Oxidative Stress-Induced STIM2 Cysteine Modiﬁcations Suppress Store-Operated Calcium Entry

SUMMARY Store-operated calcium entry (SOCE) through STIM-gated ORAI channels governs vital cellular functions. In this context, SOCE controls cellular redox signaling and is itself regulated by redox modiﬁcations. However, the molecular mechanisms underlying this calcium-redox interplay and the functional outcomes are not fully understood. Here, we examine the role of STIM2 in SOCE redox regulation. Redox proteomics identify cysteine 313 as the main redox sensor of STIM2 in vitro and in vivo . Oxidative stress sup-presses SOCE and calcium currents in cells overexpressing STIM2 and ORAI1, an effect that is abolished by mutation of cysteine 313. FLIM and FRET microscopy, together with MD simulations, indicate that oxidative modiﬁcations of cysteine 313 alter STIM2 activation dynamics and thereby hinder STIM2-medi-ated gating of ORAI1. In summary, this study establishes STIM2-controlled redox regulation of SOCE as a mechanism that affects several calcium-regulated physiological processes, as well as stress-induced pathologies. (2-Amino-2-(hydrox-ymethyl)-1,3-propanediol hydrochloride) pH 8.5, 0.5% ( w / v ) SDS (sodium dodecyl sulfate)), including 20% ( v / v ) acetonitrile in presence of light-labeled ( 12 C) ICAT (isotope-coded afﬁnity tag). Samples were precipitated and washed 80% ( v / v acetone as Proteins were fully reduced with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) for 10 min at 37 and labeled with heavy-labeled ( 13 C) ICAT following the same precipitation and washing steps. Samples were denatured using denaturing buffer (50 mM Tris-HCl, 0.1% SDS ( w / v )) and digested with trypsin (0.125 m g/ m L). Tryptic peptides were puriﬁed using cation exchange and avidin afﬁnity chromatography according to the manufacturer’s instructions. Cleavage of the biotin-tag was performed and resulting peptides were concentrated to dryness and dissolved in 0.1% ( v / v ) triﬂuoroacetic acid for LC-MS/MS analysis. Measurements were performed with a LTQ Orbitrap Elite instrument (Thermo Fisher Scientiﬁc GmbH, Schwerte, Germany) as described accessibilities of the two individual ORAI binding sites calculated for all 20 STIM1 CC1 [TM-distal] -CC2 NMR structures in ORAI bound and free conformation et al., 2013), respectively, and for the model STIM2 CC1 [TM-distal] -CC2 structures obtained in the ﬁnal 200 ns of the simulations. The binding sites in STIM1 were deﬁned based the residues interacting equivalent HA-hSTIM2- D K-EGFP and HA-hSTIM2- D K-mCherry plasmids: SpS1-HA-hSTIM2-EGFP and spS1-HA-hSTIM2-mCherry plasmids were double-digested with EcoR I-HF and SalI-HF restriction enzymes. The resulting vector backbones missing fragments encoding for hSTIM2 residues 94-746 were gel puriﬁed. YFP-hSTIM2- D K was mutagenized 5 TGG AGA GTC 5 -TTT GGC reverse primers to create a SalI restriction site after the codon of E729 residue. The mutagenized plasmid was digested with EcoR I-HF and SalI-HF restriction enzymes and an insert encoding for hSTIM2 residues 94-729 was puriﬁed. This insert was ligated into the above generated vectors. This resulted in HA-hSTIM2- D K-EGFP and HA-hSTIM2- D K-mCherry plasmids lacking last 15 residues (K-rich domain) of hSTIM2. Cysteine to alanine mutations were introduced by ampliﬁcation of STIM2 fragments with pairs of primers