CAPN1（钙蛋白酶 1）依赖性切割 STIM1（基质相互作用分子 1）导致人新生儿血小板中增强的 SOCE（储存操作钙进入）。

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

机构信息

Department of Physiology (PHYCELL group), University of Extremadura, Caceres, Spain (A.B.E., J.A.R., P.C.R.).

Institute of Molecular Biophysics, CIPMM, Saarland University, Homburg/Saar, Germany (G.R.).

出版信息

Arterioscler Thromb Vasc Biol. 2023 May;43(5):e151-e170. doi: 10.1161/ATVBAHA.122.318015. Epub 2023 Mar 16.

DOI:10.1161/ATVBAHA.122.318015

PMID:36924231

Abstract

BACKGROUND

Altered intracellular Ca homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca 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 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 imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca-dependent inactivation, so reproducing the Ca-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-dependent inactivation and, subsequently, results in an enhanced TG-evoked SOCE as observed in neonatal platelets.

摘要

背景

先前有报道称，新生儿血小板细胞内钙稳态发生改变。本研究旨在研究通过储存操作钙内流（SOCE）机制的钙内流变化在新生儿血小板中的作用。

方法

从对照女性、产妇和新生儿中分离人血小板，并在需要时进行处理后固定。通过 Western blot、qRT-PCR 和 MALDI-TOF/TOF 分析血小板样本。还测定了钙稳态。培养细胞被用作血小板的替代物，以过表达感兴趣的蛋白质，以再现在血小板中观察到的改变。

结果

与产妇和对照女性血小板相比，新生儿血小板中发现 TG（他普西胍）诱导的 SOCE、STIM1 的替代分子质量形式（基质相互作用分子 1；s-STIM1[短 STIM1 异构体（478 aa）]，约 60 kDa）和 SARAF（SOCE 相关调节因子）过表达。s-STIM1 可能是由于 CAPN1（钙蛋白酶 1）依赖性加工所致，这在血小板和 MEG01 细胞中分别使用 calpeptin 和过表达 CAPN1 得到证实。在转染了 c-STIM1（经典 STIM1[685 aa]）、s-STIM1（478）、STIM1B（540）和 CAPN1 过表达质粒的 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，除了过表达 s-STIM1（478）的细胞缺乏 CAPN1 靶位残基外。这些结果和计算机分析结果表明，CAPN1 可在 Q496 位点切割 STIM1。使用 MEG01 和 HEK293 细胞进行钙成像分析和免疫共沉淀分析，这些细胞过表达 SARAF 以及 s-STIM1（478），报告了慢 Ca 依赖性失活减少，从而再现了在新生儿血小板中观察到的 Ca 稳态模式。