| Autor: |
Garbincius, Joanne F, Salik, Oniel, Mangold, Adam S, Makhoul, Angelina D, Murray, Emma, Lazaropolous, Michael P, Tomar, Dhanendra, Jadiya, Pooja, Elrod, John W |
| Zdroj: |
Circulation (Ovid); November 2021, Vol. 144 Issue: Supplement 1 pA14153-A14153, 1p |
| Abstrakt: |
The precise control of mitochondrial calcium (mCa2+) exchange allows the cardiomyocyte to balance the regulation of mitochondrial metabolism against the risk of mCa2+overload and cell death. The mitochondrial sodium-calcium exchanger (NCLX) is a major route of mCa2+efflux and thus is one of the key proteins that govern mCa2+homeostasis. Recent studies have demonstrated that increasing NCLX-dependent mCa2+efflux is a promising therapeutic objective for pathologies characterized by deleterious mCa2+overload such as ischemia-reperfusion injury, heart failure, and neurodegeneration. A substantial barrier to the development of NCLX-based therapies is that no pharmacologic activators of NCLX are currently available, and indeed, very little is known about the fundamental mechanisms that stimulate or inhibit NCLX activity in vivo. Therefore, we generated a plasmid encoding a biotin ligase domain fused to the C-terminus of human NCLX (hNCLX-BioID2HA) and used this construct to perform an in vitroproximity biotinylation screen for the unbiased detection of proteins that interact with NCLX and/or modulate NCLX function. We identified ~120 candidate NCLX-proximal mitochondrial proteins in AC16 cardiomyocytes and ~80 in HeLa cells, including several of unknown function. Twenty-five proteins were detected in both data sets, suggestive of conserved function between these two cell types. Current studies are defining the specific molecular function of these mitochondrial proteins and their effects on NCLX expression, NCLX activity, and overall mCa2+homeostasis. We are also developing a novel NCLX-3xFLAG knock-in mouse model to enable in vivoexpression of C-terminal epitope-tagged NCLX under the control of the endogenous murine promoter. We expect this model to aid in overcoming the significant proteomic challenges of manipulating this 13-transmembrane protein. Together, these approaches will help to uncover the post-translational mechanisms that regulate NCLX-dependent mCa2+efflux and to identify new therapeutic targets for heart failure and other diseases characterized by disrupted mCa2+homeostasis. |
| Databáze: |
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