
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).


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 5-
10% of patients are known to have to have monogenic forms of the disease: autosomal dominant or autosomal recessive.
LOCUS
PARK1/PARK4
PARK2
PARK3
PARK5
PARK6
PARK7
PARK8
PARK10
PARK11 unknown unknown
CHR.
LOCATION
4q21-q23
6q25.2-q27
2p13
4p14
1p35-p36
1p36
12p11.2-q13.1
1p32
2q36-2q37
5q23.1-q23.3
2q22-q23
GENE alpha-synuclein parkin unknown
UCH-L1
PINK1
DJ-1
LRRK2 unknown
GIGYF2
Synphilin -1
NR4A2
INHERITANCE
PATTERN
AD
AR
AD
AD
AR
AR
AD unclear unclear
AD
AD

Alpha-Synuclein
Parkin
Ubiquitin Proteasome
Pathway
UCHL-1
DA Neuron
Dysfunction
DA Degeneration/Cell Death
Parkinsonism

Key:
+ATP
DUB = deubiquitinating enzyme
Ub = ubiquitin monomer
SUB = substrate
 abnormal proteins peptides

 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 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

C365 Zn binding
C Term site II
C365
C368
H373
C377
C368
H373
C377 C337
C332
90 deg
Site II
C337
C352
C360
Zn binding site I
C352
•Substitutions of coordinating residues (C332S and C65S) revealed zinc binding required for correct folding.
L2
L2
L1
Site I
L1
•L1 and L2 are perpendicular, and stabilized by the tetrahedral coordination of a zinc ion, resulting in a “scissor-like” shape.


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
N Term
C Term that an extra zinc ligand to compete
V380 residue.
•An alteration along L1 loop (G328
L331 G328
L331 interactions, and result in
R334 ubuiqitination of substrates.
R334 suggests facilitating role for protein interactions to stabilize overall geometry of RING domain orientation.
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.

•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)

Right Lobe
α7
α2
β3
α6
L9
α3
Left Lobe
P’-site
L8
P-site
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).

N159
C90
H161
E90
R178
•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.

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.

 A substitution mutation (I93M) in the UCH-L1 gene was reported to be associated with an autosomaldominant 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
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

 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.

•Humans synthesize dopamine from dietary tyrosine in L-3,4dihydroxyphenylalanine (L-
DOPA, 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, L-
DOPA (carbiDOPA or benserazide) will rapidly convert to dopamine in the blood stream.
DDC inhibitor added to slow down conversion.

carbiDOPA •DDC is a dimer that is surrounded by eight alphahelices with its cofactors (PLP) and inhibitors, carbiDOPA, in yellow.
PLP
H192 carbiDOPA


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.

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