professor Texas A&M University Houton, Texas, United States
Introduction:: Calcium release-activated calcium (CRAC) channel is a highly Ca2+-selective store-operated channel composed of two-component mediators (STIM and ORAI) and controls a wide range of cellular functions in various tissues. Gain-of-function (GoF) mutations of CRAC channel cause excessive Ca2+ entry, which can lead to serious diseases such as tubular aggregate myopathy and Stormorken syndrome (TAM/STRMK). To gain precise control of the CRAC channel and suppress the GoF phenotypes, we set out to develop genetically-encoded CRAC channel blockers that can be controlled by light or chemical (designated Opto-CRAB and Chemo-CRAB). These tools can be widely used to control Ca2+ influx and Ca2+-dependent signaling in mammalian cells, such as NFAT induced gene expression and T cell activation (Figure 1). Beyond in vitro applications, both Opto-CRAB and Chemo-CRAB have been applied in vivo using a zebrafish model of Stormorken syndrome that resulted in the mitigation of thrombocytopenia and hemorrhage in light and chemical dependent manners.
Materials and Methods:: The research used mammalian cell lines and a zebrafish models to test for calcium inhibition and T-cell regulations. HeLa cells expressing engineered CRAB was used to validate calcium influx inhibition with calcium dyes, NFAT translocation, and cellular localization. Jurkat cells and CD8+ T cells with lentivirus transduced CRAB were used for T cell regulation experiments. Zebrafish with a STIM1-R304W GoF mutant gene was used to study Stormorken Syndrome in a model organism.
Results, Conclusions, and Discussions:: Our studies revealed that a peptide derived from the ORAI channel C-terminus (designated CRAB) exerted very weak inhibition on CRAC channel when expressed in the cytosol. However, its inhibitory potency was substantially enhanced once anchored to the plasma membrane (Figure 2). Capitalizing on this unique feature, we manipulated the subcellular localization of the largely inactive cytosolic CRAB with optogenetic and chemogenetic approaches. In T-cells, both Opto-CRABs and Chemo-CRABs were able to modulate Ca2+-signaling and immune response via inhibition of T cell proliferation, down-regulation of IL2, CD69 and IFN-γ. Moreover, we h developed a light and drug inducible CRAC channel blocker that could regulate endogenous Ca2+/NFAT activity in T cells, mitigate CAR-T tonic signaling/preactivation, and reduce T-cell exhaustion. We anticipate to fill a critical gap by developing synthetic biology tools to conditionally switch off Ca2+/NFAT signaling, which could be utilized to cope with tonic signaling, uncontrolled T cell activation, and T cell exhaustion during T cell-based immunotherapy.
