RESUMEN
Inositol phosphates and their metabolites play a significant role in several biochemical pathways, gene expression regulation, and phosphate homeostasis. Among the different inositol phosphates, inositol hexakisphosphate (IP6) is a substrate of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate one or more of the IP6 phosphate groups. Pyrophosphorylation of IP6 leads to the formation of inositol pyrophosphates, high-energy signaling molecules that mediate physiological processes through their ability to modify target protein activities, either by directly binding to their target protein or by pyrophosphorylating protein serine residues. 5-diphosphoinositol pentakisphosphate, the most abundant inositol pyrophosphate in mammals, has been extensively studied and found to be significantly involved in a wide range of physiological processes. Three IP6K (IP6K1, IP6K2, and IP6K3) isoforms regulate IP7 synthesis in mammals. Here, we summarize our current understanding of IP6K1's roles in cytoskeletal remodeling, trafficking, cellular migration, metabolism, gene expression, DNA repair, and immunity. We also briefly discuss current gaps in knowledge, highlighting the need for further investigation.
Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato) , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Animales , Humanos , Fosfatos de Inositol/metabolismo , Citoesqueleto/metabolismo , Mamíferos/metabolismoRESUMEN
Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. Recent studies have identified the inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS model systems. However, the molecular mechanisms linking aberrant CL remodeling, particularly the primary, direct consequence of reduced tetralinoleoyl-CL (TLCL) levels, to PDH activity deficiency are not yet understood. In the current study, we found that remodeled TLCL promotes PDH function by directly binding to and enhancing the activity of PDH phosphatase 1 (PDP1). This is supported by our findings that TLCL uniquely activates PDH in a dose-dependent manner, TLCL binds to PDP1 in vitro, TLCL-mediated PDH activation is attenuated in the presence of phosphatase inhibitor, and PDP1 activity is decreased in Tafazzin-knockout (TAZ-KO) C2C12 myoblasts. Additionally, we observed decreased mitochondrial calcium levels in TAZ-KO cells and treating TAZ-KO cells with calcium lactate (CaLac) increases mitochondrial calcium and restores PDH activity and mitochondrial oxygen consumption rate. Based on our findings, we conclude that reduced mitochondrial calcium levels and decreased binding of PDP1 to TLCL contribute to decreased PDP1 activity in TAZ-KO cells.
Asunto(s)
Aciltransferasas , Cardiolipinas , Oxidorreductasas , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa , Aciltransferasas/genética , Aciltransferasas/metabolismo , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Calcio/metabolismo , Cardiolipinas/genética , Cardiolipinas/metabolismo , Mitocondrias/metabolismo , Oxidorreductasas/metabolismo , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa/genética , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa/metabolismo , Animales , Ratones , Técnicas de Inactivación de Genes , Unión ProteicaRESUMEN
We have examined the roles of yeast mRNA decapping-activators Pat1 and Dhh1 in repressing the translation and abundance of specific mRNAs in nutrient-replete cells using ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs, RNA Polymerase II ChIP-Seq, and TMT-mass spectrometry of mutants lacking one or both factors. Although the Environmental Stress Response (ESR) is activated in dhh1Δ and pat1Δ mutants, hundreds of non-ESR transcripts are elevated in a manner indicating cumulative repression by Pat1 and Dhh1 in wild-type cells. These mRNAs show both reduced decapping and diminished transcription in the mutants, indicating that impaired mRNA turnover drives transcript derepression in cells lacking Dhh1 or Pat1. mRNA degradation stimulated by Dhh1/Pat1 is not dictated by poor translation nor enrichment for suboptimal codons. Pat1 and Dhh1 also collaborate to reduce translation and protein production from many mRNAs. Transcripts showing concerted translational repression by Pat1/Dhh1 include mRNAs involved in cell adhesion or utilization of the poor nitrogen source allantoin. Pat1/Dhh1 also repress numerous transcripts involved in respiration, catabolism of non-preferred carbon or nitrogen sources, or autophagy; and we obtained evidence for elevated respiration and autophagy in the mutants. Thus, Pat1 and Dhh1 function as post-transcriptional repressors of multiple pathways normally activated only during nutrient limitation.
