Ubiquitin proteasome System Jacare Cardoza and Julia Bryarly

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

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

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

Step One: Tagging Substrate with Ubiquitin UBIQUITIN Primary Function: mark proteins for degradation

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. http://www.search.com/reference/Ubiquitin 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).. http://www.enzolifesciences.com/welcome/ubiquitin-and-ubiquitin-like-proteins-ubls/

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

Mechanism of Step One

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.100-102 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

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

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.

Step 2: Tagged protein degraded by 26S proteasome complex http://courses.chem.indiana.edu/c485/documents/proteasomearticleII.pdf 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.

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

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

Ubiquitin Proteasome System and its role in Parkinson’s Disease

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

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

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

Implicated Defects In PD

UPS: Dysfunctional in Parkinson’s Disease

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

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

Mutations in Alpha-Synuclein (PARK1)

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

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

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

Mutations in Parkin (PARK2)

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 )

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

Decreased Proteasomal Efficacy in Parkinson’s Disease

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

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

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

References 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

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&f eature=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 Lysia Forno/Science Photo Library http://www.sciencephoto.com/media/260700/view