Unveiling the Oncogenic Roles of ADAM10/17 by Comprehensively Profiling Proteolytic Activity on a Synthetic Human Protein Peptide Phage Library

通过全面分析合成人类蛋白肽噬菌体库的蛋白水解活性,揭示 ADAM10/17 的致癌作用

基本信息

  • 批准号:
    10061569
  • 负责人:
  • 金额:
    $ 6.86万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2018
  • 资助国家:
    美国
  • 起止时间:
    2018-12-01 至 2021-11-30
  • 项目状态:
    已结题

项目摘要

Project Summary The ADAMs (a disintegrin and metalloproteases) are transmembrane multi-domain proteins involved in multiple biological events including proteolysis, cell adhesion, fusion, proliferation and migration. Although aberrantly elevated activity of specific ADAMs has been implicated in different diseases, their best-documented role is in tumorigenesis and tumor progression, as a result of their biological functions — proteolytic “shedding” of membrane-anchored proteins (e.g., the complete ectodomain of cytokines, growth factors, receptors and adhesion molecules, etc.) and hence the rapid modulation of key cell autocrine and paracrine signaling pathways in the tumor microenvironment. Specific ADAMs that are implicated in oncogenic pathways includes ADAM8/9/10/12/15/17/19/28, among which the strongest evidences for a role in malignance exist for ADAM17 and its closest relative ADAM10. Despite major advances in our understanding of ADAM10/17 during the past decade, numerous questions have emerged regarding their expression in cancer and the mechanisms by which they contribute to tumorigenesis/progression. Substrate identification and discrimination is critical to shed light on the potential mechanisms, but little effort has focused on the identification of substrates of ADAMs, in part because of where they localize—the cell membrane. Here, we propose to develop a high-throughput method to comprehensively profile protease substrates of ADAM10/17 using phage display and massively parallel DNA next generation sequencing (NGS). By constructing two phage libraries displaying either randomized 10-mer peptides or 49-mer human protein segments covering the entire human proteome, we will be able to compare proteolysis specificities for peptides versus native human proteins, getting insights in how folding and domain structures dictate the accessibility of the cleavage site. Using this approach, we intend to define the cleavage site selectivity of ADAM10/17 proteases. In addition to conducting in vitro screening with soluble forms of ADAM10/17, which might be biased due to the location, orientation and conformation constraints imposed by their role as ecto-proteases, we will perform on-cell selection by engineering the displayed peptides on phages to have it insert into cell membranes, mimicking landscapes of membrane substrates. By identifying and characterizing the functional importance of the substrates of ADAM10/17, we hope to unveil more information regarding the roles of ADAM10/17 in cancer development and validate the hypothesis that pharmacologically targeting ADAMs would be useful. Finally, we will develop recombinant antibodies selectively targeting active forms of ADAM10/17 in hope to inhibit ADAMs involved oncogenic signaling. In the long term, this technique should be useful for studying the sequence specificity of a variety of other posttranslational modifications (PTM), including phosphorylation, citrullination, and sulfonation.
项目摘要 亚当斯(解整合素和金属蛋白酶)是跨膜多结构域蛋白,涉及多个细胞内的蛋白质。 生物学事件包括蛋白水解、细胞粘附、融合、增殖和迁移。虽然异常 特定的亚当斯活性升高与不同的疾病有关,它们的最佳记录作用是在 肿瘤发生和肿瘤进展,由于其生物学功能-蛋白水解“脱落”, 膜锚定蛋白(例如,细胞因子、生长因子、受体和 粘附分子等)从而快速调节关键细胞自分泌和旁分泌信号通路 在肿瘤微环境中。涉及致癌途径的特定亚当斯包括 ADAM 8/9/10/12/15/17/19/28,其中ADAM 17存在最强有力的证据证明其在抗肿瘤中的作用 和它最近的亲戚ADAM 10尽管过去我们对ADAM 10/17的理解取得了重大进展, 十年来,关于它们在癌症中的表达以及它们在癌症中的作用机制, 它们有助于肿瘤的发生/进展。底物识别和区分对于阐明 的潜在机制,但很少有努力集中在确定的底物的ADAM,部分 因为它们位于细胞膜上。在这里,我们建议开发一种高通量的方法, 使用噬菌体展示和大规模平行DNA对ADAM 10/17的蛋白酶底物进行全面分析 下一代测序(NGS)。通过构建两个噬菌体文库, 肽或覆盖整个人类蛋白质组的49聚体人类蛋白质片段,我们将能够比较 肽与天然人类蛋白质的蛋白质水解特异性,深入了解折叠和结构域 结构决定了切割位点的可及性。使用这种方法,我们打算定义分裂 ADAM 10/17蛋白酶的位点选择性。除了用可溶性形式的药物进行体外筛选外, ADAM 10/17,可能会有偏见,由于位置,方向和构象的限制所施加的 由于它们作为胞外蛋白酶的作用,我们将通过工程化展示的肽来进行细胞上的选择。 将其插入细胞膜,模仿膜基质的景观。通过识别和 通过对ADAM 10/17底物的功能性研究,我们希望能为进一步研究ADAM 10/17提供更多的信息。 关于ADAM 10/17在癌症发展中的作用,并验证了这一假设, 瞄准亚当斯会很有用最后,我们将开发选择性靶向活性的重组抗体, ADAM 10/17的形式,希望抑制亚当斯参与致癌信号传导。从长远来看,这项技术 应该可用于研究各种其他翻译后修饰(PTM)的序列特异性, 包括磷酸化、瓜氨酸和磺化。

项目成果

期刊论文数量(5)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2.
  • DOI:
    10.1038/s41589-020-00679-1
  • 发表时间:
    2021-01
  • 期刊:
  • 影响因子:
    14.8
  • 作者:
    Bracken CJ;Lim SA;Solomon P;Rettko NJ;Nguyen DP;Zha BS;Schaefer K;Byrnes JR;Zhou J;Lui I;Liu J;Pance K;QCRG Structural Biology Consortium;Zhou XX;Leung KK;Wells JA
  • 通讯作者:
    Wells JA
Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers.
  • DOI:
    10.1172/jci154604
  • 发表时间:
    2022-02-15
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Lim SA;Zhou J;Martinko AJ;Wang YH;Filippova EV;Steri V;Wang D;Remesh SG;Liu J;Hann B;Kossiakoff AA;Evans MJ;Leung KK;Wells JA
  • 通讯作者:
    Wells JA
Deep profiling of protease substrate specificity enabled by dual random and scanned human proteome substrate phage libraries.
通过双重随机和扫描的人类蛋白质组底物噬菌体库实现蛋白酶底物特异性的深度分析。
Targeting Phosphotyrosine in Native Proteins with Conditional, Bispecific Antibody Traps.
  • DOI:
    10.1021/jacs.0c08458
  • 发表时间:
    2020-10-14
  • 期刊:
  • 影响因子:
    15
  • 作者:
    Zhou XX;Bracken CJ;Zhang K;Zhou J;Mou Y;Wang L;Cheng Y;Leung KK;Wells JA
  • 通讯作者:
    Wells JA
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