Proteolytically Activated CRAC Effectors through Designed Intramolecular Inhibition

Abstract

Highly regulated intracellular calcium entry affects numerous cellular physiological events. External regulation of intracellular calcium signaling presents a great opportunity for the artificial regulation of cellular activity. Calcium entry can be mediated by STIM proteins interacting with Orai calcium channels; therefore, the STIM1–Orai1 pair has become a tool for artificially modulating calcium entry. We report on an innovative genetically engineered protease-activated Orai activator called PACE. CAD self-dimerization and activation were inhibited with a coiled-coil forming peptide pair linked to CAD via a protease cleavage site. PACE generated sustained calcium entry after its activation with a reconstituted split protease. We also generated PACE, whose transcriptional activation of NFAT was triggered by PPV or TEV protease. Using PACE, we successfully activated the native NFAT signaling pathway and the production of cytokines in a T-cell line. PACE represents a useful tool for generating sustained calcium entry to initiate calcium-dependent protein translation. PACE provides a promising template for the construction of links between various protease activation pathways and calcium signaling.

Synthetic Biology Meets Ca2+ Release-Activated Ca2+ Channel-Dependent Immunomodulation

“…A defined protease that can be chemically regulated was able to cleave the inhibitory coiled-coil pairs, triggering oligomerization and activation of PACE2 ( Figure 2 C), as evidenced by Ca 2+ imaging studies. Jazbec et al [ 242 ] developed a library of PACE constructs. They demonstrated that direct attachment of CAD to the TEV cleavage site and then to the coiled-coil domain leads to spontaneous activation, but it is maintained in the inactive state by the additional insertion of a peptide with a high helical propensity.…”

“…This tool was valuable to trigger Ca 2+ entry, NFAT activation, and associated downstream signaling events. The latter was demonstrated by protease cleavage-triggered expression of the cytokine IL-2, which is an essential cytokine governing T cell growth, the differentiation of regulatory T cells, and induction of cell death, as well as TNFα, which is an essential cytokine involved in the immune response, in Jurkat T cells [ 242 ]. Despite these valuable effects, further studies are needed to determine how fast PACE can be activated and whether its Ca 2+ currents correspond to those of CRAC channels.…”

“…As an alternative to adding the protease, split proteases specifically designed for the respective cleavage sites can be used. The protease-activated CAD activates Orai1 and consequently NFAT translocation to the nucleus and gene transcription (schemes adapted from references [ 211 , 242 ]).…”

Coiled Coils as Versatile Modules for Mammalian Cell Regulation

“…Using coiled coils for domain inhibition and proteolytic regulation, Jazbec et al [42] reported an innovative genetically engineered protease-activated Orai calcium channels activator called PACE-a genetically engineered mediator between proteolytic activity and calcium influx (Figure 3d). In this study, a part of STIM protein (which together with Orai1 channels modulates calcium entry) termed CAD was engineered.…”