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Proteolytically Activated CRAC Effectors through Designed Intramolecular Inhibition.

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|July 8, 2022
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Summary

Researchers developed PACE, a protease-activated Orai activator, to control cellular calcium entry. This tool enables sustained calcium influx, facilitating calcium-dependent processes like protein translation and cytokine production in T-cells.

Keywords:
OraiPPV proteaseSTIM1TEV proteasecalcium signalingcoiled-coil peptides

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Intracellular calcium (Ca2+) signaling regulates critical cellular functions.
  • Artificial control of calcium entry offers a method for modulating cellular activity.
  • The STIM1-Orai1 complex is a key mediator of calcium entry and a target for modulation.

Purpose of the Study:

  • To develop an innovative, genetically engineered tool for protease-activated regulation of calcium entry.
  • To create a system for sustained calcium influx initiated by protease activity.
  • To demonstrate the utility of this tool in activating downstream signaling pathways and cellular responses.

Main Methods:

  • Engineering a protease-activated Orai activator (PACE) by linking a coiled-coil peptide pair to the CAD domain via a protease cleavage site.
  • Utilizing reconstituted split proteases for PACE activation.
  • Generating PACE variants responsive to specific proteases (PPV, TEV) for NFAT pathway activation.
  • Assessing PACE's ability to induce sustained calcium entry and activate the NFAT signaling pathway and cytokine production in a T-cell line.

Main Results:

  • PACE successfully generated sustained calcium entry upon activation by a reconstituted split protease.
  • PACE variants enabled protease-specific transcriptional activation of NFAT.
  • The system effectively activated the native NFAT signaling pathway and cytokine production in T-cells.
  • Demonstrated PACE's role in initiating calcium-dependent protein translation.

Conclusions:

  • PACE is a novel tool for precisely controlling and sustaining calcium entry.
  • PACE provides a versatile platform for linking protease activity to calcium signaling.
  • This technology holds promise for applications in calcium-dependent cellular processes and synthetic biology.