Research of RECS1 Controlled Lysosome-mediated Cell Death

Introduction

Programmed cell death is regulated by the balance between activating and inhibitory signals. RECS1 is a member of the evolutionarily conserved TMBIM superfamily and, more specifically, the LFG family of cell death regulators. Although the role of these proteins as inhibitors of cell death is well documented, the possible existence of proapoptotic members remained elusive.

Methods

All MEF, HeLa, and human embryonic kidney (HEK) cells were maintained in Dulbecco's modified Eagle's medium. To determine the importance of RECS1 in the regulation of cell death and its regulatory role, Western blot, cell death assays, indirect immunofluorescence analysis, TEM and immunogold staining, BH3 profiling and competition assays, measurement of lysosomal pH and calcium, molecular modeling, RECS1 expression in alfalfa oocytes, electrophysiological assays, D. melanogaster and zebrafish studies were performed.

Results

The results of protein blotting analysis, cell death kinetics and fluorescence-activated cell sorting (FACS) analysis identified RECS1 as a pro-apoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. In addition, RECS1 C-terminal domain is necessary for its pro-dead function.

Fig. 1 RECS1 induces cell death through the mitochondrial pathway of apoptosis.

Unbiased screening with a large panel of cytotoxic agents revealed that RECS1 expression highly sensitizes cells to lysosomotropic agents and microtubule-destabilizing drugs. RECS1 localizes to the lysosomal membrane, where it rapidly induces cell death through LMP, involving the recruitment of active BAX to lysosomes. RECS1 also sensitizes cells to ER stress possibly via its transcriptional up-regulation.

Under basal conditions, RECS1 promotes lysosomal acidification and increases lysosomal calcium concentration. At the molecular level, RECS1 operates as a pH-dependent calcium and sodium channel, where a single mutation in the critical di-aspartyl sensor (D295Q) is sufficient to disrupt its calcium and sodium permeability, reducing cell death upon lysosomal stress.

Fig. 2 RECS1 is a pH-dependent calcium and sodium channel.

Last, we provide evidence indicating that RECS1 expression induces cell death and sensitizes whole animals to stress in fly and zebrafish models.

Fig. 3 The overexpression of the RECS1-like/CG9722 protein causes lethality and cell death during Drosophila development.

Summary

This study reveals the function of RECS1, a pro-apoptotic component of the TMBIM family, in promoting programmed cell death. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models.

Reference:

Pihán, P.; et al. Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1. Science advances, 2021, 7(46): eabe5469.

* For research use only. Not intended for any clinical use.

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Research of RECS1 Controlled Lysosome-mediated Cell Death

Introduction

Methods

Results

Summary

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