Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins
Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins
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DOI：
10.1016/j.ceca.2021.102404
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发表时间：
2021-04-05
期刊：
影响因子：
4
通讯作者：
Tokumitsu, Hiroshi
中科院分区：
文献类型：
--
作者：
Doi, Seita;Fujioka, Naoki;Tokumitsu, Hiroshi
为了阐明S100蛋白介导的信号通路，我们试图通过筛选携带19，676个重组谷胱甘肽S-转移酶（GST）融合的人蛋白与生物素化S100 A2的蛋白阵列来鉴定S100 A2的新结合伴侣。在新发现的假定的S100 A2相互作用物中，包括TMLHE、TRH、RPL 36、MRPS 34、CDR 2L、OIP 5和MED 29，我们鉴定并表征了微管蛋白聚合促进蛋白（TPPP）作为新的S100 A2结合蛋白。我们通过多种独立的方法证实了TPPP与Ca 2 +/S100 A2的相互作用，包括蛋白质阵列法，S100 A2覆盖法，体外和转染COS-7细胞的pulldown实验。基于使用各种GST-TPPP突变体的S100 A2重叠测定的结果，在参与TPPP二聚化的单体形式的TPPP的中心核心结构域的C末端（残基111-160）中鉴定了S100 A2结合区域。化学交联实验表明，S100 A2以剂量依赖性和Ca 2+依赖性方式抑制His标签TPPP的二聚体形成。除了S100 A2，TPPP二聚化被其他多个S100蛋白，包括S100 A6和S100 B，以Ca 2+依赖性方式破坏，但不被S100 A4破坏。这与S100 A6和S100 B而不是S100 A4能够在Ca 2+存在下与GST-TPPP相互作用的事实一致。综合考虑这些结果，TPPP被鉴定为S100 A2的新靶点，并且它是包括S100 A6和S100 B在内的其他多种S100蛋白的潜在结合靶点。S100蛋白与TPPP的直接结合可能导致TPPP二聚体的形成响应于细胞内Ca 2+浓度的增加而解体，从而导致TPPP的生理功能的调节，例如微管组织。
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 dosedependent 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.