Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

Department of Physiology Anatomy and Genetics University of Oxford Oxford OX1 3PT UK

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague 166 10 Czech Republic

LumiSTAR Biotechnology Inc National Biotechnology Research Park Taipei City 115 Taiwan

Sir William Dunn School of Pathology University of Oxford Oxford OX1 3RE UK

Komentář v

Mol Cell. 2021 Dec 2;81(23):4763-4765. doi: 10.1016/j.molcel.2021.11.013. PubMed

Zobrazit více v PubMed

Adrain C., Strisovsky K., Zettl M., Hu L., Lemberg M.K., Freeman M. Mammalian EGF receptor activation by the rhomboid protease RHBDL2. EMBO Rep. 2011;12:421–427. PubMed PMC

Akiyama Y., Maegawa S. Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease. Mol. Microbiol. 2007;64:1028–1037. PubMed

Baker R.P., Urban S. Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature. 2015;523:101–105. PubMed PMC

Bakowski D., Parekh A.B. Voltage-dependent Ba2+ permeation through store-operated CRAC channels: implications for channel selectivity. Cell Calcium. 2007;42:333–339. PubMed

Bassett A.R., Tibbit C., Ponting C.P., Liu J.L. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 2013;4:220–228. PubMed PMC

Battistini C., Rehman M., Avolio M., Arduin A., Valdembri D., Serini G., Tamagnone L. Rhomboid-like-2 intramembrane protease mediates metalloprotease-independent regulation of cadherins. Int. J. Mol. Sci. 2019;20:5958. PubMed PMC

Began J., Cordier B., Březinová J., Delisle J., Hexnerová R., Srb P., Rampírová P., Kožíšek M., Baudet M., Couté Y., et al. Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease. EMBO J. 2020;39:e102935. PubMed PMC

Berridge M.J., Lipp P., Bootman M.D. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 2000;1:11–21. PubMed

Brandman O., Liou J., Park W.S., Meyer T. STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Cell. 2007;131:1327–1339. PubMed PMC

Breuning J., Brown M.H. T cell costimulation by CD6 is dependent on bivalent binding of a GADS/SLP-76 complex. Mol. Cell. Biol. 2017;37 e00071-17. PubMed PMC

Burdakov D., Verkhratsky A. Biophysical re-equilibration of Ca2+ fluxes as a simple biologically plausible explanation for complex intracellular Ca2+ release patterns. FEBS Lett. 2006;580:463–468. PubMed

Cai X., Zhou Y., Nwokonko R.M., Loktionova N.A., Wang X., Xin P., Trebak M., Wang Y., Gill D.L. The Orai1 store-operated calcium channel functions as a hexamer. J. Biol. Chem. 2016;291:25764–25775. PubMed PMC

Doench J.G., Hartenian E., Graham D.B., Tothova Z., Hegde M., Smith I., Sullender M., Ebert B.L., Xavier R.J., Root D.E. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 2014;32:1262–1267. PubMed PMC

Endo Y., Noguchi S., Hara Y., Hayashi Y.K., Motomura K., Miyatake S., Murakami N., Tanaka S., Yamashita S., Kizu R., et al. Dominant mutations in ORAI1 cause tubular aggregate myopathy with hypocalcemia via constitutive activation of store-operated Ca2+ channels. Hum Mol Genet. 2015;24:637–648. PubMed

Feske S. Calcium signalling in lymphocyte activation and disease. Nat. Rev. Immunol. 2007;7:690–702. PubMed

Feske S. CRAC channelopathies. Pflugers Arch. 2010;460:417–435. PubMed PMC

Feske S., Gwack Y., Prakriya M., Srikanth S., Puppel S.H., Tanasa B., Hogan P.G., Lewis R.S., Daly M., Rao A. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441:179–185. PubMed

Fleig L., Bergbold N., Sahasrabudhe P., Geiger B., Kaltak L., Lemberg M.K. Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of membrane proteins. Mol. Cell. 2012;47:558–569. PubMed

Freeman M. The rhomboid-like superfamily: molecular mechanisms and biological roles. Annu. Rev. Cell Dev. Biol. 2014;30:235–254. PubMed

Fukushima M., Tomita T., Janoshazi A., Putney J.W. Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities. J. Cell Sci. 2012;125:4354–4361. PubMed PMC

Grynkiewicz G., Poenie M., Tsien R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 1985;260:3440–3450. PubMed

Hogan P.G., Rao A. Store-operated calcium entry: mechanisms and modulation. Biochem. Biophys. Res. Commun. 2015;460:40–49. PubMed PMC

Hogan P.G., Lewis R.S., Rao A. Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 2010;28:491–533. PubMed PMC

