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

• 批准号: 10061569
• 负责人: Jie Zhou
• 金额: $ 6.86万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10061569
• 关键词: Active Sites; Address; Affect; Autocrine Communication; Bacteriophages; Binding; Biological; Biological Process; C-terminal; CASP1 gene; CRISPR interference; Caspase; Catalytic Domain; Cell Adhesion; Cell Adhesion Molecules; Cell membrane; Cell surface; Cells; Clinical Trials; DNA; Databases; Development; Discrimination; Disease; Disintegrins; Engineering; Event; Family; Goals; Growth Factor Receptors; Human; In Vitro; Label; Libraries; Light; Lipids; Location; Malignant Neoplasms; Mammals; Membrane; Metalloproteases; Methodology; Methods; Molecular Conformation; N-terminal; Oncogenic; Paracrine Communication; Pathway interactions; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Phage Display; Pharmacology; Phosphorylation; Physiological; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Proteolysis; Proteome; Proteomics; Randomized; Recombinant Antibody; Recombinants; Reporting; Research; Resistance; Role; Signal Pathway; Signal Transduction; Site; Specificity; Structure; Substrate Specificity; Techniques; Technology; Tertiary Protein Structure; Therapeutic; Therapeutic antibodies; Toxic effect; Up-Regulation; Validation; Variant; anti-cancer; base; cancer cell; clinically relevant; cytokine; deep sequencing; design; inhibitor/antagonist; insight; member; migration; next generation sequencing; overexpression; protein purification; screening; tumor; tumor growth; tumor microenvironment; tumor progression; tumorigenesis

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 Adams（崩解蛋白和金属蛋白酶）是跨膜多域蛋白，参与多个 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 在不同的疾病中隐含了特定亚当的活动的升高，其最佳记录的作用是 肿瘤发生和肿瘤进展，由于其生物学功能 - 蛋白水解的“脱落” 膜锚定的蛋白质（例如，细胞因子，生长因子，接收器和 粘附分子等），因此钥匙细胞自分泌和旁分泌信号通路的快速调节 在肿瘤微环境中。在致癌途径中暗示的特定亚当包括 ADAM8/9/9/10/12/15/17/19/28，在ADAM17中存在强烈的恶性作用证据 以及其最接近的相对ADAM10。尽管过去我们对ADAM10/17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通过的机制出现了许多问题 它们有助于肿瘤发生/进展。底物识别和歧视对棚的光很重要 关于潜在的机制，但很少努力集中在亚当斯的基材上的识别，部分部分是 由于它们本地化的位置 - 细胞膜。在这里，我们建议开发一种高通量方法 使用噬菌体显示和大量平行DNA，全面剖面ADAM10/17的蛋白酶底物 下一代测序（NGS）。通过构建两个显示随机10-Mer的噬菌体库 涵盖整个人类蛋白质组的肽或49-mer人类蛋白段，我们将能够比较 肽与本地人类蛋白质的蛋白水解规格，对折叠和域的折叠方式有见解 结构决定了裂解位点的可及性。使用这种方法，我们打算定义乳沟 ADAM10/17蛋白酶的位点选择性。除了用固体形式进行体外筛查外 ADAM10/17，由于位置，方向和构象约束而可能存在偏见 它们作为ecto-proteases的作用，我们将通过在噬菌体上进行工程上的肽来执行在细胞中选择 要将其插入细胞膜，模仿了膜底物的景观。通过识别和 表征ADAM10/17底物的功能重要性，我们希望公布更多信息 涉及ADAM10/17在癌症发展中的作用，并验证了药物的假设 针对亚当人将很有用。最后，我们将开发重组抗体有选择地靶向活性 ADAM10/17的形式希望抑制涉及致癌信号的ADAM。从长远来看，这项技术 对于研究各种其他翻译后修饰（PTM）的序列特异性应该有用， 包括磷酸化，柠檬酸和硫化。

项目成果

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: Nature chemical biology
• 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

Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike.

• DOI: 10.1101/2020.08.08.242511
• 发表时间: 2020-08-10
• 期刊: bioRxiv : the preprint server for biology
• 作者: Bracken, Colton J;Lim, Shion A;Wells, James A
• 通讯作者: Wells, James A

Targeting Phosphotyrosine in Native Proteins with Conditional, Bispecific Antibody Traps.

• DOI: 10.1021/jacs.0c08458
• 发表时间: 2020-10-14
• 期刊: Journal of the American Chemical Society
• 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

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: The Journal of clinical investigation
• 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。

• DOI: 10.1073/pnas.2009279117
• 发表时间: 2020
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Zhou,Jie;Li,Shantao;Leung,KevinK;O'Donovan,Brian;Zou,JamesY;DeRisi,JosephL;Wells,JamesA
• 通讯作者: Wells,JamesA