Kamath Nisha D, Matreyek Kenneth A
Department of Pathology, Case Western Reserve University School of Medicine, Case Western Reserve University, Cleveland, Ohio, 44106, USA.
Genetics. 2025 Jul 30. doi: 10.1093/genetics/iyaf146.
Calcium signaling is a fundamental molecular means of cellular regulation. Store operated calcium entry (SOCE) is a major intracellular signaling module, wherein calcium release from the endoplasmic reticulum triggers transmembrane STIM1 proteins to conformationally shift and oligomerize to prompt calcium influx from the extracellular environment. STIM1 senses ER calcium concentrations with its canonical EF-hand domain, and missense variants can dysregulate SOCE and cause Tubular Aggregate Myopathy, Stormorken Syndrome or immunodeficiency. Few STIM1 EF-hand variants are characterized, obscuring how STIM1 sequence controls its function, and hampering clinical interpretation of STIM1 variants observed in patients. We leveraged fitness costs caused by overexpression of STIM1 variants in cultured human cells to functionally characterize 706 of the 720 possible single amino acid variants of the STIM1 canonical EF-hand. The calcium-coordinating EF-hand residues exhibited varying mutational patterns. The trailing helix possessed a core of immutable residues, even depleting during library propagation in bacteria, implicating residues normally restraining STIM1 aggregation. The leading helix only exhibited toxicity in cells with endogenous STIM1, implicating a multimerization-dependent STIM1 regulatory module. No cytotoxic STIM1 variants were observed in healthy human populations. Some disease-associated variants had low scores, but most pathogenic variants were not overtly cytotoxic in our assay. We demonstrate that orthogonal measurements for STIM1 oligomerization, cytoplasmic calcium influx, and cellular stress complement the cytotoxicity phenotypes to enhance variant understanding. Collectively, these data reveal the complex molecular roles embedded in the STIM1 canonical EF-hand sequence for its function in promoting calcium signaling through SOCE.
钙信号传导是细胞调节的一种基本分子方式。储存操纵性钙内流（SOCE）是一个主要的细胞内信号传导模块，其中内质网释放的钙触发跨膜的STIM1蛋白发生构象转变并寡聚化，从而促使细胞外环境中的钙内流。STIM1通过其典型的EF手结构域感知内质网钙浓度，错义变体可使SOCE失调并导致管状聚集性肌病、斯托尔莫肯综合征或免疫缺陷。很少有STIM1 EF手变体得到表征，这使得STIM1序列如何控制其功能变得模糊不清，并妨碍了对患者中观察到的STIM1变体的临床解释。我们利用培养的人类细胞中STIM1变体过表达所导致的适应性成本，对STIM1典型EF手中720个可能的单氨基酸变体中的706个进行了功能表征。钙配位的EF手残基表现出不同的突变模式。尾随螺旋拥有一个不变残基的核心，甚至在细菌文库传代过程中会减少，这表明这些残基通常会抑制STIM1聚集。领先螺旋仅在具有内源性STIM1的细胞中表现出毒性，这表明存在一个依赖多聚化的STIM1调节模块。在健康人群中未观察到细胞毒性STIM1变体。一些与疾病相关的变体得分较低，但在我们的检测中，大多数致病变体并没有明显的细胞毒性。我们证明，对STIM1寡聚化、细胞质钙内流和细胞应激的正交测量补充了细胞毒性表型，以增强对变体的理解。总的来说，这些数据揭示了STIM1典型EF手序列中所蕴含的复杂分子作用，其在通过SOCE促进钙信号传导中发挥功能。
