Protein Painting reveals hidden protein-protein interaction domains

描述（申请人提供）：我们提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 相互作用域。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分子事件的基础。目前还不存在直接分离和测序未知相互作用结构域或直接检测已知蛋白质-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的技术。跟踪相互作用蛋白质之间或错误折叠蛋白质内的界面结构域是下一代阻断驱动癌症的分子相互作用的疗法的基础。我们将创建蛋白质涂料化学，一种新型的合成有机小分子面板，以高亲和力结合蛋白质分子，并掩盖蛋白质的所有蛋白酶切割位点。我们将使用我们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-蛋白质相互作用结构域。溶液中相互作用的天然蛋白质被描绘成 用颜料分子的调色板覆盖蛋白质的暴露表面，但不能进入内部蛋白质-蛋白质接触域。因此，如果两个天然蛋白质结合在一起，界面结构域将保持非着色。蛋白质涂料涂层具有高分辨率（<3个氨基酸）。在绘画之后，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不可接近的非绘画相互作用结构域。即使在解离后，着色区域也被蛋白酶切割或抗体识别掩蔽。然后可以对解离的着色蛋白进行蛋白酶切割（例如胰蛋白酶）和ms测序或抗体探测。由于涂料阻断不接触的蛋白酶切割位点，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涂料区域产生。本发明的目的在于我们发现了通过磺化蒽有机染料分子“涂料”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 -磺酸二钠掩蔽胰蛋白酶切割位点（精氨酸和赖氨酸的羧基侧），其实现了高度的胰蛋白酶切割位点覆盖。我们通过直接对白细胞介素-1结合其受体的隐藏相互作用域进行质谱测序，验证了蛋白质绘画能力。在过去，这个畴只能用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中广泛而关键的未满足需求。我们设想，该技术可以在未来用于破译来自肿瘤活检样本或用配体或疗法处理的细胞培养物的相互作用域。抗体结合测定可用于定量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，并将涂料应用于3种癌症相关的蛋白质-蛋白质相互作用模型：1）受体-配体，2）磷酸化介导的缔合，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.