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ABSTRACT: The pathogenic Huntington's disease (HD) mutation causes polyglutamine (polyQ) tract expansion of the 348 kDa HTT protein above a critical threshold of ~35 glutamines. HD mutation effect on HTT is poorly understood, partly due to difficulties in performing biochemical studies with this large protein. To facilitate such studies, we generated resources for HTT production in multiple eukaryotic expression systems, comprising constructs with polyQ lengths representing general population, HD patients, juvenile HD patients and the more extreme expansions used in some tissue and animal models. These reagents yield milligram quantities of pure HTT protein. We biophysically characterised HTT samples produced using this HD resource, gleaning insight into the nature of full-length HTT in its apo form and when bound to its binding partner HAP40. Work outlined in this manuscript and the tools generated, lay a foundation for further biochemical study of the HTT protein and its functional interactions with other biomolecules. ; ACKNOWLEDGEMENTS We thank Dr. Xiaobing Zuo (Argonne National Laboratory) for expert support with SAXS measurements, and acknowledge the use of the SAXS Core facility of Center for Cancer Research (CCR), National Cancer Institute (NCI) which is funded by Frederick National Laboratory for Cancer Research under contract HHSN261200800001E and the intramural research program of the NIH, NCI, CCR. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. This research used 12-ID-B beamline of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We also acknowledge Dr. Pravin Mahajan who constructed the pBMDEL vector. This research was supported by a Huntington's Disease ...

ABSTRACT: The pathogenic Huntington's disease (HD) mutation causes polyglutamine (polyQ) tract expansion of the 348 kDa HTT protein above a critical threshold of ~35 glutamines. HD mutation effect on HTT is poorly understood, partly due to difficulties in performing biochemical studies with this large protein. To facilitate such studies, we generated resources for HTT production in multiple eukaryotic expression systems, comprising constructs with polyQ lengths representing general population, HD patients, juvenile HD patients and the more extreme expansions used in some tissue and animal models. These reagents yield milligram quantities of pure HTT protein. We biophysically characterised HTT samples produced using this HD resource, gleaning insight into the nature of full-length HTT in its apo form and when bound to its binding partner HAP40. Work outlined in this manuscript and the tools generated, lay a foundation for further biochemical study of the HTT protein and its functional interactions with other biomolecules. NB: HTT_Manuscript_20190319.zip should contain all of the raw data used for this manuscript including raw images for the Western blots, missing in the previous upload. ; ACKNOWLEDGEMENTS We thank Dr. Xiaobing Zuo (Argonne National Laboratory) for expert support with SAXS measurements, and acknowledge the use of the SAXS Core facility of Center for Cancer Research (CCR), National Cancer Institute (NCI) which is funded by Frederick National Laboratory for Cancer Research under contract HHSN261200800001E and the intramural research program of the NIH, NCI, CCR. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. This research used 12-ID-B beamline of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory ...

Computational approaches in drug discovery and development hold great promise, with artificial intelligence methods undergoing widespread contemporary use, but the experimental validation of these new approaches is frequently inadequate. We are initiating Critical Assessment of Computational Hit-finding Experiments (CACHE) as a public benchmarking project that aims to accelerate the development of small molecule hit-finding algorithms by competitive assessment. Compounds will be identified by participants using a wide range of computational methods for dozens of protein targets selected for different types of prediction scenarios, as well as for their potential biological or pharmaceutical relevance. Community-generated predictions will be tested centrally and rigorously in an experimental hub(s), and all data, including the chemical structures of experimentally tested compounds, will be made publicly available without restrictions. The ability of a range of computational approaches to find novel compounds will be evaluated, compared, and published. The overarching goal of CACHE is to accelerate the development of computational chemistry methods by providing rapid and unbiased feedback to those developing methods, with an ancillary and valuable benefit of identifying new compound-protein binding pairs for biologically interesting targets. The initiative builds on the power of crowd sourcing and expands the open science paradigm for drug discovery. ; ACKNOWLEDGEMENTS The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 875510. The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA and Ontario Institute for Cancer Research, Royal Institution for ...