ABSTRACT

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 Tmbim6^+/+ mice compared with that in the counter tmbim6^−/- 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.Abbreviations: 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

摘要

溶酶体 Ca ²⁺有助于巨自噬/自噬，这是一种细胞内过程，用于降解溶酶体中的细胞质材料和细胞器，以保护细胞免受应激反应。TMBIM6（包含 6 的跨膜 BAX 抑制剂基序）是一种 Ca ²⁺通道样蛋白，已知可调节内质网应激反应和细胞凋亡。在这项研究中，我们研究了 TMBIM6 在调节溶酶体 Ca ²⁺水平方面的未知作用。发现通过 TMBIM6 从 ER 流出的 Ca ²⁺增加了静息溶酶体 Ca ²⁺水平，其中观察到ITPR 独立调节 Ca ²⁺状态。此外，TMBIM6 调控 Ca ²⁺的局部释放通过在营养饥饿或 MTOR 抑制下的溶酶体 MCOLN1/TRPML1 通道。发现通过 MCOLN1 通道的局部 Ca ²⁺流出可激活 PPP3/钙调神经磷酸酶，触发 TFEB（转录因子 EB）核易位、自噬诱导和溶酶体生物发生。TMBIM6 基因失活后，溶酶体 Ca ²⁺和相关的 TFEB 核易位减少。此外，与反tmbim6 ^-/-小鼠相比，饥饿的Tmbim6 ^+/+小鼠的肝脏或肾脏中的自噬通量显着增强。总之，我们的观察表明，在压力条件下，TMBIM6 增加溶酶体 Ca ²⁺释放，导致 PPP3/钙调神经磷酸酶介导的 TFEB 激活并随后增强自噬。因此，内质网膜蛋白 TMBIM6 被认为是一种溶酶体 Ca ²⁺调节剂，可与自噬协调以减轻代谢压力。缩写：AVs：自噬泡；CEPIA：钙测量细胞器包裹的蛋白质指示剂；ER：内质网；GPN：甘氨酰-L-苯丙氨酸-β-萘酰胺；ITPR/IP3R：肌醇 1,4,5-三磷酸受体；LAMP1：溶酶体相关膜蛋白 1；MCOLN/TRPML：粘蛋白；MEF：小鼠胚胎成纤维细胞；ML-SA1：粘蛋白合成激动剂 1；MTORC1：雷帕霉素激酶复合物 1 的机制靶点；RPS6KB1：核糖体蛋白S6激酶B1；SQSTM1：螯合体 1；TFEB：转录因子EB；TKO：三重淘汰赛；TMBIM6/BI-1：包含 6 个的跨膜 BAX 抑制剂基序