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207 results for “Huntingtin”
Search for huntingtin interactors in online databases – 2018/08/08
<p><strong>Project</strong> - Huntingtin structure-function open lab notebook. </p> <p><strong>Rationale</strong> - To identify different huntingtin interaction partners. </p> <p><strong>Overview</strong> - Different online databases which detail protein interaction partners were searched for huntingtin protein interaction partners. Data detailing huntingtin interaction partners from 9 different databases was extracted and simplified – worksheets 1-15. The information from each database was collated – worksheet 16. Huntingtin protein interaction partners were ranked according to the number of databases they were found in as well as the number of different experiments detailing the interaction with huntingtin – worksheet 17. </p>
Comparative analysis of huntingtin interaction partner database searches with Dr. Maiuri's work - 2018/08/31
<p><strong>Project </strong>- Huntingtin structure-function open lab notebook. </p> <p><strong>Rationale</strong> - To compare the ROS-specific huntingtin interaction partners identified by Dr. Tamara Maiuri with those detailed in existing databases. </p> <p><strong>Overview</strong> - Previously, different online databases which detail protein interaction partners were searched for huntingtin protein interaction partners. Following completion of this initial analysis, Dr. Tamara Maiuri posted in her open notebook a detailed list of high and medium confidence ROS-specific huntingtin interacting proteins: <a href="https://zenodo.org/record/1319540">https://zenodo.org/record/1319540</a>. A comparison of these interactors with those identified in the previously mined databases is briefly detailed. </p>
ROS-specific Huntingtin Interactions: In vitro poly-ADP ribose binding assays
<p>Poly-ADP ribose overlay assay with purified huntingtin protein to test ability of full-length and fragment huntingtin to bind poly-ADP ribose.</p>
ROS-Specific Huntingtin Interactions: Peptide poly ADP ribose overlay assay
<p>In vitro testing of peptides representing potential poly ADP ribose binding motifs within the huntingtin sequence.</p>
Analysis of Huntingtin BioID Datasets 2019/04/09
<p><strong>Project: </strong>Investigation of putative HTT interacting proteins</p> <p><strong>Experiment: </strong>Analysis of HTT BioID datasets </p> <p><strong>Date completed:­ </strong>2019/04/09</p> <p><strong>Rationale: </strong>BioID technology employs a promiscuous biotin ligase (BirA) fused to the terminus of the target protein, huntingtin, allowing proximal proteins to be biotinylated and then subsequently identified through mass spectrometry experiments. This technique has not been applied to assess huntingtin interactors to date in the published literature, so will provide a novel methodology to characterize the huntingtin interactome. As huntingtin is a large protein molecule and the precise location of the N and C-termini remain unresolved due to their flexible nature, both N and C terminally BirA-tagged constructs for full-length huntingtin will be generated for overexpression as well as a truncated construct spanning amino acids 80-3100, the region of the protein resolved in the recent cryo-electron microscopy structure which omits the flexible termini. Huntingtin fusion proteins will be overexpressed in cells subjected to different ROS stresses as well as control conditions. Resultant cell lysates will be analysed through collaboration with Prof. Anne-Claude Gingras (Lunenfeld Tanenbaum Research Institute, University of Toronto). From this work, we hope to obtain a list of putative huntingtin interactors which will be compared to previously published findings and assessed for stable complex formation with huntingtin</p>
ROS-Specific Huntingtin Interactions: Chromatin Retention Assay Set-up
<p>Optimization of an assay to measure huntingtin chromatin retention in response to oxidative stress, using the YFP-tagged huntingtin-specific intrabody nucHCB2.</p>
ROS-Specific Huntingtin Interactions: Chromatin Retention of Huntingtin in PARP KO Cells
<p>Huntingtin chromatin retention in response to oxidative stress in wild type, PARP1 knockout, PARP2 knockout, and PARP1/PARP2 knockout RPE1 cells.</p>
ROS-Specific Huntingtin Interactions: Poly ADP Ribose Levels in STHdh Cells
<p>Comparison of poly ADP ribose levels in Q7/Q7 versus Q111/Q111 STHdh cells in response to oxidative stress.</p>
ROS-Specific Huntingtin Interactions: Huntingtin chromatin retention dynamics by FRAP with veliparib
<p>Measurement of huntingtin chromatin recruitment dynamics by fluorescence recovery after photobleaching (FRAP) of the YFP-tagged huntingtin-specific intrabody, nucHCB2, under conditions of oxidative stress and PARP inhibition</p>
Huntingtin-RNA complex generation for cryoEM analysis 2019/07/24
<p><strong>Project: </strong>High resolution structural analysis of HTT-nucleic acid complexes</p> <p><strong>Experiment: </strong>Huntingtin-RNA complex generation for cryoEM analysis</p> <p><strong>Date: </strong>2019/07/24</p> <p><strong>Background: </strong>Huntingtin has been shown to copurify with nucleic acid material as well as bind different nucleic acid species in band shift assays. To more thoroughly characterise this interaction, HTT-nucleic acid complex generation and structure solution should provide insight into how huntingtin performs this function. </p> <p><strong>Rationale: </strong>Previous attempts to capture HTT-nucleic complexes have been difficult and maintaining the complex throughout purification of the sample has been tricky. These four approaches are different ways to try and form a stable complex between Q54 HTT-HAP40 and CAG<sub>8</sub>RNA. </p>
ROS-Specific Huntingtin Interactions: PAR overlay assay with PBM3 peptide mutant
<p>Poly-ADP ribose overlay assay with huntingtin PBM peptides to test ability of a PBM3 alanine mutant to bind poly-ADP ribose.</p>
ROS-Specific Huntingtin Interactions: Chromatin retention assay with huntingtin fragments containing PBM3
<p>Huntingtin amino acids 1790-1798 make up a potential PAR binding motif (PBM3). Two huntingtin fragments (1208-1810 and 1775-2413) were tested for chromatin retention upon oxidative stress.</p>
ROS-Specific Huntingtin Interactions: Summary of PAR binding for different huntingtin protein preps
<p>PAR overlay assay with different preparations of huntingtin has yielded variable results. These results are summarized.</p>
ROS-Specific Huntingtin Interactions: PAR overlay slot blotting optimization
<p>Optimization to find conditions allowing quantification of huntingtin PAR binding in the PAR overlay assay.</p>
ROS-Specific Huntingtin Interactions: Competition of huntingtin PAR binding by PBM3 peptide
<p>Attempts to use the PBM3 peptide in a competition assay to measure the on-off rates of huntingtin PAR binding.</p>
ROS-Specific Huntingtin Interactions: Testing PARG activity in HD patient fibroblasts
<p>Comparison nuclear PAR levels in wild type and HD (TruHD) fibroblasts in response to a PARG inhibitor concentration gradient (as a measure of PARG activity).</p>
Study to Measure Cerebrospinal Fluid Mutant Huntingtin Protein in Participants With Early Manifest Stage I or Stage II Huntington's Disease
ClinicalTrials.gov study NCT03664804. IPD Sharing: Not stated. Countries: 4. Publications: 0.
Limited Proteolysis of Huntingtin (2016/02/01)
<p>Open lab notebook write up for project: huntingtin structural studies. </p>
Mass Spectrometry of Huntingtin Fragments (2016/02/06)
<p>Open lab notebook write up for project: huntingtin structural studies.</p>
Mass Spectrometry .raw Files and Methods for Huntingtin Limited Proteolysis (2016/02/11)
<p>Open lab notebook for project: huntingtin structural studies</p>
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