Asunto(s)
Saccharomyces cerevisiae , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Nutrientes , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The mitochondrial phospholipid cardiolipin (CL) is critical for numerous essential biological processes, including mitochondrial dynamics and energy metabolism. Mutations in the CL remodeling enzyme TAFAZZIN cause Barth syndrome, a life-threatening genetic disorder that results in severe physiological defects, including cardiomyopathy, skeletal myopathy, and neutropenia. To study the molecular mechanisms whereby CL deficiency leads to skeletal myopathy, we carried out transcriptomic analysis of the TAFAZZIN-knockout (TAZ-KO) mouse myoblast C2C12 cell line. Our data indicated that cardiac and muscle development pathways are highly decreased in TAZ-KO cells, consistent with a previous report of defective myogenesis in this cell line. Interestingly, the muscle transcription factor myoblast determination protein 1 (MyoD1) is significantly repressed in TAZ-KO cells and TAZ-KO mouse hearts. Exogenous expression of MyoD1 rescued the myogenesis defects previously observed in TAZ-KO cells. Our data suggest that MyoD1 repression is caused by upregulation of the MyoD1 negative regulator, homeobox protein Mohawk, and decreased Wnt signaling. Our findings reveal, for the first time, that CL metabolism regulates muscle differentiation through MyoD1 and identify the mechanism whereby MyoD1 is repressed in CL-deficient cells.
Asunto(s)
Síndrome de Barth , Cardiolipinas , Proteína MioD , Animales , Ratones , Aciltransferasas/genética , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Cardiolipinas/genética , Cardiolipinas/metabolismo , Ratones Noqueados , Músculos/metabolismo , Factores de Transcripción/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismoRESUMEN
Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-inositol-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme. Binding of IP6K1 to phosphatidic acid (PA) is required for this repression. Here, we elucidate the role of PA in IP6K1 repression. Our results indicate that increasing PA levels through pharmacological stimulation of phospholipase D (PLD) or direct supplementation of 18:1 PA induces nuclear translocation of IP6K1 and represses expression of the MIPS protein. We found that this effect was specific to PA synthesized in the plasma membrane, as endoplasmic reticulum-derived PA did not induce IP6K1 translocation. Furthermore, we determined that PLD-mediated PA synthesis can be stimulated by the master metabolic regulator 5' AMP-activated protein kinase (AMPK). We show that activation of AMPK by glucose deprivation or by treatment with the mood-stabilizing drugs valproate or lithium recapitulated IP6K1 nuclear translocation and decreased MIPS expression. This study demonstrates for the first time that modulation of PA levels through the AMPK-PLD pathway regulates IP6K1-mediated repression of MIPS.
Asunto(s)
Ácidos Fosfatidicos , Fosfolipasa D , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Fibroblastos/metabolismo , Glucosa , Humanos , Inositol/metabolismo , Inositol/farmacología , Litio , Mamíferos/metabolismo , Ratones , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato) , Ácido ValproicoRESUMEN
Inositol is an essential nutrient, obtained either by uptake from the environment or by de novo synthesis from glucose. Inositol and its derivatives exhibit tumor-suppressive effects, potentially mediated by inhibition of the ERK-MAPK or PI3K-Akt pathways. Accordingly, many cancers have been documented to silence expression of the ISYNA1 gene, which encodes the rate-limiting enzyme of inositol synthesis. Paradoxically, recent studies have also reported upregulation of ISYNA1 in some cancers. Upregulation may reflect a compensatory response brought about by defective inositol uptake or oncogenic mutations that preclude its tumor-suppressive effects. In these scenarios, de novo synthesis of inositol may be upregulated to promote cell proliferation. The role of inositol in cancer is further complicated by its ability to inhibit the master metabolic regulator AMPK, which upon activation can either decrease cell proliferation and metastasis or promote cell survival. Due to its potential dual role in cancer, inositol homeostasis must be tightly regulated in tumor cells. Thus, whether inositol acts to suppress or promote tumor progression is determined by the metabolic profile and oncogenic background of the cancer.
Asunto(s)
Inositol , Neoplasias , Proliferación Celular , Humanos , Neoplasias/genética , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-aktRESUMEN
Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pHc) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pHc by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pHc causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pHc in regulating the metabolic program of yeast cells.
Asunto(s)
Citosol/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Ácido Valproico/farmacología , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Glucólisis/efectos de los fármacos , Glucólisis/genética , Concentración de Iones de Hidrógeno , Proteínas Serina-Treonina Quinasas/genética , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cardiolipin (CL) is the signature phospholipid (PL) of mitochondria and plays a pivotal role in mitochondrial and cellular function. Disruption of the CL remodeling gene tafazzin (TAZ) causes the severe genetic disorder Barth syndrome (BTHS). Our current understanding of the function of CL and the mechanism underlying the disease has greatly benefited from studies utilizing the powerful yeast model Saccharomyces cerevisiae. In this review, we discuss important findings on the function of CL and its remodeling from yeast studies and the implications of these findings for BTHS, highlighting the potential physiological modifiers that may contribute to the disparities in clinical presentation among BTHS patients.
