Sensor, channel, pump and leak - TMBIM6 assembles a multi-protein complex that governs the Ca2+ content of the endoplasmic reticulum
Final Report Abstract
Ca2+ release from the 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. In previous work, we 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. The aim of this project was to clarify whether 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. Unfortunately, this problem is still unsolved because we could not reliably establish a procedure to quantify protein-protein interaction due to the lack of suitable antibodies and problems caused by the extreme hydrophobicity of TMBIM6. In addition, our KO mice acquired breeding problems. We therefore reverted to another species where TMBIM6 and its evolutionarily conserved N-terminus is conserved, the fruitfly Drosophila melanogaster. We identified the fly orthologue of TMBIM6 and demonstrated that its knockdown is lethal due to an increased ER Ca2+ content. In contrast, we did not observe any changes in Xbp1 splicing, a hallmark of ER stress which is exactly what we found in the mouse system. Using this novel model, we identified WFS1, which, similar to TMBIM6 is a transmembrane protein primarily located in the ER that is a component of the unfolded protein response, modulates free Ca2+ in ER and is involved in apoptosis. Once we have established reliable methods to measure protein-protein interaction with TMBIM6 and demonstrated a genetic and physical interaction with WFS1 (and SERCA2b), we will aim for a renewal of this project.
Publications
- (2020) Bax Inhibitor-1 Deficiency Leads to Obesity by Increasing Ca2+-Dependent Insulin Secretion. Journal of Molecular Medicine 98 (6): 849–62
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, and Axel Methner
(See online at https://doi.org/10.1007/s00109-020-01914-x)
