Transcript UCH-L1
Parkinson’s Disease Introduction Parkinson’s disease is a chronic, progressive neurological disorder estimated to affect approx 1% of the population above 65 years. After Alzheimer’s disease, Parkinson’s disease is the second most frequent neurodegenerative disease linked to age. As compared to Lou Gehrig’s disease, Multiple Sclerosis, and Musular Dystrophy, Parkinson’s disease affects more individuals combined. In the next 25 years, the global burden of care for the disease is expected to increase markedly. A study was conducted in 2005 that estimated there were over 1 million individuals with PD in Western Europe and the USA, but by 2030, the value was projected to more than double (Dorsey et al 2007). An Overview of Parkinson’s Disease The symptoms of Parkinson’s disease have been known since the medieval times, but was not formally recognized until James Parkinson formally documented it in 1817. Characterized clinically by tremor, bardykinesia, rigidity, and postural instability. Pathological hallmarks are the loss of dopaminergic neurons in the substantia nigra of the brain and the increased accumulation of intracytoplasmic inclusion bodies (Lewy bodies). Parkinson’s disease is mostly of idiopathic origin, but 510% of patients are known to have to have monogenic forms of the disease: autosomal dominant or autosomal recessive. LOCUS CHR. LOCATION GENE INHERITANCE PATTERN PARK1/PARK4 4q21-q23 alpha-synuclein AD PARK2 6q25.2-q27 parkin AR PARK3 2p13 unknown AD PARK5 4p14 UCH-L1 AD PARK6 1p35-p36 PINK1 AR PARK7 1p36 DJ-1 AR PARK8 12p11.2-q13.1 LRRK2 AD PARK10 1p32 unknown unclear PARK11 2q36-2q37 GIGYF2 unclear unknown 5q23.1-q23.3 Synphilin-1 AD unknown 2q22-q23 NR4A2 AD Parkinson’s Disease Pathway Alpha-Synuclein Parkin UCHL-1 Ubiquitin Proteasome Pathway DA Neuron Dysfunction DA Degeneration/Cell Death Parkinsonism The ubiquitin-proteasome system Key: E1 DUB = deubiquitinating enzyme E2 Ub E1 Ub E2 Ub Ub = ubiquitin monomer +ATP SUB = substrate abnormal proteins DUB Parkin Ub Ubl Ring1 SUB Ub peptides 26S proteasome IBR UCH-L1 Ring2 E2 Ub Poly-ubiquitination Non-proteasomal functions Parkin A large region spanning chromosome 6q25.2-q27 was linked to a autosomal recessive juvenile parkinsonism (ARJP) in consanguineous Japanese families, and the gene was designated parkin (Matsumine et al. 1997). ARJP is an early-onset (<40 years old) form of the disease that is caused by hereditary factors. Parkin mutations cause dopaminergic neural cell death through the accumulation of proteins without the formation of Lewy bodies. Parkin function may contribute to the formation of Lewy bodies or that Lewy body formation may not be required for the development of PD. Parkin function/structure Parkin was identified a ubiquitin-protein ligase (E3) that targets specific protein substrates for proteasomal degradation. The parkin gene encodes a protein that contains an N-terminal ubiquitin-like (UBL) domain, a central linker region, and a C-terminal RING domain comprising two RING finger motifs separated by an inbetween-RING (IBR) domain. C terminal N terminal Central linker region Facilities transfer of polyubiquitinated substrates to 26S proteasome RING finger domain involved in interactions with E2 Isolated IBR domain from Parkin C TermN Term C Term H373 C365 Zn binding site II C368 C365 Site II Site II C377 C368 C332 H373 C377C337 90 deg C360C332 C337 Zn binding C352 site II C360 Zn C352 binding site I •Substitutions of coordinating residues (C332S and C65S) revealed zinc binding required for correct folding. L1 L2 Site SiteI I L1 L2 •L1 and L2 are perpendicular, and stabilized by the tetrahedral coordination of a zinc ion, resulting in a “scissor-like” shape. Mutations in Parkin ARJP patients have been discovered to have one or more missense or truncation mutations in Parkin. In particular four missense mutations (G328E, R334C, T351P, and V380L) in the IBR domain have been identified. V380 V380 •R334C protein possibly contributes that an extra zinc ligand to compete against nearby coordinating V380 residue. •An alteration along L1 loop (G328 and R334) interfereR334 with protein interactions, and result in R334 decreased binding and ubuiqitination of substrates. R334 •Proximity of N and C termini suggests facilitating role for protein interactions to stabilize overall geometry of RING domain orientation. N Term G328 G328 G328 C Term V380 L331 G328 L331 L331 R334 L1 UCH-L1 Ubiqutin is recycled by proteolytic removal from its conjugating protein by dubiquitinating enzymes (DUBs). DUBs catalyze the hydrolysis of C-terminal ubiquityl esters and amides, which is critical to recycle free ubuiqitin and continue protein degradation. UCH-L1 is highly abundant in the brain, constituting up to 2% of the total protein, and has been shown to be exclusively localized in the neurons. Mutations in the UCH-L1 have been reported to be linked to both susceptibility to and protection from Parkinson’s disease. Proposed Functions of UCH-L1 1. Hydrolyze Ub Ub Ub 2. Ub α Synuclein UCH-L1 Conjugate