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

传感器、通道、泵和泄漏 - TMBIM6 组装控制内质网 Ca2 含量的多蛋白复合物

• 批准号: 333214844
• 负责人: Professor Dr. Axel Methner
• 金额: --
• 依托单位: Institut für Molekulare Medizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/333214844?language=en
• 关键词: Sensor; channel; pump; leak; TMBIM6

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.

从内质网（ER）释放的Ca 2+控制大量的细胞功能，并且受到肌质/内质网Ca 2 +-ATP酶（SERCA）的活性的限制，所述肌质/内质网Ca 2 +-ATP酶（SERCA）逆着陡峭的浓度梯度泵回Ca 2+。除了作为Ca 2+储存的功能外，ER还在分泌途径的蛋白质的合成，折叠和分选中起关键作用。在ER功能障碍的情况下，错误折叠的蛋白质的数量增加，这触发了未折叠的蛋白质反应，这是一种通过应力传感器肌醇需要蛋白1a（IRE 1a）协调ER折叠能力增加和折叠负荷减少的机制。TMBIM 6（Transmembrane Bax inhibitor motif containing 6）是一种进化上保守的pH依赖性ER Ca 2+渗漏通道，与配体诱导的Ca 2+通道、1，4，5-三磷酸肌醇受体1和3（IP 3R 1/3）以及ER膜上的IRE 1a相互作用。因此，TMBIM 6连接了ER的Ca 2+储存和蛋白质折叠功能。我们最近报道，来自缺乏TMBIM 6的环结构域的小鼠（所谓的TMBIM 6敲除小鼠）的淋巴细胞具有增加的胞质和ER Ca 2+水平，这严重削弱了它们的功能并增加了它们对细胞死亡的易感性。虽然对于缺乏重要的ER Ca 2+泄漏通道的细胞可以预期ER Ca 2+浓度增加，但仍然不清楚为什么缺乏TMBIM 6会导致细胞溶质Ca 2+浓度增加。该项目基于初步数据，该数据表明在上述小鼠中仍然表达的TMBIM 6的进化上保守的N-末端通过直接相互作用抑制普遍存在的SERCA 2b泵。通过TMBIM 6和IP 3R 1/3的SERCA活性和Ca 2+泄漏的这种物理整合可能导致在生理条件下更快地重新填充储存，而与IRE 1a的相互作用允许以增加ER折叠能力的方式微调ER Ca 2+含量。在这里，我们将阐明SERCA 2b，IRE 1a和TMBIM 6如何以及何时存在于同一蛋白质复合物中。我们将研究缺乏全长TMBIM 6而不仅仅是环结构域的细胞中的Ca 2+渗漏和SERCA活性。我们将进一步完善TMBIM 6的N-末端部分对这些机制的抑制活性，以鉴定活性所需的氨基酸或基序，并产生缺乏这些基序的TMBIM 6变体。通过比较这些TMBIM 6变体对蛋白质相互作用，Ca 2+稳态和ER应激的影响与先前确定的缺乏通道活性的点突变体，我们将推进我们对错误折叠蛋白质引起的细胞生物学和人类疾病的重要方面的理解。

项目成果

Bax inhibitor-1 deficiency leads to obesity by increasing Ca2+-dependent insulin secretion

Bax抑制剂-1缺乏通过增加Ca2依赖性胰岛素分泌导致肥胖

• DOI: 10.1007/s00109-020-01914-x
• 发表时间: 2020
• 期刊: Journal of Molecular Medicine (Berlin, Germany)
• 作者: Koenraad Philippaert;Michael Roden;Dmitrij Lisak;Diones Bueno;Tomas Jelenik;Konstantin Radyushkin;Teresa Schacht;Marion Mesuere;Verena Wüllner;Ann-Kathrin Herrmann;Jan Baumbart;Rudi Vennekens;Axel Methner
• 通讯作者: Axel Methner