1. Ubiquitin proteasome System
Jacare Cardoza 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
UBIQUITIN Primary 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
Ubiquitin is a highly conserved small regulatory protein that is ubiquitous in eukaryotes. Ubiquitination (or Ubiquitylation) refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. The most prominent function of Ubiquitin is labeling proteins for proteasomal degradation (see: Proteasome). Besides this function, ubiquitination also controls the stability, function, and intracellular localization of a wide variety of proteins.
The central element of this process is the small protein ubiquitin that can be attached to proteins as a "mark" so that they can be recognized, transported to where in the cell they are needed or eliminated. The molecules that destroy these marked ("ubiquitinated") proteins are called proteases that can chop them into small pieces (peptides and amino acids)..

7. Step One: Tagging Substrate with Ubiquitin- A 3 Part Mechanism
ATP AMP+ 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
Proteins are tagged with ubiquitin
Activating Enzyme, E1 primes ubiquitin for action
Ub is transferred to Conjugating Enzyme, E2
E2 acts as an escort to Ligase Enzyme, E3
E3 acts as a platform, a place where target protein substrate and active E2/ub complex can meet and interact
E3 is very specific for which E2 and protein can interact
E2/Ub attaches to E3, brought close enough for Ub to be transferred to the target protein substrate
Result: polyubiquitin chain= death sentence
The chain provides a clear signal to the cell’s disposal unit- the proteosome
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/ removal Base: substrate unfolding
20S core
Contains 4 heptameric rings
Function: processive proteolysis
he 19S regulatory subunit acts as a gate agent to limit entry to the proteasome to targeted proteins. The 19S subunit is also essential for proteolytic activity because the 20S subunit alone is inactive.37,100 The 19S regulatory particle is composed of two substructures, a lid and base, and is involved in substrate selection, preparation, and protein translocation into the catalytic 20S chamber for degradation Each 19S particle is composed of numerous subunits, including six ATPases, that most likely provide the energy necessary for substrate unfolding that is required before entry into the 20S chamber.103 The outer-lid subcomplex of the 19S component is involved in the recognition and ubiquitin chain processing before substrate translocation and degradation.104,10

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

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
One catalytic b subunit has a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 (peptide cabonyl) position.
One has a trypsin-like activity with preference for arginine or lysine at the P1 position.
One has a post-glutamyl activity with preference for glutamate or other acidic residue at the P1 position.

12. Step 2: Tagged protein degraded by 26S proteasome complex
One catalytic b subunit has a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 (peptide cabonyl) position.
One has a trypsin-like activity with preference for arginine or lysine at the P1 position.
One has a post-glutamyl activity with preference for glutamate or other acidic residue at the P1 position.
Different variants of the three catalytic subunits, with different substrate specificity, are produced in cells of the immune system that cleave proteins for antigen display.
The proteasome hydrolases constitute a unique family of threonine proteases. A conserved N-terminal threonine is involved in catalysis at each active site. The three catalytic b subunits are synthesized as pre-proteins. They are activated when the N-terminus is cleaved off, making threonine the N-terminal residue. Catalytic threonines are exposed at the lumenal surface.

13. Summary: Substrate Degradation by the Proteasome
a. Recognition of ub chain by Rpt5/S6’
b. ATPases in the base unfold substrate
c. Translocation of substrate into the pore of 20S proteasome
d. Substrate hydrolysis
e. Peptides exit the catalytic chamber
f. Rpn11 of the lid hydrolyses isopeptide bond that links ub to substrate

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
The UPS prototypically recognizes specific protein substrates and places polyubiquitin chains on them for subsequent destruction by the proteasome. This system is in place to degrade not only misfolded and damaged proteins, but is essential also in regulating a host of cell signaling pathways involved in proliferation, adaptation to stress, regulation of cell size, and cell death.
The majority of intracellular proteins are proteolyzed by the proteasomes. In most cultured mammalian cells, these account for 80-90% of protein breakdown

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)
Lost DA neurons from the SNc
Neurodegenerative, motor disorder
Involves preferential degeneration of dopamine neurons of the substantia nigra pars compacta (SNc) of the midbrain

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. Decreased hydrolytic activity of the monomeric form of UCH-L1
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
Describes different aspects related to protein misfolding and neurodegenerative diseases
(1)Triggers for the accumulation of misfolded proteins—both mutations and epigenetic factors
(2) The primary responses to accumulating misfolded proteins—these are related to the reduced capacity of the UPS that is the consequence of proteinoverload/inhibition by aggregated substrates. The misfolded proteins that accumulate may be refolded by chaperones or accumulate in
aggregates in the cytoplasm, nucleus, or extracellular space. The aggregates may sequester additional proteins
(3) LBs are a types of neuropathological intracellular protein deposits

34. References
Burn, David J., et al. Chapter 62: Dementia with Lewy Bodies. “Neurobiology of Mental Illness”. 2011;
Ciechanover, Aaron. The ubiquitin proteasome system in neurodegenerative diseases: Sometimes the chicken, sometimes the egg. Neuron (40)
Fornai, Francesco, et al. Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitin proteasome system and α-synuclein. PNAS (102)
McNaught, Kevin St. P., et al. Altered proteasomal function in sporadic Parkinson’s disease. Experimental Neurology 2003; (179)
McNaught, Kevin St. P., et al. Failure of the ubiquitin-proteasome system in Parkinson’s disease. Nature Reviews: Neuroscience 2001; (2)
Taylor, J. Paul, et al. Toxic Proteins in Neurodegenerative disease. Science 2002; (296)
Mani, A., Gelmann, Edward P., The Ubiquitin-Proteasome Pathway and Its Role in Cancer. J. of Clinical Oncology 2005; (23, 21)
Marques, Antonio J., et al. Cstslytic Mechanism and the Assembly of the Proteasome. Chem. Review 2009; (109)
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”.

35. Images references
Parkin: pages/perspectives/perspectives-detail/the-shaking- palsy-and-the-hunt-for-a-cure/
Proteasome:
UPS system on YouTube:
eature=youtu.be
Alpha Synuclein Aggregation Pathway and Monomeric Form:
Implicated Defects in PD:
Credit to Jane Hand of Rutgers
TEM image of a LB:
Credit to Lysia Forno/Science Photo Library