Engineered antibody fragments for cocrystallization with signal peptide peptidase

用于与信号肽肽酶共结晶的工程化抗体片段

基本信息

  • 批准号:
    8149925
  • 负责人:
  • 金额:
    $ 29.7万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2010
  • 资助国家:
    美国
  • 起止时间:
    2010-09-30 至 2014-07-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): 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 hitocompatibility 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 cocrystallization 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. PUBLIC HEALTH RELEVANCE: The integral membrane protein protease, SPP, is an essential protein implicated in a wide variety of diseases, including Alzheimer's and Hepatitis C virus. The work proposed here aims to develop a suite of novel technologies to aid in determination of the three-dimensional structure of SPP and other membrane proteins of pharmacological interest. An SPP structure will help to understand its role in disease and aid in development of specific inhibitors as potential Alzheimer's and Hepatitis C therapies.
描述(由申请人提供):疏水膜蛋白在细胞中执行多种功能,但它们的结构众所周知难以解决。因此,获得用于结构测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分子工具箱,并使用它们来结晶和解决两个信号肽肽酶(SPP)的从头,高分辨率结构,其使用催化的三磷酸盐在脂质膜内进行水解。与采用对感兴趣的膜蛋白具有特异性的亲和试剂的工作相反,我们潜在的变革性方法使用可高度结晶的单链抗体片段(scFv)。我们的分子伴侣被设计为与可以插入任何膜蛋白的短表位紧密结合。我们期望我们的紧密结合的scFv分子伴侣将取代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)以将它们从内质网膜释放。SPP底物包括来源于新的组织相容性复合物1b(MHC-1b)分子的SP残余物。作为先天免疫的一部分,这些经过加工的肽被呈递在细胞表面,以被自然杀伤细胞识别,从而表明细胞是健康的。此外,SPP底物包括来自参与免疫应答和肌肉收缩的蛋白质的SP。SPP也被丙型肝炎病毒(HCV)劫持以进行复制,并且与早老素有关,早老素使用类似的化学物质来产生阿尔茨海默病中的淀粉样蛋白生成肽。SPP和早老素组成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,受调节的膜内蛋白水解的细节既具有基本的生化重要性,又具有潜在的治疗应用。然而,底物如何在脂膜的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,SPP是保守的通量生物学,但还没有晶体结构。 我们将首先解决与我们的伴侣分子复合的古同源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。到目前为止,我们已经设计了我们的第一个伴侣,并通过凝胶过滤分离出与SPP的亲和复合物。独立地,我们已经生长了分子伴侣和SPP的晶体。然而,SPP的晶体不能很好地进行结构测定,因此共结晶技术是关键。 预期的成果是结晶分子伴侣的工具箱以及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。总之,该项目不仅有助于免疫调节和膜内蛋白水解的生物学,而且还拓宽了我们对膜蛋白的知识,并使其他膜蛋白结构得以解决。 公共卫生关系:整合膜蛋白蛋白酶SPP是一种与多种疾病(包括阿尔茨海默病和丙型肝炎病毒)有关的必需蛋白质。本文提出的工作旨在开发一套新技术,以帮助确定SPP和其他具有药理学意义的膜蛋白的三维结构。SPP结构将有助于了解其在疾病中的作用,并有助于开发特异性抑制剂作为潜在的阿尔茨海默氏症和丙型肝炎疗法。

项目成果

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JENNIFER A MAYNARD其他文献

JENNIFER A MAYNARD的其他文献

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{{ truncateString('JENNIFER A MAYNARD', 18)}}的其他基金

Structure, function and antigenicity of B. pertussis virulence factors
百日咳博德特氏菌毒力因子的结构、功能和抗原性
  • 批准号:
    10298432
  • 财政年份:
    2021
  • 资助金额:
    $ 29.7万
  • 项目类别:
Structure, function and antigenicity of B. pertussis virulence factors
百日咳博德特氏菌毒力因子的结构、功能和抗原性
  • 批准号:
    10448307
  • 财政年份:
    2021
  • 资助金额:
    $ 29.7万
  • 项目类别:
Structure, function and antigenicity of B. pertussis virulence factors
百日咳博德特氏菌毒力因子的结构、功能和抗原性
  • 批准号:
    10656458
  • 财政年份:
    2021
  • 资助金额:
    $ 29.7万
  • 项目类别:
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
  • 财政年份:
    2010
  • 资助金额:
    $ 29.7万
  • 项目类别:
Instrument to quantify solution binding kinetics for drug discovery & development
用于药物发现的溶液结合动力学量化仪器
  • 批准号:
    7794532
  • 财政年份:
    2010
  • 资助金额:
    $ 29.7万
  • 项目类别:
Engineered antibody fragments for cocrystallization with signal peptide peptidase
用于与信号肽肽酶共结晶的工程化抗体片段
  • 批准号:
    8780816
  • 财政年份:
    2010
  • 资助金额:
    $ 29.7万
  • 项目类别:
Engineered antibody fragments for cocrystallization with signal peptide peptidase
用于与信号肽肽酶共结晶的工程化抗体片段
  • 批准号:
    8520337
  • 财政年份:
    2010
  • 资助金额:
    $ 29.7万
  • 项目类别:
Engineered antibody fragments for cocrystallization with signal peptide peptidase
用于与信号肽肽酶共结晶的工程化抗体片段
  • 批准号:
    8028124
  • 财政年份:
    2010
  • 资助金额:
    $ 29.7万
  • 项目类别:
Engineered antibody fragments for cocrystallization with signal peptide peptidase
用于与信号肽肽酶共结晶的工程化抗体片段
  • 批准号:
    8309972
  • 财政年份:
    2010
  • 资助金额:
    $ 29.7万
  • 项目类别:
Antibody-mediated protection against whooping cough
抗体介导的百日咳保护
  • 批准号:
    6962314
  • 财政年份:
    2005
  • 资助金额:
    $ 29.7万
  • 项目类别:

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