Asunto(s)
Aciltransferasas/genética , Síndrome de Barth/metabolismo , Cardiolipinas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Síndrome de Barth/genética , Cardiolipinas/genética , Humanos , Mitocondrias/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.
Asunto(s)
Cardiolipinas/metabolismo , Animales , Drosophila melanogaster/metabolismo , Membrana Dobles de Lípidos/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Músculos/metabolismo , Fosforilación Oxidativa , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays an important role in mitochondrial bioenergetics. Previous studies in the yeast model have indicated that CL is required for optimal iron homeostasis, which is disrupted by a mechanism not yet determined in the yeast CL mutant, crd1Δ. This finding has implications for the severe genetic disorder, Barth syndrome (BTHS), in which CL metabolism is perturbed because of mutations in the CL-remodeling enzyme, tafazzin. Here, we investigate the effects of tafazzin deficiency on iron homeostasis in the mouse myoblast model of BTHS tafazzin knockout (TAZ-KO) cells. Similarly to CL-deficient yeast cells, TAZ-KO cells exhibited elevated sensitivity to iron, as well as to H2O2, which was alleviated by the iron chelator deferoxamine. TAZ-KO cells exhibited increased expression of the iron exporter ferroportin and decreased expression of the iron importer transferrin receptor, likely reflecting a regulatory response to elevated mitochondrial iron. Reduced activities of mitochondrial iron-sulfur cluster enzymes suggested that the mechanism underlying perturbation of iron homeostasis was defective iron-sulfur biogenesis. We observed decreased levels of Yfh1/frataxin, an essential component of the iron-sulfur biogenesis machinery, in mitochondria from TAZ-KO mouse cells and in CL-deleted yeast crd1Δ cells, indicating that the role of CL in iron-sulfur biogenesis is highly conserved. Yeast crd1Δ cells exhibited decreased processing of the Yfh1 precursor upon import, which likely contributes to the iron homeostasis defects. Implications for understanding the pathogenesis of BTHS are discussed.
Asunto(s)
Síndrome de Barth/metabolismo , Cardiolipinas/metabolismo , Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Mioblastos/metabolismo , Aciltransferasas , Animales , Síndrome de Barth/genética , Síndrome de Barth/patología , Cardiolipinas/genética , Línea Celular , Eliminación de Gen , Técnicas de Inactivación de Genes , Proteínas de Unión a Hierro/genética , Ratones , Mioblastos/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , FrataxinaRESUMEN
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a â¼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.
Asunto(s)
Cardiolipinas/fisiología , Ciclo del Ácido Cítrico , Lípidos/biosíntesis , Mitocondrias/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Acetilcoenzima A/biosíntesis , Aciltransferasas , Animales , Carbono/metabolismo , Línea Celular , Metabolismo Energético , Activación Enzimática , Ratones , Ratones Noqueados , Piruvato Carboxilasa/metabolismo , Factores de Transcripción/genéticaRESUMEN
Inositol is the precursor for all inositol compounds and is essential for viability of eukaryotic cells. Numerous cellular processes and signaling functions are dependent on inositol compounds, and perturbation of their synthesis leads to a wide range of human diseases. Although considerable research has been directed at understanding the function of inositol compounds, especially phosphoinositides and inositol phosphates, a focus on regulatory and homeostatic mechanisms controlling inositol biosynthesis has been largely neglected. Consequently, little is known about how synthesis of inositol is regulated in human cells. Identifying physiological regulators of inositol synthesis and elucidating the molecular mechanisms that regulate inositol synthesis will contribute fundamental insight into cellular processes that are mediated by inositol compounds and will provide a foundation to understand numerous disease processes that result from perturbation of inositol homeostasis. In addition, elucidating the mechanisms of action of inositol-depleting drugs may suggest new strategies for the design of second-generation pharmaceuticals to treat psychiatric disorders and other illnesses.
Asunto(s)
Inositol/biosíntesis , Homeostasis , Humanos , FosfatidilinositolesRESUMEN
Phosphatidic acid (PA) and the conserved integral ER membrane protein Scs2p regulate localization of the transcriptional repressor Opi1p, which controls expression of phospholipid biosynthesis genes, but the mechanisms conducting Opi1p localization are not fully understood. A new study suggests the existence of a distinct pool of PA in the ER that is required for regulation of Opi1p localization and thus phospholipid metabolism in yeast.
