Sensor, channel, pump and leak - TMBIM6 assembles a multi-protein complex that governs the Ca2+ content of the endoplasmic reticulum

内质网(ER)的钙释放控制着一系列的细胞功能，并受到肌浆/内质网钙-ATPase(SERCA)活性的限制，SERCA在陡峭的浓度梯度下将钙泵回。除了作为钙离子储存库的功能外，内质网还在分泌途径中蛋白质的合成、折叠和分类中发挥关键作用。在内质网功能障碍的情况下，错误折叠的蛋白质数量增加，从而触发未折叠的蛋白质反应，这是一种通过应力传感器肌醇要求蛋白1a(IRE1a)协调内质网折叠能力的增加和折叠负荷的减少的机制。内质网钙离子释放失调、SERCA活性降低或错误折叠蛋白过多都会导致细胞死亡。TMBIM6(Trans膜Bax Inhibitor Motif Containing 6)是一个进化保守的pH依赖性内质网钙泄漏通道，它与配体诱导的钙通道三磷酸肌醇1，4，5-三磷酸受体1和3(IP3R1/3)以及IRE1a在内质网膜上相互作用。因此，TMBIM6连接了内质网的钙存储和蛋白质折叠功能。我们最近报道，来自缺乏TMBIM6环结构域的小鼠的淋巴细胞，即TMBIM6基因敲除小鼠，胞浆和内质网钙离子水平增加，严重削弱了它们的功能，增加了它们对细胞死亡的敏感性。虽然对于缺乏重要的内质网钙离子泄漏通道的细胞来说，内质网钙离子浓度的增加是可以预期的，但尚不清楚为什么缺乏TMBIM6会导致胞浆内钙离子浓度的增加。该项目基于初步数据，表明TMBIM6在进化上保守的N末端通过直接相互作用抑制了无处不在的SERCA2b泵，TMBIM6在上述小鼠中仍有表达。TMBIM6和IP3R1/3对SERCA活性和钙离子泄漏的物理整合可能会导致生理条件下更快的储藏重新填充，而与IRE1a的相互作用则允许以一种增加ER折叠能力的方式微调ER钙含量。在这里，我们将阐明SERCA2b、IRE1a和TMBIM6如何以及何时出现在同一蛋白质复合体中。我们将研究缺乏全长TMBIM6的细胞的钙泄漏和SERCA活性，而不仅仅是环状结构域。我们将进一步提炼TMBIM6的N-末端对这些机制的抑制活性，以确定活性所需的氨基酸或基序，并产生缺少这些基序的TMBIM6变体。通过比较这些TMBIM6突变体与之前发现的缺乏通道活性的点突变在蛋白质相互作用、钙稳态和内质网应激中的作用，我们将加深对错误折叠蛋白引起的细胞生物学和人类疾病的重要方面的理解。

Ca2+ release from the endoplasmic reticulum (ER) controls a vast array of cellular functions and is limited by the activity of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) which pump back Ca2+ against a steep concentration gradient. Besides this function as a Ca2+ store, the ER also plays a key role in the synthesis, folding and sorting of proteins destined for the secretory pathway. In the event of dysfunction of the ER, the number of misfolded proteins increases which triggers the unfolded protein response, a mechanism that coordinates an increase in the ER folding capacity and a decrease in folding load via the stress sensor inositol-requiring protein 1a (IRE1a). Dysregulated release of ER Ca2+, reduced SERCA activity or an excess of misfolded proteins all cause cell death.TMBIM6 (Transmembrane Bax inhibitor motif containing 6) is an evolutionarily conserved pH-dependent ER Ca2+ leak channel that interacts with ligand-induced Ca2+ channels inositol 1,4,5-trisphosphate receptors 1 and 3 (IP3R1/3) and IRE1a at the ER membrane. TMBIM6 therefore connects the Ca2+ store and protein-folding functions of the ER. We recently reported that lymphocytes from mice lacking the loop domain of TMBIM6, alleged TMBIM6 knockout mice, have increased cytosolic and ER Ca2+ levels that critically attenuate their function and increase their susceptibility to cell death. While an increased ER Ca2+ concentration can be expected for cells lacking an important ER Ca2+ leak channel, it remains unclear why lack of TMBIM6 should result in an increased cytosolic Ca2+ concentration. This project is based on preliminary data that suggest that the evolutionarily conserved N-terminus of TMBIM6, which is still expressed in the above-mentioned mice, inhibits the ubiquitous SERCA2b pump by direct interaction. Such a physical integration of SERCA activity and Ca2+ leakage by TMBIM6 and IP3R1/3 probably results in faster refilling of the store under physiological conditions while the interaction with IRE1a allows fine-tuning of the ER Ca2+ content in a way that increases the ER folding capacity. Here, we will clarify how and when SERCA2b, IRE1a and TMBIM6 are present in the same protein complex. We will study Ca2+ leakage and SERCA activity in cells lacking full-length TMBIM6 and not just the loop domain. We will further refine the inhibitory activity of the N-terminal part of TMBIM6 on these mechanisms to identify amino acids or motifs necessary for activity and generate TMBIM6 variants lacking these motifs. By comparing the effect of these TMBIM6 variants on protein interaction, Ca2+ homeostasis and ER stress with those of a previously identified point-mutant lacking channel activity, we will advance our understanding of important aspects of cell biology and human disease caused by misfolded proteins.