Protein Painting reveals hidden protein-protein interaction domains

描述(申请人提供)：我们建议创造一种全新的技术“蛋白质涂抹”，用于快速、直接地分离和测序隐藏的天然蛋白质 相互作用域。蛋白质-蛋白质相互作用是驱动癌细胞信号和基因调控的几乎所有功能分子事件的基础。目前还没有直接分离和测序未知相互作用结构域的技术，也没有直接检测已知的蛋白质结合结构域是否与天然蛋白质的细胞或组织样本接触的技术。追踪相互作用的蛋白质之间或错误折叠的蛋白质之间的界面结构域，是下一代阻断导致癌症的分子相互作用的治疗方法的基础。我们将创建蛋白质涂料化学，这是一种新型的合成有机小分子面板，以高亲和力结合到蛋白质分子上，并掩盖蛋白质的所有蛋白酶裂解位点。我们将使用我们新的蛋白质绘制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。绘制了溶液中相互作用的天然蛋白质 具有覆盖在暴露的蛋白质表面的油漆分子的调色板，但不能接触到内部的蛋白质-蛋白质接触域。因此，如果两个天然蛋白质结合在一起，界面结构域将保持不被绘制。该蛋白质涂料涂层具有高分辨率(&lt；3个氨基酸)。在涂装之后，相互作用的蛋白质被解离，以揭示和暴露当蛋白质在其自然状态下相互作用时，涂料分子无法接触到的非涂装相互作用域。即使在解离之后，绘制的区域也被遮盖，使其不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(例如胰酶)和MS测序，或抗体探测。由于油漆阻止了没有接触的蛋白酶裂解位点，所以用于MS测序的蛋白酶片段将仅从仅包括相互作用结构域的非油漆区域产生。这些目标是在我们发现磺化蒽类有机染料分子“涂料”掩盖胰酶裂解位点(精氨酸和赖氨酸的羧基)之后进行的，例如1-氨基-9，10-二氧代-4-[3-(2-磺酰氧乙基磺酰基)苯基]菲-2-磺酸盐，它们实现了高度的胰酶裂解位点覆盖率。我们通过直接对白介素1与其受体结合的隐藏的相互作用区域进行MS测序来验证蛋白质的绘制能力。在过去，这一领域只能通过X射线结晶学来预测。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得到满足的需求。我们设想，这项技术在未来可以用于破译肿瘤活检样本或用配体或疗法处理的细胞培养的相互作用域。抗体结合分析可以用来量化细胞裂解物中占据的结合部位的数量/类型。我们将创建一个由12个蛋白质涂料组成的小组，并将这些涂料应用于3个与癌症相关的蛋白质-蛋白质相互作用模型：1)受体-配体，2)磷酸化介导的结合，以及3)转录因子复合体。

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.