Returning to the workforce supplement request for R01 GM095638

返回 R01 GM095638 的劳动力补充请求

• 批准号: 8670457
• 负责人: JENNIFER A MAYNARD
• 金额: $ 7.4万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8670457
• 关键词: Active Sites; Address; Affinity; Alzheimer' s Disease; Antibodies; Aspartate; Binding; Binding Proteins; Biochemical; Bioinformatics; Biological; Biology; Cell surface; Cells; Chemistry; Cocrystallography; Complex; Crystallization; Development; Disease; Distant; Docking; Endoplasmic Reticulum; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Escherichia coli; Gel Chromatography; Health; Hepatitis C; Hepatitis C Therapy; Hepatitis C virus; His-His-His-His-His-His; Homologous Gene; Hydrolysis; Immune response; Immunity; Immunoglobulin Fragments; Knowledge; Location; Major Histocompatibility Complex; Membrane; Membrane Lipids; Membrane Proteins; Methods; Molecular; Molecular Chaperones; Molecular Structure; Muscle Contraction; Mutagenesis; Natural Immunity; Natural Killer Cells; Outcome; Parents; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Phage Display; Process; Proteins; Proteolysis; Randomized; Reagent; Resolution; Role; Route; Signal Transduction; Site-Directed Mutagenesis; Structure; Technology; Therapeutic; Variant; Virus Diseases; Virus Replication; Work; adaptive immunity; directed evolution; experience; immunoregulation; inhibitor/antagonist; interest; new technology; presenilin; protein structure; signal peptide peptidase; virus pathogenesis

Hydrophobic membrane proteins perform a variety of functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. The objectives of this proposal are to develop a toolbox of chaperones and use them to crystallize and solve the de novo, high resolution structure of two signal peptide peptidases (SPPs), which use catalytic aspartates to conduct hydrolysis within the lipid membrane. In contrast to work employing affinity reagents specific to the membrane protein of interest, our potentially transformative approach uses hypercrystallizable single chain antibody fragments (scFvs). Our chaperones are engineered for tight binding to a short epitope that can be inserted into any membrane protein. We expect that our tightly bound scFv chaperone will immobilize an SPP loop and provide a stable crystal lattice, leading to better diffracting crystals. SPPs trim signal peptides (SPs) to liberate them from the endoplasmic reticulum membrane. SPP substrates include SPs remnants derived from new histocompatibility complex 1b (MHC-1b) molecules. As a part of innate immunity, these processed peptides are presented on cell surfaces for recognition by Natural Killer cells to indicate that the cell is healthy. In addition, SPP substrates include SPs from proteins involved in immune response and muscle contraction. SPP is also hijacked by the Hepatitis C virus (HCV) for replication, and is related to presenilin, which uses similar chemistry to generate amyloidogenic peptides in Alzheimer Disease. SPP and presenilin comprise one of just three superfamilies of intramembrane proteases. The details of regulated intramembrane proteolysis, from cell biological signaling to active site chemistry, are of both fundamental biochemical importance and potential therapeutic application. How substrates are presented and hydrolyzed within the confines of the hydrophobic space of the lipid membrane, however, remain largely a mystery. At least 5 SPP variants have been sequenced, located in different regions of ER, and SPPs are conserved throughput biology, but there is no crystal structure yet. We will start by solving the structure an archeal homolog in complex with our chaperones as proof-of principle, and then expand to a eukaryotic SPP, whose biomedical relevant activity is known. To date, we have engineered our first chaperone and isolated an affinity complex with SPP by gel filtration. Independently, we have grown crystals of the chaperone and SPP. However, the crystals of SPP do not diffract well enough for structure determination, and thus the cocrystalllization technology is critical. The expected outcomes are a toolbox of crystallization chaperones as well as the first molecular picture of SPP, including the location of the active site and substrate-docking patches. Taken together, this project will contribute not only to the biology of immunoregulation and intramembrane proteolysis, but also broaden our knowledge of membrane proteins and enable other membrane protein structures to be solved.

疏水膜蛋白在细胞中执行多种功能，但众所周知，它们的结构很难求解。因此，获得结构确定的膜蛋白晶体的新策略至关重要。该提案的目标是开发一个伴侣的工具箱，并使用它们来结晶和解决两种信号肽肽酶（SPP）的高分辨率结构，这些结构使用催化天然病型在脂质膜内进行水解。与采用针对感兴趣的膜蛋白的亲和力试剂的工作相反，我们的潜在转化方法使用了可染定的单链抗体片段（SCFV）。我们的伴侣经过精心设计，可与可以插入任何膜蛋白的短期表位紧密结合。我们期望我们紧密结合的SCFV伴侣会固定SPP环并提供稳定的晶体晶格，从而导致更好的衍射晶体。 SPPS修剪信号肽（SP）将其从内质网膜中解放出来。 SPP底物包括源自新的组织兼容复合物1B（MHC-1B）分子的SPS残留物。作为先天免疫的一部​​分，这些加工的肽被呈现在细胞表面上，以识别自然杀伤细胞，以表明细胞健康。此外，SPP底物包括来自免疫反应和肌肉收缩的蛋白质的SP。 SPP也被丙型肝炎病毒（HCV）劫持，并与Presenilin有关，后者使用类似的化学物质在阿尔茨海默氏病中产生淀粉样蛋白生成肽。 SPP和Presenilin构成了膜内蛋白酶的三个超家族之一。 从细胞生物信号传导到活性位点化学的受调节膜内蛋白水解的细节既具有生化重要性，又具有潜在的治疗应用。然而，如何在脂质膜的疏水空间的范围内呈现底物和水解，这在很大程度上仍然是一个谜。已经在ER的不同区域进行了至少5个SPP变体，并且SPP是保守的吞吐量生物学，但尚无晶体结构。 我们将首先解决与伴侣作为原理证明的复杂的结构，然后扩展到真核生物属，其生物医学相关活性是已知的。迄今为止，我们已经设计了第一个伴侣，并通过凝胶过滤隔离了与SPP的亲和力综合体。独立地，我们已经种植了伴侣和属的晶体。但是，SPP的晶体对于结构确定不足以衍射，因此共晶技术至关重要。 预期的结果是结晶伴侣的工具箱以及SPP的第一分子图片，包括活性位点的位置和底物船坞斑块。综上所述，该项目不仅将有助于免疫调节和膜内蛋白水解的生物学，而且还扩大了我们对膜蛋白的了解，并使其他膜蛋白结构能够解决。