Suchergebnisse

© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ; The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus, which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators. ; We thank CNPq Brazil (fellowship 200456/2015-6 to J.B.B. and grants 454507/2014-3 and 300606/2010-9 to H.T.), the Fundação para a Ciência e a Tecnologia (FCT Investigator award IF/00624/2015 to G.J.L.B.), the European Union (Marie-Sklodowska Curie Innovative Training Network Protein Conjugates; Marie Skłodowska-Curie Individual Fellowship 743640 to T.R.; Marie-Curie Intra-European Fellowship 626890 to O.B.), the Ministerio de Economía, Industria, y Competitividad (project CTQ2015-67727-R to F.C.), and the Biotechnology and Biological Sciences Research Council (PhD studentship to L.D.) for funding. G.J.L.B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt, 676832). We also acknowledge funding by LISBOA-01-0145-FEDER-007391, co-financed ...

The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus, which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators. ; e thank CNPq Brazil (fellowship 200456/2015-6 to J.B.B. and grants 454507/2014-3 and 300606/2010-9 to H.T.), the Fundação para a Ciência e a Tecnologia (FCT Investigator award IF/00624/2015 to G.J.L.B.), the European Union (Marie-Sklodowska Curie Innovative Training Network Protein Conjugates; Marie Skłodowska-Curie Individual Fellowship 743640 to T.R.; Marie-Curie Intra-European Fellowship 626890 to O.B.), the Ministerio de Economía, Industria, y Competitividad (project CTQ2015-67727-R to F.C.), and the Biotechnology and Biological Sciences Research Council (PhD studentship to L.D.) for funding. G.J.L.B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt, 676832 ). We also acknowledge funding by LISBOA-01-0145-FEDER-007391, co-financed by FEDER through the Programa Operacional Regional de Lisboa (Lisboa 2020) of PORTUGAL 2020 and by FCT Portugal.

This is the author accepted manuscript. The final version is avialble via PNAS at http://www.pnas.org/content/112/16/E1994.long#ack-1. ; We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species. ; We thank Dr. Katherine Stott, from the Biophysics Facility, Department of Biochemistry, University of Cambridge, for her assistance in using these facilities. This work was supported by the Agency for Science, Technology and Research, Singapore (S.W.C.), the "La Caixa" foundation (S.D.), Wellcome/MRC (Medical Research Council) Parkinson's Disease Consortium Grant WT089698 (to E.D. and N.W.W.), National Institute for Health Research Biomedical Research Centres funding at University College London (to N.W.W.), the BBSRC through Grants BB/H003843/1 (to M.O.) and BB/E019927/1 (to C.M.D.), the Spanish Ministry of Economy and Competitiveness through Grants SAF 2012-39720 (to C.R.), BFU2013-44202 (to J.M.V.), and BIO2011-28941-C03-03 (to C.A. and G.R.), the Spanish Ministry of Health with cofunding by The European Regional Development Fund through Grant CP10/00527 (to C.R.), the Madrid Regional Government through Grant S2013/MIT-2807 (to J.M.V.), Parkinson's UK through Grant H-0903 (to T.G.), the Wellcome Trust, the Leverhulme Trust, the European Commission through project LSHM-CT-2006-037525 (to C.M.D.), the Medical Research Council through Grant MRC G1002272 (to E.J.D.-G. and C.M.D.), and the Engineering and Physical Sciences Research Council (C.M.D.). A.Y.A. was a Parkinson's UK Senior Research Fellow. N.C. is a Royal Society Research Fellow and also acknowledges financial support by the Human Frontier Science Program from Long-Term Fellowship LT000795/2009.

12 pags, 3 figs. -- The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acschembio.8b00466. ; The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders. ; This work was supported by the Boehringer Ingelheim Fonds (P.F.), the Studienstiftung des Deutschen Volkes (P.F.), Gates Cambridge Scholarships (R.L. and G.T.H) and a St. John's College Benefactors' Scholarship (R.L.), the UK Biotechnology and Biochemical Sciences Research Council (M.V. and C.M.D.), a Senior Research Fellowship award from the Alzheimer's Society, UK, grant number (317, AS-SF-16-003) (F.A.A.), the Wellcome Trust (C.M.D., M.V., and T.P.J.K.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Regione Toscana—FAS Salute—Supremal project (R.C., C.C., and F.C.), a Marie Skłodowska-Curie Actions—Individual Fellowship (C.G.), Sidney Sussex College Cambridge (G.M.), the Spanish Government—MINECO (N.C.), and by the Cambridge Centre for Misfolding Diseases (M.P., P.F., R.L., F.A.A., C.G., G.T.H., S.W.C., J.R.K., T.P.J.K., M.V., and C.M.D).