Identifying relevant HLA-F ligands

鉴定相关的 HLA-F 配体

• 批准号: 10171782
• 负责人: Roland K Strong
• 金额: $ 15.95万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10171782
• 关键词: Activated Lymphocyte; Alleles; Amino Acids; Amyotrophic Lateral Sclerosis; Antigen Presentation Pathway; Antigens; Antiviral Response; Applications Grants; Binding; Biochemical; Biological; CD8-Positive T-Lymphocytes; Cancerous; Cell Surface Proteins; Cell surface; Cells; Circular Dichroism Spectroscopy; Complex; Correlative Study; Coupled; Cross Presentation; Crystallization; Data; Development; Disease; Future; Glioblastoma; Glycoconjugates; Glycopeptides; Glycoproteins; Goals; HLA-A2 Antigen; Histocompatibility Antigens Class I; Human; Immune; Immune response; Immune system; Immunity; Inflammatory Response; Insecta; KIR3DS1; Ligand Binding; Ligands; Lipids; MHC Class I Genes; MHC Interaction; Mediating; Methods; Microbe; Modeling; Modification; Nasopharynx Carcinoma; Natural Killer Cells; Oligosaccharides; Output; Pathogenesis; Pathway interactions; Patient-Focused Outcomes; Peptides; Peptidoglycan; Peripheral Nervous System; Physiological; Plasmodium falciparum; Play; Polysaccharides; Pregnancy; Prevention; Protein Fragment; Proteins; Recombinants; Research; Role; Sampling; Seminal; Side; Source; Specificity; Structure; Surface; Surface Plasmon Resonance; System; T-Lymphocyte; Testing; Toxoplasma gondii; Tryptophan; Vaccines; Validation; X-Ray Crystallography; base; conformer; electron density; extracellular; improved; insight; interest; molecular recognition; novel; pathogen; programs; protein expression; screening; small molecule; success; tumor; uptake

Project Abstract/Summary · Our underlying hypothesis is that, unlike human classical and other non-classical MHC class I proteins, HLA- F ligand recognition is focused on glycans, either as components of oligosaccharides, proteo/peptidoglycans, or glycoproteins/glycopeptides – representing a paradigm shift in current understanding. This hypothesis is based on a rigorous reevaluation of recent, otherwise seminal crystallographic and mass-spec results (1). · Echoing FOA PA-19-066, our long-term overall goal is “to characterize antigen processing and presentation […] of novel peptidic and non-peptidic ligands presented by [HLA-F], and to determine the contribution of these unique antigenic ligands to: protective immune responses to infectious pathogens and/or vaccines; pathogen- associated immune pathogenesis; and/or in the induction/progression or prevention of immune-mediated dis- eases.” Our first steps towards accomplishing this goal in this R21 will be to: (a) identify physiologically- relevant ligand/s for HLA-F using rigorous biochemical approaches, focusing on glycopeptides and glycoconju- gates; and (b) determine conditions for growing diffraction-quality co-crystals to support future crystallographic structure determinations. These results will comprise the necessary preliminary results to support follow-on grant applications to structurally, biologically, and functionally validate candidate HLA-F glycoligands. · We will achieve these goals, and rigorously test our hypothesis, through the following Specific Aim: we will use our novel ARTEMIS mass-spec peptide discovery platform, glycan arrays, and fragment screening ap- proaches, to fully parse HLA-F ligand specificity for glycosylated ligands. Candidate ligands will be used to screen crystallization conditions to support follow-on crystallographic studies to eventually structurally characterize recognition mechanisms to fully parse specificity. Rigor will ultimately be achieved through careful biochemical and structural validation, and the likelihood of success will be maximized by incorporating multiple, parallel ap- proaches and experimental methods into the proposed scope to overcome potential confounders and pitfalls. · Significance (from PA-19-066): “The classic understanding of antigen processing and presentation begins with a protein fragment (peptide) associating with MHC molecules. […] These long-accepted paradigms regard- ing antigen processing and presentation and T cell recognition are incomplete. Approximately 10% of antigenic peptides can be derived from unconventional sources […]. Additionally, the antigen components that trigger non- classical MHC I restricted CD8 T cells and unconventional or innate T cells include lipids and small-molecule metabolites but are not well characterized. […] The objective of this research program is to promote the discovery of unique antigenic ligands (peptidic and non-peptidic) and understand the immune responses to these ligands: peptidic and non-peptidic ligands presented by non-classical MHC molecules: [HLA-F].”

项目摘要/摘要 ·我们的基本假设是，与人类经典和其他非经典MHC I类蛋白不同，HLA- F配体识别集中在聚糖上，要么是寡糖，蛋白含量/肽二聚糖的组成部分或 糖蛋白/糖肽 - 代表当前理解的范式转移。该假设是基于 严格重新评估了最近的半晶体学和质谱结果（1）。 与FOA PA-19-066相呼应，我们的长期总体目标是“表征抗原处理和表现 […] [HLA-F]提出的新型肽和非肽配体，并确定这些配体的贡献 独特的抗原配体至：对感染性病原体和/或疫苗的保护性免疫回应；病原- 相关的免疫病变；和/或在诱导/进展或预防免疫介导的疾病中 轻松。”在R21中实现这一目标的第一步将是：（a）在物理上确定 使用严格的生化方法，用于HLA-F的相关配体，重点是糖肽和糖糖肽 - 大门（b）确定生长衍射质量共晶体以支持未来晶体学的条件 结构确定。这些结果将包括支持后续赠款的必要初步结果 在结构，生物学和功能上验证候选HLA-F糖胶的应用。 我们将通过以下具体目的实现这些目标，并严格检验我们的假设：我们将 使用我们新颖的Artemis质谱肽发现平台，聚糖阵列和碎片筛选 促进，以完全解析糖基化配体的HLA-F配体特异性。候选配体将用于筛选 结晶条件以支持后续晶体学研究，以最终在结构上表征 完全解析特异性的识别机制。最终将通过仔细的生化来实现严峻 和结构验证以及成功的可能性将通过合并多个平行的ap- 在提出的范围中，采用和实验方法克服了潜在的混杂因素和陷阱。 意义（来自PA-19-066）：“对抗原处理和表现的经典理解开始 与MHC分子相关的蛋白质片段（肽）。 […]这些长期被接受的范式关注 - 抗原加工和表现以及T细胞识别是不完整的。大约10％的抗原 肽可以源自非常规的来源[…]。此外，触发非 - 的抗原成分 经典MHC I限制了CD8 T细胞以及非常规或先天T细胞包括脂质和小分子 代谢物，但表征不佳。 […]该研究计划的目的是促进发现 独特的抗原配体（肽和非肽），并了解对这些配体的免疫反应： 非经典MHC分子提出的肽和非肽配体：[HLA-F]。