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发挥多种功能，但其结构却非常难以解析。因此，获得膜蛋白晶体用于结构测定的新策略至关重要。该提案的目标是开发一个伴侣工具箱，并使用它们结晶和解析两种信号肽肽酶 (SPP) 的从头高分辨率结构，这两种信号肽肽酶使用催化天冬氨酸在脂质膜内进行水解。与使用针对感兴趣的膜蛋白的亲和试剂的工作相比，我们潜在的变革性方法使用超结晶单链抗体片段（scFv）。我们的分子伴侣经过精心设计，可与可插入任何膜蛋白的短表位紧密结合。我们期望紧密结合的 scFv 伴侣能够固定 SPP 环并提供稳定的晶格，从而产生更好的衍射晶体。 SPP 修剪信号肽 (SP)，将其从内质网膜中释放出来。 SPP 底物包括源自新组织相容性复合体 1b (MHC-1b) 分子的 SP 残余物。作为先天免疫的一部​​分，这些加工过的肽呈现在细胞表面，被自然杀伤细胞识别，表明细胞是健康的。此外，SPP 底物包括来自参与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) 劫持进行复制，并且与早老素相关，早老素使用类似的化学物质在阿尔茨海默病中产生淀粉样肽。 SPP 和早老素是膜内蛋白酶的三个超家族之一。 受调节的膜内蛋白水解的细节，从细胞生物信号传导到活性位点化学，具有基本的生化重要性和潜在的治疗应用。然而，底物如何在脂膜疏水空间的范围内呈现和水解，在很大程度上仍然是一个谜。至少有 5 个 SPP 变体已被测序，位于 ER 的不同区域，并且 SPP 是保守的通量生物学，但尚无晶体结构。 我们将首先解决与我们的分子伴侣复合物的结构，作为原理证明，然后扩展到真核 SPP，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分子伴侣，并通过凝胶过滤分离出具有 SPP 的亲和复合物。我们独立地生长了伴侣分子和 SPP 的晶体。然而，SPP晶体的衍射性能不足以确定结构，因此共结晶技术至关重要。 预期结果是结晶伴侣工具箱以及 SPP 的第一个分子图片，包括活性位点和底物对接补丁的位置。总而言之，该项目不仅将为免疫调节和膜内蛋白水解的生物学做出贡献，而且还将拓宽我们对膜蛋白的知识，并使其他膜蛋白结构得到解决。