Jane Hand of Rutgers http://maptest.rutgers.edu/drupal/?q=node/399 TEM image of a LB: Credit to LysiaForno/Science Photo Library http://www.sciencephoto.com/media/260700/view