CAPN1 (Calpain1) 依赖性 STIM1（基质相互作用分子 1）裂解导致人新生儿血小板 SOCE（钙库操纵的钙进入）增强,Arteriosclerosis, Thrombosis, and Vascular Biology

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CAPN1 (Calpain1) 依赖性 STIM1（基质相互作用分子 1）裂解导致人新生儿血小板 SOCE（钙库操纵的钙进入）增强
Arteriosclerosis, Thrombosis, and Vascular Biology ( IF 7.4 ) Pub Date : 2023-03-16 , DOI: 10.1161/atvbaha.122.318015
Alejandro Berna-Erro ₁ , Girish Ramesh ₂ , Elena Delgado ₃ , Antonio J Corbacho ₃ , Francisca Ferrer-Marín ₄ , Raul Teruel ₄ , María P Granados ₅ , Juan A Rosado ₁ , Pedro C Redondo ₁

Affiliation

背景：细胞内钙改变2+新生儿血小板的稳态先前已有报道。本研究旨在探讨 Ca 的变化2+通过新生儿血小板中的钙库操纵钙进入（SOCE）机制进入。方法：分离来自对照妇女、母亲和新生儿的人类血小板，并根据需要进行处理后进行固定。通过蛋白质印迹、qRT-PCR 和 MALDITOF/TOF 分析血小板样本。钙2+还确定了体内平衡。培养细胞用作血小板的替代物，以过表达感兴趣的蛋白质，以重现在血小板中观察到的变化。结果：改变的 TG（毒胡萝卜素）诱发的 SOCE、STIM1 的替代分子量形式（基质相互作用分子 1；s-STIM1 [短 STIM1 亚型（478 个氨基酸）]，约 60 kDa）和 SARAF（SOCE 相关调节因子）的过表达与母体和对照女性血小板相比，在新生儿血小板中发现了这一点。s-STIM1 可能是由于 CAPN1 (calpain1) 依赖性加工导致的，这一点分别通过使用 calpeptin 和过表达 CAPN1 在血小板和 MEG01 细胞中得到证实。在用 c-STIM1（经典 STIM1 [685 个氨基酸]）、s-STIM1 (478)、STIM1B (540) 和 CAPN1 过表达质粒转染的 HEK293（STIM1 和 STIM2 [基质相互作用分子 2] 双敲除）细胞中，我们发现s-STIM1 和 c-STIM1，但过表达 s-STIM1 (478) 且缺少 CAPN1 靶残基的细胞除外。这些结果和计算机分析使我们得出结论，STIM1 在 Q496 处被 CAPN1 切割。钙2+使用过表达 SARAF 和 s-STIM1 (478) 的 MEG01 和 HEK293 细胞进行成像分析和共免疫沉淀测定，报告慢 Ca 减少2+–依赖性失活，因此复制 Ca2+-在新生儿血小板中观察到的稳态模式。结论：CAPN1 可能会裂解新生儿血小板中的 STIM1，从而在 SOCE 激活后损害 SARAF 偶联。s-STIM1 可以避免慢 Ca2+– 依赖性失活，随后导致 TG 诱发的 SOCE 增强，如在新生儿血小板中观察到的那样。

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CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets

Background: Altered intracellular Ca 2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca 2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. Methods: Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca 2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. Results: Altered TG (thapsigargin)-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor) were found in neonatal platelets as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca 2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca 2+ –dependent inactivation, so reproducing the Ca 2+ -homeostasis pattern observed in neonatal platelets. Conclusions: CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. s-STIM1 may avoid slow Ca 2+ –dependent inactivation and, subsequently, results in an enhanced TG-evoked SOCE as observed in neonatal platelets.

更新日期：2023-03-16

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