Asunto(s)
Retículo Endoplásmico/metabolismo , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Ácidos Fosfatidicos/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Retículo Endoplásmico/enzimología , Mitocondrias/enzimología , Mitocondrias/metabolismo , Fosfolípidos/biosíntesis , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Bipolar disorder (BD) is a severe psychiatric illness affecting â¼1% of the world population. Valproate (VPA) and lithium, widely used for the treatment of BD, are not universally effective. These drugs have been shown to cause inositol depletion, but translating this observation to a specific therapeutic mechanism has been difficult, hampering the development of more effective therapies. We have shown previously in yeast that chronic VPA treatment induces the unfolded protein response due to increasing ceramide levels. To gain insight into the mechanisms activated during acute VPA treatment, we performed a genome-wide expression study in yeast treated with VPA for 30 min. We observed increased mRNA and protein levels of RSB1, which encodes an exporter of long chain bases dihydrosphingosine (DHS) and phytosphingosine (PHS), and further saw that VPA increased sensitivity of an rsb1Δ mutant to PHS, suggesting that VPA increases long chain base levels. Consistent with this, PHS levels were elevated in wild type and, to a greater extent, in rsb1Δ cells. Expression of ORM genes (negative regulators of PHS synthesis) and of fatty acid elongase genes FEN1 and SUR4 were decreased, and expression of YOR1 (exporter of PHS-1P) and DPL1 (lyase that degrades DHS-1P and PHS-1P) was increased. These effects were more pronounced in medium lacking inositol, and were mirrored by inositol starvation of an ino1Δ mutant. These findings provide a metabolic explanation as to how VPA-mediated inositol depletion causes increased synthesis of PHS and further support the therapeutic relevance of inositol depletion as a bipolar disorder treatment.
Asunto(s)
Antimaníacos/farmacología , Inositol/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Esfingosina/análogos & derivados , Ácido Valproico/farmacología , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Trastorno Bipolar/tratamiento farmacológico , Ceramidas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingosina/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, plays an important role in mitochondrial processes and bioenergetics. CL is synthesized de novo and undergoes remodeling in the mitochondrial membranes. Perturbation of CL remodeling leads to the rare X-linked genetic disorder Barth syndrome, which shows disparities in clinical presentation. To uncover biochemical modifiers that exacerbate CL deficiency, we carried out a synthetic genetic array screen to identify synthetic lethal interactions with the yeast CL synthase mutant crd1Δ. The results indicated that crd1Δ is synthetically lethal with mutants in pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA. Acetyl-CoA levels were decreased in the mutant. The synthesis of acetyl-CoA depends primarily on the PDH-catalyzed conversion of pyruvate in the mitochondria and on the PDH bypass in the cytosol, which synthesizes acetyl-CoA from acetate. Consistent with perturbation of the PDH bypass, crd1Δ cells grown on acetate as the sole carbon source exhibited decreased growth, decreased acetyl-CoA, and increased intracellular acetate levels resulting from decreased acetyl-CoA synthetase activity. PDH mRNA and protein levels were up-regulated in crd1Δ cells, but PDH enzyme activity was not increased, indicating that PDH up-regulation did not compensate for defects in the PDH bypass. These findings demonstrate for the first time that CL is required for acetyl-CoA synthesis, which is decreased in CL-deficient cells as a result of a defective PDH bypass pathway.
Asunto(s)
Acetilcoenzima A/biosíntesis , Cardiolipinas/metabolismo , Coenzima A Ligasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/genética , Cardiolipinas/genética , Coenzima A Ligasas/genética , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, is important for cardiovascular health, and perturbation of CL metabolism is implicated in cardiovascular disease. Although the role of CL in mitochondrial function, biogenesis, and genome stability has been studied, recent findings indicate that it is essential for functions apart from mitochondrial bioenergetics. In this study, we report that mitophagy is perturbed in CL-deficient yeast cells. Mutants of autophagy/mitophagy genes ATG8, ATG18, and ATG32 synthetically interact with CL synthase mutant crd1Δ. CL-deficient cells exhibited decreased GFP-tagged mitochondrial proteins inside the vacuole and decreased free GFP, consistent with decreased mitophagy. Both PKC and high osmolarity glycerol (HOG) MAPK pathways were shown previously to be required for mitophagy. Activation of both MAPKs was defective in CL-deficient cells. Deletion of HOG pathway genes SHO1, SSK1, STE50, and HOG1 exacerbated crd1Δ growth. 1 m sorbitol and 0.2 m NaCl, which induce the HOG pathway, rescued growth of the mutant. Activation of the MAPK Slt2p was defective in crd1Δ cells, and up-regulation of the PKC pathway by expression of the PKC1R398P gene, which encodes constitutively activated Pkc1p, rescued crd1Δ growth and mitophagy defects. These findings indicate that loss of CL impairs MAPK pathway activation, and decreased activation of the PKC pathway leads to defective mitophagy.
