TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

Lysosomal Ca²⁺ contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca²⁺ channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca²⁺ levels. The Ca²⁺ efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca²⁺ level, in which ITPR-independent regulation of Ca²⁺ status was observed. Further, TMBIM6 regulated the local release of Ca²⁺ through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca²⁺ efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca²⁺ and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved <i>Tmbim6</i>^+/+ mice compared with that in the counter <i>tmbim6</i>^−/- mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca²⁺ release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca²⁺ modulator that coordinates with autophagy to alleviate metabolism stress.<b>Abbreviations</b>: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6溶酶体Ca²⁺参与巨自噬（macroautophagy，又称自噬），即一种将胞质物质与细胞器转运至溶酶体内进行降解，以保护细胞抵御应激反应的胞内过程。TMBIM6（含BAX抑制基序跨膜蛋白6，transmembrane BAX inhibitor motif containing 6）是一类类Ca²⁺通道蛋白，已知其可调控内质网（ER，endoplasmic reticulum）应激反应与细胞凋亡。本研究探究了TMBIM6在调控溶酶体Ca²⁺水平中尚未明确的作用。 研究发现，经由TMBIM6从内质网流出的Ca²⁺可提升静息态溶酶体Ca²⁺水平，且该过程存在不依赖于ITPR（肌醇1,4,5-三磷酸受体，inositol 1,4,5-trisphosphate receptor）的Ca²⁺稳态调控机制。进一步研究显示，在营养匮乏或MTOR（雷帕霉素靶蛋白，mechanistic target of rapamycin）抑制条件下，TMBIM6可通过溶酶体MCOLN1/TRPML1（黏脂蛋白1，mucolipin 1）通道介导Ca²⁺的局部释放。 研究证实，经由MCOLN1通道的局部Ca²⁺外流可激活PPP3/钙调磷酸酶（calcineurin），进而触发TFEB（转录因子EB，transcription factor EB）的核转位、自噬诱导与溶酶体生物发生。当TMBIM6发生遗传失活时，溶酶体Ca²⁺水平及相关的TFEB核转位均显著降低。此外，与纯合敲除的<i>Tmbim6</i>^−/−小鼠相比，营养匮乏状态下野生型<i>Tmbim6</i>^+/+小鼠的肝脏与肾脏组织中的自噬流显著增强。 综上，本研究结果表明，在应激条件下，TMBIM6可促进溶酶体Ca²⁺释放，进而介导PPP3/钙调磷酸酶依赖的TFEB激活，最终增强自噬过程。因此，作为一种内质网膜蛋白，TMBIM6被证实是一类溶酶体Ca²⁺调控因子，可通过协调自噬通路以缓解代谢应激。 **缩写说明**： AVs：自噬泡（autophagic vacuoles）； CEPIA：钙离子捕获细胞器包埋蛋白指示剂（calcium-measuring organelle-entrapped protein indicator）； ER：内质网（endoplasmic reticulum）； GPN：甘氨酰-L-苯丙氨酸-β-萘酰胺（glycyl-L-phenylalanine-beta-naphthylamide）； ITPR/IP3R：肌醇1,4,5-三磷酸受体（inositol 1,4,5-trisphosphate receptor）； LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）； MCOLN/TRPML：黏脂蛋白（mucolipin）； MEF：小鼠胚胎成纤维细胞（mouse embryonic fibroblast）； ML-SA1：黏脂蛋白合成激动剂1（mucolipin synthetic agonist 1）； MTORC1：雷帕霉素激酶复合物1（mechanistic target of rapamycin kinase complex 1）； RPS6KB1：核糖体蛋白S6激酶B1（ribosomal protein S6 kinase B1）； SQSTM1：自噬衔接蛋白1（sequestosome 1，又称p62）； TFEB：转录因子EB（transcription factor EB）； TKO：三基因敲除（triple knockout）； TMBIM6/BI-1：含BAX抑制基序跨膜蛋白6（transmembrane BAX inhibitor motif containing 6）