Division of Oncology Research, Department of Oncology, Mayo Clinic, Rochester, MN 55905, USA.
Biochem Biophys Res Commun. 2011 Oct 28;414(3):581-6. doi: 10.1016/j.bbrc.2011.09.125. Epub 2011 Oct 1.
The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric μ- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of μ- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)-PIN1 fusion protein. Adding GST-PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains the ability of calpastatin to inhibit calpain, maintaining calpain activity in endothelial cells. PIN1 may act directly via phosphorylated serine/threonine-proline motifs in calpastatin, or indirectly via other PIN1 substrates that control calpastatin.
肽基脯氨酰顺反异构酶，蛋白永不在有丝分裂基因 A 相互作用-1（PIN1）结合并异构化丝氨酸/苏氨酸磷酸化的蛋白质，然后是脯氨酸。先前发现，在鼠主动脉内皮细胞（MAEC）中，通过降低钙蛋白酶活性，耗尽 PIN1 可大大增加环氧化酶-2 和诱导型一氧化氮合酶的诱导。在这里，我们研究了 PIN1 对异二聚体μ和 m-钙蛋白酶内源性抑制剂钙蛋白酶抑制剂的影响。MAEC 用短发夹（sh）RNA 转导以敲低 PIN1（KD）或无活性对照 shRNA。细胞也用非靶向双链小干扰 RNA（siRNA）或设计耗尽钙蛋白酶抑制剂的 siRNA 处理。尽管降低钙蛋白酶活性，但与对照 shRNA 相比，PIN1 KD 对 μ 和 m-钙蛋白酶或钙蛋白酶抑制剂的表达没有显著影响。相反，耗尽 PIN1 增加了钙蛋白酶抑制剂的抑制活性。钙蛋白酶抑制剂与内源性 PIN1 共免疫沉淀，并与谷胱甘肽-S-转移酶（GST）-PIN1 融合蛋白拉下。将 GST-PIN1 添加到缺乏 PIN1 的 KD 细胞提取物中降低了钙蛋白酶抑制剂的活性。PIN1 的底物结合和催化结构域突变体不能做到这一点。这些结果表明，蛋白质相互作用和 PIN1 的脯氨酸异构酶功能是其抑制钙蛋白酶抑制剂所必需的。此外，钙蛋白酶抑制剂耗尽使钙蛋白酶活性升高，并使 KD 和对照 MAEC 中的钙蛋白酶抑制活性降低到相似水平，表明钙蛋白酶抑制剂是 PIN1 耗尽降低钙蛋白酶活性所必需的。因此，PIN1 显然限制了钙蛋白酶抑制剂抑制钙蛋白酶的能力，从而维持内皮细胞中的钙蛋白酶活性。PIN1 可能通过钙蛋白酶抑制剂中的磷酸化丝氨酸/苏氨酸-脯氨酸基序直接作用，或通过其他控制钙蛋白酶抑制剂的间接作用的 PIN1 底物作用。
