To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca²⁺/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111–160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca²⁺-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca²⁺-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca²⁺. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca²⁺, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

为了阐明 S100 蛋白质介导的信号通路，我们试图通过筛选携带 19,676 个重组谷胱甘肽 S 转移酶 (GST) 融合人类蛋白质与生物素化 S100A2 的蛋白质阵列来鉴定 S100A2 的新型结合配偶体。在新发现的假定 S100A2 相互作用体中，包括 TMLHE、TRH、RPL36、MRPS34、CDR2L、OIP5 和 MED29，我们将微管蛋白聚合促进蛋白 (TPPP) 鉴定并表征为一种新型 S100A2 结合蛋白。我们通过多种独立的方法证实了 TPPP 与 Ca ²⁺ /S100A2的相互作用，包括蛋白质阵列法、S100A2 叠加和体外下拉测定在转染的 COS-7 细胞中。根据使用各种 GST-TPPP 突变体的 S100A2 重叠测定的结果，在涉及 TPPP 的单体形式的 TPPP 的中央核心域的 C 末端（残基 111-160）中鉴定了 S100A2 结合区二聚化。化学交联实验表明，S100A2 以剂量依赖性和 Ca ²⁺依赖性方式抑制带有 His 标签的 TPPP 的二聚体形成。除了 S100A2，TPPP 二聚化被其他多种 S100 蛋白（包括 S100A6 和 S100B）以 Ca ²⁺依赖性方式破坏，但不被 S100A4破坏。这与 S100A6 和 S100B 而不是 S100A4 在 Ca ²⁺存在下能够与 GST-TPPP 相互作用的事实一致. 综合考虑这些结果，TPPP 被确定为 S100A2 的新靶点，并且它是其他多种 S100 蛋白（包括 S100A6 和 S100B）的潜在结合靶点。S100 蛋白与 TPPP 的直接结合可能会导致 TPPP 二聚体的解组装，以响应细胞内 Ca ²⁺浓度的增加，从而导致调节 TPPP 的生理功能，如微管组织。