via K63 UCH-L1 UCH-L1 α Synuclein •UCH-L1 is usually considered to be monomeric, but it was found its asymmetric unit contained two proteins. •When UCH-L1 dimerizes in vitro, the protein can additionally function as a ubiquitin protein ligase in addition to its hydrolase activity. UCH-L1 Dimer (PDB 2etl X Ray) UCH-L1 Monomer Right Lobe α2 Left Lobe Left Lobe α7 β3 α1 αβ α6 Left Lobe P’-site L8 P-site α3 L9 Right Lobe α4 α5 •The secondary structures of the two lobes, one consisting of five α-helices (α1, α3, α4, α5, and α6) and the other (α2 and α7 and the β strands), form a helix-βhelix sandwich fold. •Between the two lobes is the active site cleft where the hydrolysis reaction occurs through a catalytic triad. •The active-site left is covered by a loop, L8. •On the interfaces of the two lobes are the probable binding sites for ubiquitin (P site) and the protein conjugate (P’ site). UCH-L1 Active Site Cleft N159 W4 C90 H161 W3 E90 R178 W2 W1 •Column of four water molecules splits H161 apart from C90, disrupting the classical catalytic His-Cys diad. •W3 and W4, as well as H-bonding network between E60, N159, H161, D176, and R178 are absent in homologues. In vitro activity of UCH-L1 significantly lower in comparison to homologues. UCH-L1 Active Site Cleft C90 H161 W4 •To form a productive catalytic triad, H161 must move closer towards C90, and this requires a large degree of plasticity in the noncovalent bonds. •W4 is positioned in the exact location that the imidazolium nitrogen of H161 needs to be to form a productive catalytic triad. •Waters loosely hold together the active site, and when triggered, the plasticity of water-mediated H bonds allows conformational change. UCH-L1 Mutations A substitution mutation (I93M) in the UCH-L1 gene was reported to be associated with an autosomal-dominant form of Parkinson’s disease, whereas a polymorphism (S18Y) was reported to be associated with reduced susceptibility to Parkinson’s disease. Researchers speculate that this dichotomy may be explained by the discovery that UCH-L1 exhibits dual activities: a ubiquitin hydrolase ativity and a ubiquitin ligase activity. I93M Mutation in UCH-L1 Isoleucine 93 to Methionine amino acid mutation Cys90 Hydrophobic Pocket Pocket Hydrophobic Cys90 I93 I93 •The side chain of I93 is in the hydrophobic pocket that holds right lobe together. Methionine Amino Acid structures from http://www.biochem.northwestern.edu/holmgren/Glossary/Images/pics/amino_acids/Isoleucine.gif Parkinson’s Disease Treatment There is no treatment to stop or reverse the progressive degeneration of dopaminergic neurons in the brain. However, drugs can help to alleviate some of the motor symptoms of Parkinson’s disease. Two general approaches to treatment: to either impede the loss of dopamine in the brain or improve the symptoms of Parkinson’s disease by other means. The “gold standard” for Parkinson’s disease treatment is levodopa as compared to dopamine agonists. Drug-inhibited DOPA decarboxylase (DDC) •Humans synthesize dopamine from dietary tyrosine in L-3,4dihydroxyphenylalanine (LDOPA, or levodopa) via decarboxylation by DDC. •Since dopamine cannot cross the blood-brain barrier, L-DOPA must be administered to increase the amount of dopamine in neurons. •If administered as a drug, LDOPA (carbiDOPA or benserazide) will rapidly convert to dopamine in the blood stream. DDC inhibitor added to slow down conversion. L-DOPA DOPA decarboxylase (DDC) Dopamine blood-brain barrier DDC in complex with carbiDOPA carbiDOPA •DDC is a dimer that is surrounded by eight alphahelices with its cofactors (PLP) and inhibitors, carbiDOPA, in yellow. PLP H192 carbiDOPA Conclusion Research suggests that Parkinson’s disease affects approximately 500,000 people in the United States each year. The total annual cost of Parkinson’s disease to the nation is estimated to exceed $6 billion annually. Parkinson's research has advanced to the point that halting the progression of PD, lost function restoration, and disease prevention are all considered realistic goals. However, we cannot yet cure any major neurodegenerative disorder, and defeating PD remains a significant challenge. Bibliography 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Bell J, lark AJ. 1926. A pedigree of paralysis agitans. Ann. Eugen. 1:455-62 Bonifati V, Ostra BA, Heutink P. 2004. Linking DJ-1 to neurodegeneration offers novel insights for understanding the pathogenesis of Parkinson’s disease. J. Mol. Med. 82:163-74. Burkhard, P. et al. J. Mol. Biol. 283, 121-133 (1998). Larson, E.M., Larimer, F.W. & Hartman, F.. Biohemistry 34, 4531-4537 (1995). Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Ata Crystallogr. D 53, 240-255 (1997). Kraulis, P.J. J. Appl. Crsytallogr. 24, 956-950 (1991). Merritt, E.A. & Bacon, D.J. RASTER3D: Photorealistic molecular graphics. 505524 (Academic Press, San Diego; 1997) Yahr, M.D. et al: Trans. Amer. Neurol. Ass., 93: 56, 1968. Dery, J. P. et al.: Un Med. Canada, 91:842, 843 Cotzias, G.C., Papavasiliou, P.S. and Gellene, R.: New Eng. J. Med., 280: 337, 1969.