Publications (79) View all
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Article: Analysis of LRRK2 accessory repeat domains: prediction of repeat length, number and sites of Parkinson's disease mutations.
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ABSTRACT: Various investigators have identified the major domain organization of LRRK2 (leucine-rich repeat kinase 2), which includes a GTPase ROC (Ras of complex proteins) domain followed by a COR (C-terminal of ROC) domain and a protein kinase domain. In addition, there are four domains composed of structural repeat motifs likely to be involved in regulation and localization of this complex protein. In the present paper, we report our bioinformatic analyses of the human LRRK2 amino acid sequence to predict the repeat size, number and likely boundaries for the armadillo repeat, ankyrin repeat, the leucine-rich repeat and WD40 repeat regions of LRRK2. Homology modelling using known protein structures with similar domains was used to predict structures, exposed residues and location of mutations for these repeat regions. We predict that the armadillo repeats, ankyrin repeats and leucine-rich repeats together form an extended N-terminal flexible 'solenoid'-like structure composed of tandem repeat modules likely to be important in anchoring to the membrane and cytoskeletal structures as well as binding to other protein ligands. Near the C-terminus of LRRK2, the WD40 repeat region is predicted to form a closed propeller structure that is important for protein complex formation.Biochemical Society Transactions 10/2012; 40(5):1086-9. · 3.71 Impact Factor -
Article: Analysis of the regulatory and catalytic domains of PTEN-induced kinase-1 (PINK1).
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ABSTRACT: Mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene can cause early-onset familial Parkinson disease (PD). PINK1 encodes a neuroprotective protein kinase localized at the mitochondria, and its involvement in regulating mitochondrial dynamics, trafficking, structure, and function is well documented. Owing to the lack of information on structure and biochemical properties for PINK1, exactly how PINK1 exerts its neuroprotective function and how the PD-causative mutations impact on PINK1 structure and function remain unclear. As an approach to address these questions, we conducted bioinformatic analyses of the mitochondrial targeting, the transmembrane, and kinase domains of PINK1 to predict the motifs governing its regulation and function. Our report sheds light on how PINK1 is targeted to the mitochondria and how PINK1 is cleaved by mitochondrial peptidases. Moreover, it includes a potential optimal phosphorylation sequence preferred by the PINK1 kinase domain. On the basis of the results of our analyses, we predict how the PD-causative mutations affect processing of PINK1 in the mitochondria, PINK1 kinase activity, and substrate specificity. In summary, our results provide a conceptual framework for future investigation of the structural and biochemical basis of regulation and the neuroprotective mechanism of PINK1. Hum Mutat 33:1408-1422, 2012. © 2012 Wiley Periodicals, Inc.Human Mutation 05/2012; 33(10):1408-22. · 5.69 Impact Factor -
Article: Amyloid precursor protein processing and retinal pathology in mouse models of Alzheimer’s disease
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ABSTRACT: BackgroundRetinal ganglion cell loss is considered to be a cause of visual impairment in Alzheimer`s patients. Alterations in amyloid precursor protein (APP) processing and amyloid-β (Aβ) accumulation, key molecules associated with Alzheimer`s disease pathogenesis, may therefore contribute to retinal damage. We therefore investigated retinal APP processing and eye morphology in Alzheimer`s transgenic mouse models. MethodsEyes and brain samples of 2- to 18-month-old transgenic mice expressing human APP with the double Swedish mutation (APPswe) (APP K595N/M596L)(Tg2576) were compared with eyes and brain tissue from wild-type background C57BL6xSJL controls. In addition, 6- to 12-month-old double transgenic mice over-expressing human APPswe and mutant presenilin 1 with exon 9 deletion (APPswe/PS1-dE9) were compared with background controls of C57BL6xC3H strain. Tissue samples were fixed in formalin for immunohistochemistry, and dissected retinal and cerebellar extracts were frozen for Western blotting and enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies 1E8 and WO2 were used for immunohistochemical detection of APP and Aβ, whereas Aβ 42/40 levels were assayed by ELISA. APP and processed fragments were detected biochemically by Western blotting with domain-specific antibodies, using antibody WO2 (Aβ) and rabbit antibody 369 to the C-terminal domain of APP. ResultsImmunocytochemistry revealed strong cytoplasmic expression of APP and possibly Aβ in retinal ganglion cells and inner nuclear layer cells, and in lens and corneal epithelia for APP transgenic mice. Retinas from the APP transgenic mouse strains contained 18 to 70kDa APP proteolytic products that were not detected in the cerebellum. We found a higher proportion of APP α-secretase generated C-terminal fragments in transgenic retinal tissues than β-secretase-generated C-terminal fragments. Very low level Aβ was detected in transgenic retinas by ELISA; retinal Aβ 42 was 75 times less than for transgenic brain. Aβ was not detected in mouse retina by Western blotting in our study, indicating much less generation of Aβ in retina than brain tissue. ConclusionsAlzheimer’s mouse model retinas present with different APP proteolytic products and have a significantly lower production of amyloidogenic Aβ than found in brain.Albrecht von Graæes Archiv für Ophthalmologie 04/2012; 247(9):1213-1221. · 2.17 Impact Factor -
Article: Diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)) protects against peroxynitrite-induced nitrosative damage and prolongs survival in amyotrophic lateral sclerosis mouse model.
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.Journal of Biological Chemistry 12/2011; 286(51):44035-44. · 4.77 Impact Factor -
Article: Defining the substrate specificity determinants recognized by the active site of C-terminal Src kinase-homologous kinase (CHK) and identification of β-synuclein as a potential CHK physiological substrate.
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ABSTRACT: C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed β-synuclein as a potential CHK substrate, and Y127 in β-synuclein as the preferential phosphorylation site. Using peptides derived from β-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides β-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.Biochemistry 06/2011; 50(31):6667-77. · 3.42 Impact Factor
