Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are currently recognized as the most common genetic cause of parkinsonism. Among the large number of LRRK2 mutations identified to date, the G2019S variant is the most common. In Asia, however, another LRRK2 variant, G2385R, appears to occur more frequently. To better understand the contribution of different LRRK2 variants toward disease pathogenesis, we generated transgenic Drosophila over-expressing various human LRRK2 alleles, including wild type, G2019S, Y1699C, and G2385R LRRK2. We found that transgenic flies harboring G2019S, Y1699C, or G2385R LRRK2 variant, but not the wild-type protein, exhibit late-onset loss of dopaminergic (DA) neurons in selected clusters that is accompanied by locomotion deficits. Furthermore, LRRK2 mutant flies also display reduced lifespan and increased sensitivity to rotenone, a mitochondrial complex I inhibitor. Importantly, coexpression of human parkin in LRRK2 G2019S-expressing flies provides significant protection against DA neurodegeneration that occurs with age or in response to rotenone. Together, our results suggest a potential link between LRRK2, parkin, and mitochondria in the pathogenesis of LRRK2-related parkinsonism.

A common genetic form of Parkinson's disease (PD) is caused by mutations in LRRK2. We identify WSB1 as a LRRK2 interacting protein. WSB1 ubiquitinates LRRK2 through K27 and K29 linkage chains, leading to LRRK2 aggregation and neuronal protection in primary neurons and a Drosophila model of G2019S LRRK2. Knocking down endogenous WSB1 exacerbates mutant LRRK2 neuronal toxicity in neurons and the Drosophila model, indicating a role for endogenous WSB1 in modulating LRRK2 cell toxicity. WSB1 is in Lewy bodies in human PD post-mortem tissue. These data demonstrate a role for WSB1 in mutant LRRK2 pathogenesis, and suggest involvement in Lewy body pathology in sporadic PD. Our data indicate a role in PD for ubiquitin K27 and K29 linkages, and suggest that ubiquitination may be a signal for aggregation and neuronal protection in PD, which may be relevant for other neurodegenerative disorders. Finally, our study identifies a novel therapeutic target for PD.

Protein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the ␤-subunit of Rab geranylgeranyltransferase II (␤GGT II). The specific interaction of ␤GGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with ␤GGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for ␤GGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for ␤GGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the ␣-subunit (␣) of GGT II, ␤GGT II, and PRL-2 resulted in ␣/␤GGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous ␣/␤GGT II activity in HeLa cells. Together, these results indicate that the binding of ␣GGT II and PRL-2 to ␤GGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity.

The two tandem homologous catalytic domains of PTP␣ possess different kinetic properties, with the membrane proximal domain (D1) exhibiting much higher activity than the membrane distal (D2) domain. Sequence alignment of PTP␣-D1 and -D2 with the D1 domains of other receptor-like PTPs, and modeling of the PTP␣-D1 and -D2 structures, identified two nonconserved amino acids in PTP␣-D2 that may account for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTP␣-D1 positively affected the catalytic efficiency of PTP␣-D2 toward the in vitro substrates para-nitrophenylphosphate and the phosphotyrosyl-peptide RR-src. Together, they synergistically transformed PTP␣-D2 into a phosphatase with catalytic efficiency for paranitrophenylphosphate equal to PTP␣-D1 but not approaching that of PTP␣-D1 for the more complex substrate RR-src. In vivo, no gain in D2 activity toward p59 fyn was effected by the double mutation. Alteration of the two corresponding invariant residues in PTP␣-D1 to those in D2 conferred D2-like kinetics toward all substrates. Thus, these two amino acids are critical for interaction with phosphotyrosine but not sufficient to supply PTP␣-D2 with a D1-like substrate specificity for elements of the phosphotyrosine microenvironment present in RR-src and p59fyn . Whether the structural features of D2 can uniquely accommodate a specific phosphoprotein substrate or whether D2 has an alternate function in PTP␣ remains an open question.Protein tyrosine phosphorylation status is a key determinant of nearly all eukaryotic cell processes and is controlled by the protein-tyrosine kinases and phosphatases (PTPs).1 Phosphotyrosine hydrolysis is catalyzed by members of the large and diverse PTP superfamily, and although the specific roles of most of these enzymes have yet to be determined, they can positively or negatively regulate cellular signaling pathways (1, 2). The PTPs include enzymes with absolute specificity for phosphotyrosine as well as dual specificity enzymes that can also dephosphorylate serine and threonine residues. Most do not share significant sequence identity, with the exception of the phosphotyrosine-specific receptor and non-receptor-like PTPs, which have stretches of amino acid identity throughout their catalytic domains. Nevertheless, despite dissimilarities in primary sequence, PTPs from diverse subgroups have a remarkably conserved tertiary structure and predicted catalytic mechanism (3, 4).Enzymological and mutational studies have elucidated several features of the catalytic mechanism of PTPs (5). All have an absolutely conserved CX 5 R motif in the active site and nucleophilic attack on the phosphate ester by this essential cysteine results in the formation of a covalent thiophosphate intermediate (6 -8). This phosphate transfer is facilitated by proton donation to the phenolic oxygen of phosphotyrosine from a general acid, identified in the tyrosine-specific PTP1 and Yop51 from Yersinia as the aspartate residue within a conserved WPD moti...

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM) models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP) scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7–11.2 μM) as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5–40.2 μM). Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 μM), hepatotoxicity (up to 30 μM) or cardiotoxicity (up to 30 μM).