Protein Painting reveals hidden protein-protein interaction domains

描述（由申请人提供）：我们建议创建一种全新的技术“蛋白质绘画”，用于快速、直接分离和测序隐藏的天然蛋白质-蛋白质 交互域。蛋白质-蛋白质相互作用是几乎所有驱动癌细胞信号传导和基因调控的功能性分子事件的基础。目前还没有技术可以直接分离和测序未知的相互作用结构域，或者直接检测已知的蛋白质-蛋白质结合结构域是否在天然蛋白质的细胞或组织样品中接触。追踪相互作用蛋白质之间或错误折叠蛋白质内的界面域，是阻止导致癌症的分子相互作用的下一代疗法的基础。我们将创建蛋白质涂料化学物质，这是一组新型的合成有机小分子，它以高亲和力与蛋白质分子结合，并掩盖蛋白质的所有蛋白酶切割位点。我们将使用新的蛋白质绘画化学来分离和测序癌症相关蛋白质的蛋白质-蛋白质相互作用域。溶液中相互作用的天然蛋白质被涂漆 用一系列油漆分子覆盖蛋白质的暴露表面，但无法进入内部蛋白质-蛋白质接触域。因此，如果两个天然蛋白质结合在一起，界面域将保持未绘制状态。蛋白质涂料具有高分辨率（<3 个氨基酸）。涂漆后，相互作用的蛋白质被解离，以揭示和暴露当蛋白质在其天然状态下相互作用时油漆分子无法接近的未涂漆的相互作用域。即使在解离后，绘制的区域也不会被蛋白酶切割或抗体识别。然后可以对解离的绘制蛋白进行蛋白酶切割（例如胰蛋白酶）和 MS 测序或抗体探测。由于油漆阻挡了不接触的蛋白酶切割位点，因此用于 ms 测序的蛋白酶片段将仅从仅包含相互作用结构域的非油漆区域产生。我们的目标是通过磺化蒽有机染料分子“涂料”（例如 1-氨基-9,10-二氧代-4-[3-（2-磺酰氧基乙基磺酰基）苯胺基]蒽-2-磺酸二钠）来掩盖胰蛋白酶裂解位点（精氨酸和赖氨酸的羧基侧），从而实现高度的胰蛋白酶裂解位点覆盖范围。我们通过直接 MS 测序验证了白细胞介素 1 与其受体结合的隐藏相互作用域的蛋白质绘制能力。过去，这个区域只能通过 X 射线晶体学来预测。我们的变革性技术解决了癌症生物学和癌症治疗领域广泛且关键的未满足需求。我们设想该技术将来可用于破译肿瘤活检样本或用配体或疗法处理的细胞培养物中的相互作用域。抗体结合测定可用于量化细胞裂解物中占据的结合位点的数量/类型。我们将创建一组 12 种蛋白质涂料，并将这些涂料应用于 3 个癌症相关蛋白质-蛋白质相互作用模型：1) 受体-配体，2) 磷酸化介导的关联，以及 3) 转录因子复合物。

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.