Hoover P.J., Lewis R.S. Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1) Proc. Natl. Acad. Sci. USA. 2011;108:13299–13304. PubMed PMC

Hoth M. Calcium and barium permeation through calcium release-activated calcium (CRAC) channels. Pflugers Arch. 1995;430:315–322. PubMed

Hou X., Pedi L., Diver M.M., Long S.B. Crystal structure of the calcium release-activated calcium channel Orai. Science. 2012;338:1308–1313. PubMed PMC

Hou X., Burstein S.R., Long S.B. Structures reveal opening of the store-operated calcium channel Orai. eLife. 2018;7:e36758. PubMed PMC

Hsu P.D., Scott D.A., Weinstein J.A., Ran F.A., Konermann S., Agarwala V., Li Y., Fine E.J., Wu X., Shalem O., et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 2013;31:827–832. PubMed PMC

Ji W., Xu P., Li Z., Lu J., Liu L., Zhan Y., Chen Y., Hille B., Xu T., Chen L. Functional stoichiometry of the unitary calcium-release-activated calcium channel. Proc. Natl. Acad. Sci. U S A. 2008;105:13668–13673. PubMed PMC

Johnson N., Březinová J., Stephens E., Burbridge E., Freeman M., Adrain C., Strisovsky K. Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis. Sci. Rep. 2017;7:7283. PubMed PMC

Kar P., Parekh A. STIM proteins, Orai1 and gene expression. Channels (Austin) 2013;7:374–378. PubMed PMC

Kar P., Nelson C., Parekh A.B. Selective activation of the transcription factor NFAT1 by calcium microdomains near Ca2+ release-activated Ca2+ (CRAC) channels. J. Biol. Chem. 2011;286:14795–14803. PubMed PMC

Koch L., Kespohl B., Agthe M., Schumertl T., Düsterhöft S., Lemberg M.K., Lokau J., Garbers C. Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling. FASEB J. 2021;35:e21380. PubMed

Kong S.K., Lee C.Y. The use of Fura 2 for measurement of free calcium concentration. Biochem. Educ. 1995;23:97–98.

Kreutzberger A.J.B., Ji M., Aaron J., Mihaljević L., Urban S. Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion. Science. 2019;363:eaao0076. PubMed PMC

Krogh A., Larsson B., von Heijne G., Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–580. PubMed

Lastun V.L., Grieve A.G., Freeman M. Substrates and physiological functions of secretase rhomboid proteases. Semin. Cell Dev. Biol. 2016;60:10–18. PubMed

Lemberg M.K., Freeman M. Functional and evolutionary implications of enhanced genomic analysis of rhomboid intramembrane proteases. Genome Res. 2007;17:1634–1646. PubMed PMC

Lemberg M.K., Martoglio B. Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis. Mol. Cell. 2002;10:735–744. PubMed

Liou J., Fivaz M., Inoue T., Meyer T. Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion. Proc. Natl. Acad. Sci. U S A. 2007;104:9301–9306. PubMed PMC

Lohi O., Urban S., Freeman M. Diverse substrate recognition mechanisms for rhomboids; thrombomodulin is cleaved by Mammalian rhomboids. Curr. Biol. 2004;14:236–241. PubMed

Mercer J.C., Dehaven W.I., Smyth J.T., Wedel B., Boyles R.R., Bird G.S., Putney J.W., Jr. Large store-operated calcium selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1. J. Biol. Chem. 2006;281:24979–24990. PubMed PMC

Moin S.M., Urban S. Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics. eLife. 2012;1:e00173. PubMed PMC

Nesin V., Wiley G., Kousi M., Ong E.C., Lehmann T., Nicholl D.J., Suri M., Shahrizaila N., Katsanis N., Gaffney P.M., et al. Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis. Proc. Natl. Acad. Sci. U S A. 2014;111:4197–4202. PubMed PMC

Orci L., Ravazzola M., Le Coadic M., Shen W.W., Demaurex N., Cosson P. From the Cover: STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum. Proc. Natl. Acad. Sci. U S A. 2009;106:19358–19362. PubMed PMC

Palty R., Stanley C., Isacoff E.Y. Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating. Cell Res. 2015;25:963–980. PubMed PMC

Parekh A.B., Putney J.W., Jr. Store-operated calcium channels. Physiol. Rev. 2005;85:757–810. PubMed

Park C.Y., Hoover P.J., Mullins F.M., Bachhawat P., Covington E.D., Raunser S., Walz T., Garcia K.C., Dolmetsch R.E., Lewis R.S. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell. 2009;136:876–890. PubMed PMC

Peinelt C., Vig M., Koomoa D.L., Beck A., Nadler M.J., Koblan-Huberson M., Lis A., Fleig A., Penner R., Kinet J.P. Amplification of CRAC current by STIM1 and CRACM1 (Orai1) Nat. Cell Biol. 2006;8:771–773. PubMed PMC

