Proapoptotic RECS1: a requisite gateway to lysosomal dysfunction and death

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For a long time since their discovery by Christian de Duve in the 1950s, lysosomes have been referred to almost exclusively as passive garbage bags; the endpoint in the degradation of intra- and extracellular cargo. The catabolic function of lysosomes is accomplished by an array of more than 60 acid hydrolases, which together break down a wide variety of biological macromolecules, including proteins, lipids, carbohydrates, and nucleic acids, for reutilization in the metabolic processes of the cell. For their optimal function, these enzymes require an acidic intraluminal pH of ~4.5, which is maintained by the joint action of a proton pump, the vacuolar H+-ATPase, and several ion channels embedded in the lysosomal limiting membrane. Nowadays, lysosomes are envisioned as complex signaling hubs, integrating diverse stimuli about the cell’s metabolic status to coordinate different adaptive responses (Ballabio and Bonifacino, 2020). The lysosome can also induce cell death signals in response to certain conditions, such as infections and treatment with lysosomotropic drugs, which leads to lysosomal membrane permeabilization (LMP) and the release of cathepsins, resulting in lysosomal-mediated cell death (Figure 1A, left). Lysosomes are also important intracellular calcium reservoirs. Lysosomal calcium plays essential functions in several cellular processes, such as lysosomal fusion with other vesicles, lysosomal biogenesis, and exocytosis (Figure 1A, right). In addition, lysosomal calcium is critical for lysosomal acidification, probably through the establishment of physical contacts with the endoplasmic reticulum. As a signaling molecule, calcium release from the lysosome through the transient receptor potential cation channel, mucolipin subfamily member 1 (TRPML1) activates the autophagic signaling pathway through the transcription factor EB (TFEB), which upregulates genes involved in autophagy and lysosomal biogenesis. Only three main types of lysosomal Ca2+ channels have been identified: the transient receptor potential channels of the mucolipin family, two-pore channels (TPC), and the trimeric Ca2+ two-transmembrane channel P2X4. However, the lysosomal membrane comprises dozens of integral and peripheral proteins of unknown functions. The identification of new regulators of lysosomal biology is essential to better understand the role of lysosomes in the global regulation of adaptive and pro-dead responses, and their close connection to cell metabolism.Figure 1: The newly discovered functions of RECS1 in the regulation of lysosomal calcium and cell death.(A) Lysosomes are membrane-enclosed cytosolic organelles, functioning as the main intracellular catabolic compartment. In the lysosomal lumen, biomolecules are degraded by hydrolase enzymes that function at very acidic pH (~4.5). Under stress conditions, LMP leads to the cytosolic translocation of lysosomal enzymes causing cell death. Lysosomes are also intracellular calcium reservoirs. Lysosomal calcium signaling is mediated by at least three different families of calcium channels and is essential for many processes, including lysosomal fusion with different organelles and the induction of the autophagic pathway. (B) RECS1 overexpression or starvation results in decreased lysosomal pH and increased intraluminal calcium concentration, leading to imbalances in lysosomal calcium homeostasis. Abnormal calcium signaling is involved in the etiology of many diseases, including lysosomal storage disorders, neurodegenerative diseases and cancer. RECS1 overexpression sensitizes cells to various chemotherapeutic drugs, including the lysosomotropic agents CQ and HCQ. The treatment of RECS1 overexpressing cells with these drugs correlates with BAX translocation to the lysosomal membrane and LMP, leading to lysosomal-dependent cell death. Lysosomal membrane damage also results in autophagy inhibition, which under certain conditions, can lead to cell death. BAX: Bcl-2-associated X protein; CQ: chloroquine; HCQ: hydroxychloroquine; LMP: lysosomal membrane permeabilization; P2X4: trimeric Ca2+ two-transmembrane channel; RECS1: responsive to centrifugal force and shear stress 1; TPC: two-pore channel; TRPML1: transient receptor potential channels of the mucolipin family 1.RECS1 (responsive to centrifugal force and shear stress 1) [also known as transmembrane bcl-2-associated X protein (BAX) inhibitor motif containing 1 (TMBIM1)] is a member of the TMBIM superfamily, which consists of at least six evolutionarily conserved proteins with homologs in viruses, bacteria and plants. The proteins of the TMBIM family localize to different intracellular membranes, including the endoplasmic reticulum, Golgi apparatus, and mitochondria, and they regulate stress-induced cell death and calcium homeostasis (Rojas-Rivera and Hetz, 2015). The resolution of the crystal structure of the TMBIM ortholog from Bacillus subtilis, BsYetJ, together with additional functional studies in human cells, have revealed that the proteins of the TMBIM family are seven-transmembrane, pH-sensitive calcium channels (Chang et al., 2014). RECS1 localizes to endosomes and lysosomes, suggesting that it may be involved in the regulation of lysosomal calcium homeostasis. RECS1 also regulates apoptosis in response to external stimuli; raising the possibility that its location at the lysosome membrane is implicated in the regulation of cell death. In our recent study (Pihan et al., 2021a), we have uncovered a novel function for RECS1 in the regulation of lysosomal pH, calcium homeostasis, and cell death. RECS1 overexpression triggered cell death through a crosstalk with the canonical mitochondrial pathway of apoptosis. These results suggested the identification of the first proapoptotic component of the TMBIM family. One of the main problems to accurately measure lysosomal intraluminal calcium concentration is that the affinity of commonly used calcium fluorescent probes (i.e. Fura-2) varies non-linearly with proton concentration at pH below 6. To circumvent this issue, calcium and pH must be determined simultaneously in each lysosome, allowing the precise assessment of the effects of luminal pH on the affinity of the calcium probe (Kd) (Christensen et al., 2002). Using a simplified version of this method (Pihan et al., 2021b), we measured lysosomal pH and calcium in cells overexpressing RECS1. We found that RECS1 overexpression increased lysosomal acidification, correlating with increased lysosomal intraluminal calcium concentration. These results suggest that RECS1 may be a novel lysosomal calcium channel, regulating resting lysosomal Ca2+ and H+ concentrations (Figure 1B, left). To directly determine RECS1’s channel conductance, we expressed the protein in Xenopus laevis oocytes and carried out electrophysiological studies at different pH values. Interestingly, we found that RESC1 exhibited spontaneous single-channel current spikes at neutral pH, which decreased dramatically when the pH was lowered to 6.5. Our results showed that RECS1 is permeable to Ca2+ and Na+, identifying a new lysosomal cation channel that regulates intraluminal pH and calcium concentration. Interestingly, as also shown in other members of the TMBIM family, the calcium conductivity of RECS1 is regulated by a conserved di-aspartyl pH sensor motif contained in its C-terminal domain. Ablation of this motif by mutagenesis significantly reduces the conductivity of the RECS1 channel. What cellular processes are regulated by RECS1 through calcium signals? We found that RECS1 overexpressing cells showed an increased accumulation of autophagosomes and a higher susceptibility to stress. Lysosomal-mediated cell death pathways are poorly understood. However, lysosomes are considered an attractive therapeutic target for cancer, because the induction of LMP represents an effective strategy against a variety of different cancers. Since RECS1 is a member of the TMBIM family of apoptosis regulators and lo

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