Asunto(s)
Cardiolipinas/fisiología , Mitofagia/fisiología , Proteína Quinasa C/metabolismo , Mitofagia/genética , Fosforilación , Saccharomyces cerevisiae/metabolismo , Regulación hacia ArribaRESUMEN
Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.
Asunto(s)
Síndrome de Barth/patología , Cardiolipinas/metabolismo , Diferenciación Celular/genética , Lisofosfolípidos/metabolismo , Mioblastos/patología , Factores de Transcripción/metabolismo , Aciltransferasas , Animales , Síndrome de Barth/genética , Sistemas CRISPR-Cas , Línea Celular , Técnicas de Inactivación de Genes , Humanos , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Modelos Biológicos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mioblastos/citología , Mioblastos/metabolismo , Factores de Transcripción/genéticaRESUMEN
Valproate (VPA), an FDA approved anti-epileptic drug with a half-life of 12-18 h in humans, has been shown to perturb the vacuolar proton pump (vH+-ATPase) function in yeasts by inhibiting myo-inositol phosphate synthase, the first and rate-limiting enzyme in inositol biosynthesis, thereby resulting in inositol depletion. vH+-ATPase transfers protons (H+) across cell membranes, which help maintain pH gradients within cells necessary for various cellular functions including secretion. This proton pump has a membrane (V0) and a soluble cytosolic (V1) domain, with C-subunit associated with V1. In secretory cells such as neurons and insulin-secreting beta cells, vH+-ATPase acidifies vesicles essential for secretion. In this study, we demonstrate that exposure of insulin-secreting Min6 cells to a clinical dose of VPA results in inositol depletion and loss of co-localization of subunit C of vH+-ATPase with insulin-secreting granules. Consequently, a reduction of glucose-stimulated insulin secretion is observed following VPA exposure. These results merit caution and the reassessment of the clinical use of VPA.
Asunto(s)
Glucosa/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ácido Valproico/farmacología , Animales , Secreción de Insulina , Ratones , Células Tumorales Cultivadas , Ácido Valproico/químicaRESUMEN
myo-Inositol, the precursor of all inositol compounds, has pivotal roles in cell metabolism and signaling pathways. Although physiological studies indicate a strong correlation between abnormal intracellular inositol levels and neurological disorders, very little is known about the regulation of inositol synthesis in mammalian cells. In this study, we report that IP6K1, an inositol hexakisphosphate kinase that catalyzes the synthesis of inositol pyrophosphate, regulates inositol synthesis in mammalian cells. Ip6k1 ablation led to profound changes in DNA methylation and expression of Isyna1 (designated mIno1), which encodes the rate-limiting enzyme inositol-3-phosphate synthase. Interestingly, IP6K1 preferentially bound to the phospholipid phosphatidic acid, and this binding was required for IP6K1 nuclear localization and the regulation of mIno1 transcription. This is the first demonstration of IP6K1 as a novel negative regulator of inositol synthesis in mammalian cells.
Asunto(s)
Inositol/biosíntesis , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Metilación de ADN , Técnicas de Inactivación de Genes , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Ratones , Modelos Biológicos , Ácidos Fosfatidicos/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/deficiencia , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Bipolar disorder (BD), which is characterized by depression and mania, affects 1-2% of the world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproate (VPA) is one of the most widely used drugs for the treatment of BD, but the therapeutic mechanism of action of this drug is not understood. This knowledge gap has hampered the development of effective treatments. To identify candidate pathways affected by VPA, we performed a genome-wide expression analysis in yeast cells grown in the presence or absence of the drug. VPA caused up-regulation of FEN1 and SUR4, encoding fatty acid elongases that catalyze the synthesis of very long chain fatty acids (C24 to C26) required for ceramide synthesis. Interestingly, fen1Δ and sur4Δ mutants exhibited VPA sensitivity. In agreement with increased fatty acid elongase gene expression, VPA increased levels of phytoceramide, especially those containing C24-C26 fatty acids. Consistent with an increase in ceramide, VPA decreased the expression of amino acid transporters, increased the expression of ER chaperones, and activated the unfolded protein response element (UPRE), suggesting that VPA induces the UPR pathway. These effects were rescued by supplementation of inositol and similarly observed in inositol-starved ino1Δ cells. Starvation of ino1Δ cells increased expression of FEN1 and SUR4, increased ceramide levels, decreased expression of nutrient transporters, and induced the UPR. These findings suggest that VPA-mediated inositol depletion induces the UPR by increasing the de novo synthesis of ceramide.