Prakriya M., Lewis R.S. Store-operated calcium channels. Physiol. Rev. 2015;95:1383–1436. PubMed PMC

Prakriya M., Feske S., Gwack Y., Srikanth S., Rao A., Hogan P.G. Orai1 is an essential pore subunit of the CRAC channel. Nature. 2006;443:230–233. PubMed

Ran F.A., Hsu P.D., Lin C.Y., Gootenberg J.S., Konermann S., Trevino A.E., Scott D.A., Inoue A., Matoba S., Zhang Y., et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380–1389. PubMed PMC

Rao A., Luo C., Hogan P.G. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 1997;15:707–747. PubMed

Roux K.J., Kim D.I., Raida M., Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 2012;196:801–810. PubMed PMC

Sahin U., Weskamp G., Kelly K., Zhou H.M., Higashiyama S., Peschon J., Hartmann D., Saftig P., Blobel C.P. Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands. J. Cell Biol. 2004;164:769–779. PubMed PMC

Sanjana N.E., Shalem O., Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods. 2014;11:783–784. PubMed PMC

Schneider C.A., Rasband W.S., Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–675. PubMed PMC

Scrimgeour N., Litjens T., Ma L., Barritt G.J., Rychkov G.Y. Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins. J. Physiol. 2009;587:2903–2918. PubMed PMC

Shen Y., Dana H., Abdelfattah A.S., Patel R., Shea J., Molina R.S., Rawal B., Rancic V., Chang Y.F., Wu L., et al. A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578. BMC Biol. 2018;16:9. PubMed PMC

Soboloff J., Spassova M.A., Tang X.D., Hewavitharana T., Xu W., Gill D.L. Orai1 and STIM reconstitute store-operated calcium channel function. J. Biol. Chem. 2006;281:20661–20665. PubMed

Strisovsky K., Sharpe H.J., Freeman M. Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates. Mol. Cell. 2009;36:1048–1059. PubMed PMC

Subedi K.P., Ong H.L., Son G.Y., Liu X., Ambudkar I.S. STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions. Cell Rep. 2018;23:522–534. PubMed

Urban S., Freeman M. Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain. Mol. Cell. 2003;11:1425–1434. PubMed

Vig M., Peinelt C., Beck A., Koomoa D.L., Rabah D., Koblan-Huberson M., Kraft S., Turner H., Fleig A., Penner R., Kinet J.P. CRACM1 is a plasma membrane protein essential for SOCE. Science. 2006;312:1220–1223. PubMed PMC

Wu M.M., Buchanan J., Luik R.M., Lewis R.S. Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J. Cell Biol. 2006;174:803–813. PubMed PMC

Yamashita M., Yeung P.S., Ing C.E., McNally B.A., Pomès R., Prakriya M. STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate. Nat. Commun. 2017;8:14512. PubMed PMC

Ye J., Davé U.P., Grishin N.V., Goldstein J.L., Brown M.S. Asparagine-proline sequence within membrane-spanning segment of SREBP triggers intramembrane cleavage by site-2 protease. Proc. Natl. Acad. Sci. U S A. 2000;97:5123–5128. PubMed PMC

Yeh Y.C., Lin Y.P., Kramer H., Parekh A.B. Single nucleotide polymorphisms in Orai1 associated with atopic dermatitis inhibit protein turnover, decrease calcium entry and disrupt calcium-dependent gene expression. Hum. Mol. Genet. 2020;29:1808–1823. PubMed PMC

Yeung P.S., Yamashita M., Ing C.E., Pomès R., Freymann D.M., Prakriya M. Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating. Proc. Natl. Acad. Sci. USA. 2018;115:E5193–E5202. PubMed PMC

Yeung P.S., Yamashita M., Prakriya M. Molecular basis of allosteric Orai1 channel activation by STIM1. J. Physiol. 2020;598:1707–1723. PubMed PMC

Zhang S.L., Yeromin A.V., Zhang X.H., Yu Y., Safrina O., Penna A., Roos J., Stauderman K.A., Cahalan M.D. Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity. Proc. Natl. Acad. Sci. U S A. 2006;103:9357–9362. PubMed PMC

Ziegler S.F., Ramsdell F., Alderson M.R. The activation antigen CD69. Stem Cells. 1994;12:456–465. PubMed

Zimmermann L., Stephens A., Nam S.Z., Rau D., Kübler J., Lozajic M., Gabler F., Söding J., Lupas A.N., Alva V. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J. Mol. Biol. 2018;430:2237–2243. PubMed

4-Oxo-β-lactams as Covalent Inhibitors of the Mitochondrial Intramembrane Protease PARL