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1.
Cell Metab ; 2024 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-39153480

RESUMEN

To examine the roles of mitochondrial calcium Ca2+ ([Ca2+]mt) and cytosolic Ca2+ ([Ca2+]cyt) in the regulation of hepatic mitochondrial fat oxidation, we studied a liver-specific mitochondrial calcium uniporter knockout (MCU KO) mouse model with reduced [Ca2+]mt and increased [Ca2+]cyt content. Despite decreased [Ca2+]mt, deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux, α-ketoglutarate dehydrogenase flux, and succinate dehydrogenase flux in vivo. Rates of [14C16]palmitate oxidation and intrahepatic lipolysis were increased in MCU KO liver slices, which led to decreased hepatic triacylglycerol content. These effects were recapitulated with activation of CAMKII and abrogated with CAMKII knockdown, demonstrating that [Ca2+]cyt activation of CAMKII may be the primary mechanism by which MCU deletion promotes increased hepatic mitochondrial oxidation. Together, these data demonstrate that hepatic mitochondrial oxidation can be dissociated from [Ca2+]mt and reveal a key role for [Ca2+]cyt in the regulation of hepatic fat mitochondrial oxidation, intrahepatic lipolysis, gluconeogenesis, and lipid accumulation.

2.
bioRxiv ; 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38853984

RESUMEN

Metabolic adaptations in response to changes in energy supply and demand are essential for survival. The mitochondrial calcium uniporter coordinates metabolic homeostasis by regulating TCA cycle activation, mitochondrial fatty acid oxidation and cellular calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial metabolic pathways has remained unexplored. Here, we investigate the metabolic consequences of uniporter loss- and gain-of-function, and identify a key transcriptional regulator that mediates these effects. Using gene expression profiling and proteomic, we find that loss of uniporter function increases the expression of proteins in the branched-chain amino acid (BCAA) catabolism pathway. Activity is further augmented through phosphorylation of the enzyme that catalyzes this pathway's committed step. Conversely, in the liver cancer fibrolamellar carcinoma (FLC)-which we demonstrate to have high mitochondrial calcium levels- expression of BCAA catabolism enzymes is suppressed. We also observe uniporter-dependent suppression of the transcription factor KLF15, a master regulator of liver metabolic gene expression, including those involved in BCAA catabolism. Notably, loss of uniporter activity upregulates KLF15, along with its transcriptional target ornithine transcarbamylase (OTC), a component of the urea cycle, suggesting that uniporter hyperactivation may contribute to the hyperammonemia observed in FLC patients. Collectively, we establish that FLC has increased mitochondrial calcium levels, and identify an important role for mitochondrial calcium signaling in metabolic adaptation through the transcriptional regulation of metabolism.

3.
J Transl Med ; 22(1): 441, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38730481

RESUMEN

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.


Asunto(s)
Muerte Celular , Células Gigantes , Interfase , Microtúbulos , Poliploidía , Humanos , Interfase/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Línea Celular Tumoral , Muerte Celular/efectos de los fármacos , Células Gigantes/efectos de los fármacos , Células Gigantes/metabolismo , Células Gigantes/patología , Dinámicas Mitocondriales/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Glioblastoma/patología , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/genética , Neoplasias/patología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos
4.
ACS Chem Biol ; 19(2): 407-418, 2024 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-38301282

RESUMEN

Mixed lineage kinase domain-like (MLKL) is a key signaling protein of necroptosis. Upon activation by phosphorylation, MLKL translocates to the plasma membrane and induces membrane permeabilization, which contributes to the necroptosis-associated inflammation. Membrane binding of MLKL is initially initiated by electrostatic interactions between the protein and membrane phospholipids. We previously showed that MLKL and its phosphorylated form (pMLKL) are S-acylated during necroptosis. Here, we characterize the acylation sites of MLKL and identify multiple cysteines that can undergo acylation with an interesting promiscuity at play. Our results show that MLKL and pMLKL undergo acylation at a single cysteine, with C184, C269, and C286 as possible acylation sites. Using all-atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane levels. Through investigations of the S-palmitoyltransferases that might acylate pMLKL in necroptosis, we showed that zDHHC21 activity has the strongest effect on pMLKL acylation, inactivation of which profoundly reduced the pMLKL levels in cells and improved membrane integrity. These results suggest that blocking the acylation of pMLKL destabilizes the protein at the membrane interface and causes its degradation, ameliorating the necroptotic activity. At a broader level, our findings shed light on the effect of S-acylation on MLKL functioning in necroptosis and MLKL-membrane interactions mediated by its acylation.


Asunto(s)
Necroptosis , Proteínas Quinasas , Proteínas Quinasas/metabolismo , Fosforilación , Membrana Celular/metabolismo , Apoptosis
5.
bioRxiv ; 2024 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-38293219

RESUMEN

Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by lack of high-throughput, unbiased, and quantitative methods to identify proteins-calcium engagement. To address this, we adapted protein thermostability assays in the budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of novel signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-CoA reductase DECR1 binds calcium at physiologically-relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These unbiased, proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

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