Protein Painting reveals hidden protein-protein interaction domains

蛋白质绘画揭示了隐藏的蛋白质-蛋白质相互作用域

基本信息

批准号：
8728792
负责人：
Lance Allen Liotta
金额：
$ 23.73万
依托单位：
GEORGE MASON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8728792
关键词：
Address Affinity Affinity Chromatography Alkanesulfonates Alkylation Amino Acids Anthracenes Antibodies Antibody Specificity Area Automobile Driving Binding Binding Sites Biological Assay Biopsy Specimen Cancer Biology Cancer Model Cell Culture Techniques Cells Chemicals Chemistry Complex Cytoplasmic Protein Data Development Digestion Dissociation Drug Targeting Dyes Event Future Gene Expression Regulation Genetic Individual Interleukin-1 Ligands Malignant Neoplasms Masks Mass Spectrum Analysis Mediating Membrane Proteins Methods Molecular Nuclear Proteins Paint Peptide Hydrolases Phosphorylation Population Protein Binding Protein Binding Domain Protein Microchips Protein Region Proteins Proteomics Resolution Side Signal Transduction Site Solutions Technology Therapeutic Time Tissue Sample Transcription Factor 3 Trypsin Western Blotting X-Ray Crystallography X-Ray Tomography arginyllysine base cancer cell chymotrypsin crosslink innovation member new technology next generation novel protein folding protein misfolding protein protein interaction public health relevance receptor screening small molecule tumor yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): We propose the creation of a wholly novel technology "protein painting", for the rapid, direct isolation and sequencing of hidden native protein-protein interaction domains. Protein-protein interactions are the basis of virtually all functional moleculr events driving cancer cell signaling and gene regulation. Currently no technology exists to directly isolate and sequence unknown interaction domains, or to directly detect whether known protein-protein binding domains are in contact in a cellular or tissue sample of native proteins. Tracking the interface domains between interacting proteins, or within misfolded proteins, is the basis for the next generation of therapies that block the molecular interactions driving cancer. We will create protein paint chemistries, a novel panel of synthetic organic small molecules that bind to protein molecules with high affinity and mask all the protease cleavage sites of the protein. We will use our new protein painting chemistry to isolate and sequence protein-protein interaction domains of cancer related proteins. Interacting native proteins in solution are painted with a palette of paint molecules that coat the exposed surfaces of the proteins but do not have access to the internal protein-protein contact domains. Thus if two native proteins are bound together, the interface domains will remain non-painted. The protein paints coat with a high resolution (<3 amino acids). Following painting, the interacting proteins are dissociated, to reveal and expose the non-painted interaction domains that were inaccessible to the paint molecules when the proteins were interacting in their native state. Painted regions are masked from proteinase cleavage or antibody recognition, even after dissociation. The dissociated painted proteins can then be subjected to proteinase cleavage (e.g. trypsin) and ms sequencing, or antibody probing. Since the paint blocks the proteinase cleavage sites that are not in contact, proteinase fragments for ms sequencing will only be generated from the non-painted areas exclusively comprising the interaction domains. The Aims follow our discovery of the masking of trypsin cleavage sites (carboxyl side of arginine & lysine) by sulfonated anthracene organic dye molecule "paints" such as disodium 1-amino-9,10- dioxo-4- [3- (2- sulfonatooxyethylsulfonyl) anilino] anthracene -2 -sulfonate, which achieve a high degree of trypsin cleavage site coverage. We verified protein painting capabilities by direct ms sequencing of the hidden interaction domain of interleukin-1¿ bound to its receptor. In the past this domain could only be predicted by X- Ray crystallography. Our transformative technology addresses a broad and critical unmet need in cancer biology and cancer therapeutics. We envision that the technology can be used in the future to decipher interaction domains from tumor biopsy samples or cell cultures treated with a ligand or a therapy. Antibody binding assays can be used to quantify the number/type of occupied binding sites in a cell lysate. We will create a panel of 12 protein paints, and apply the paints to 3 model cancer related protein-protein interactions:1) receptor-ligand, 2) phosphorylation mediated association, and 3) transcription factor complexes.

描述（通过应用程序提供）：我们提出了一种全新的技术“蛋白绘画”的创建，用于快速，直接隔离和测序隐藏的本机蛋白质 - 蛋白质 - 交互域。蛋白质 - 蛋白质相互作用几乎是驱动癌细胞信号传导和基因调节的所有功能分子事件的基础。当前，尚无技术直接分离和序列未知的相互作用域，或直接检测到天然蛋白质的细胞或组织样品中是否接触已知的蛋白质蛋白结合结构域。跟踪相互作用蛋白之间的界面结构域，或在错误折叠的蛋白质中，是阻止驱动癌症的分子相互作用的下一代疗法的基础。我们将创建蛋白质油漆化学，这是一个新型的合成有机小分子，它们与具有高亲和力的蛋白质分子结合，并掩盖了蛋白质的所有蛋白质切割位点。我们将使用我们的新蛋白绘画化学来隔离癌症相关蛋白的蛋白质蛋白质相互作用结构域。在溶液中的相互作用的本机蛋白被绘制带有涂层蛋白质表面但无法进入内部蛋白质 - 蛋白质接触结构域的涂料分子的调色板。如果两个天然蛋白结合在一起，则界面域将保持未绘制。该蛋白质以高分辨率（<3氨基酸）涂上涂层。绘画后，相互作用的蛋白被解离，以揭示和揭示当蛋白在本地状态相互作用时油漆分子无法接近的未涂色相互作用域。即使分离后，涂漆的区域也会从蛋白酶裂解或抗体识别中掩盖。然后可以将分解的涂漆蛋白进行蛋白酶裂解（例如胰蛋白酶）和MS测序或抗体探测。由于油漆阻止了未接触的蛋白酶裂解位点，因此仅从非涂漆区域才能从专门完成相互作用域的非绘画区域生成用于MS测序的蛋白酶片段。 The Aims Follow our discovery of the masking of trypsin cleavage sites (carboxyl side of arginine & lysine) by sulfonated anthracene organic dye molecule "paints" such as disodium 1-amino-9,10-dioxo-4- [3- (2- sulfonatooxyethylsulfonyl) anilino] anthracene -2 -sulfonate, which achieves高度的胰蛋白酶切割部位覆盖范围。我们通过直接的MS测序来验证蛋白质绘画能力，该蛋白质测序是白介素1的隐藏相互作用域与其接收器结合。过去，这个域只能通过X射线晶体学预测。我们的变革性技术解决了癌症生物学和癌症治疗方面的广泛而关键的未满足需求。我们设想，将来可以将该技术用于从肿瘤活检样品或用配体或治疗治疗的细胞培养物中解解相互作用结构域。抗体结合测定可用于量化细胞裂解物中占据结合位点的数量/类型。我们将创建一个由12种蛋白质油漆的面板，并将这些油漆应用于3型模型癌症相关的蛋白质 - 蛋白质相互作用：1）受体配体，2）磷酸化介导的关联和3）转录因子复合物。