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Trametinib is a MEK1 (mitogen-activated extracellular signal-related kinase kinase 1) inhibitor used in the treatment of BRAF (rapid accelerated fibrosarcoma B-type)-mutated metastatic melanoma. Roughly 11% of patients develop cardiomyopathy following long-term trametinib exposure. Although described clinically, the molecular landscape of trametinib cardiotoxicity has not been characterized.The aim of this study was to test the hypothesis that trametinib promotes widespread transcriptomic and cellular changes consistent with oxidative stress and impairs cardiac function.Mice were treated with trametinib (1 mg/kg/d). Echocardiography was performed pre- and post-treatment. Gross, histopathologic, and biochemical assessments were performed to probe for molecular and cellular changes. Human cardiac organoids were used as an in vitro measurement of cardiotoxicity and recovery.Long-term administration of trametinib was associated with significant reductions in survival and left ventricular ejection fraction. Histologic analyses of the heart revealed myocardial vacuolization and calcification in 28% of animals. Bulk RNA sequencing identified 435 differentially expressed genes and 116 differential signaling pathways following trametinib treatment. Upstream gene analysis predicted interleukin-6 as a regulator of 17 relevant differentially expressed genes, suggestive of PI3K/AKT and JAK/STAT activation, which was subsequently validated. Trametinib hearts displayed elevated markers of oxidative stress, myofibrillar degeneration, an 11-fold down-regulation of the apelin receptor, and connexin-43 mislocalization. To confirm the direct cardiotoxic effects of trametinib, human cardiac organoids were treated for 6 days, followed by a 6-day media-only recovery. Trametinib-treated organoids exhibited reductions in diameter and contractility, followed by partial recovery with removal of treatment.These data describe pathologic changes observed in trametinib cardiotoxicity, supporting the exploration of drug holidays and alternative pharmacologic strategies for disease prevention.

Yeast ISC1 (Yer019w) encodes inositolphosphosphingolipid-phospholipase C and is activated by phosphatidylserine (PS) and cardiolipin (CL) (Sawai, H., Okamoto, Y., Lubert, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793–39798). In this study, the structural requirements for anionic phospholipid-selective binding of ISC1 were determined using site-directed and deletion mutants. FLAG-tagged Isc1p was activated by PS, CL, and phosphatidylglycerol (PG) in a dose-dependent manner. Using lipid-protein overlay assays, Isc1p interacted specifically and directly with PS/CL/PG. Lipid-protein binding studies of a series of deletion mutants demonstrated that the second transmembrane domain (TMII) and the C terminus were required for PS binding. Moreover, the TMII and the C terminus domain were sufficient to impart PS binding to a heterologous protein, green fluorescence protein. In addition, mutations of positively charged amino acid residues at the C terminus of ISC1 reduced the activating effects of PS, suggesting involvement of these amino acids in interaction with PS/CL/PG and in the activation of the enzyme. Finally, when separate fragments containing the N terminus-TMI and TMII-C terminus were expressed heterologously, enzyme activity was reconstituted, demonstrating that the interaction of the N terminus and the C terminus is required for activity of Isc1p. These results raise the hypothesis that in the presence of PS/CL/PG, the catalytic domain in the N terminus of Isc1p is “pulled” to the membrane to interact with substrate. These studies provide unique insights into the properties ofISC1 and define a novel mechanism for activation of enzymes by lipids cofactors.

Growth of Saccharomyces cerevisiae following glucose depletion (the diauxic shift) depends on a profound metabolic adaptation accompanied by a global reprogramming of gene expression. In this study, we provide evidence for a heretofore unsuspected role for Isc1p in mediating this reprogramming. Initial studies revealed that yeast cells deleted in ISC1, the gene encoding inositol sphingolipid phospholipase C, which resides in mitochondria in the post-diauxic phase, showed defective aerobic respiration in the post-diauxic phase but retained normal intrinsic mitochondrial functions, including intact mitochondrial DNA, normal oxygen consumption, and normal mitochondrial polarization. Microarray analysis revealed that the Δisc1 strain failed to up-regulate genes required for nonfermentable carbon source metabolism during the diauxic shift, thus suggesting a mechanism for the defective supply of respiratory substrates into mitochondria in the post-diauxic phase. This defect in regulating nuclear gene induction in response to a defect in a mitochondrial enzyme raised the possibility that mitochondria may initiate diauxic shift-associated regulation of nucleus-encoded genes. This was established by demonstrating that in respiratory-deficient petite cells these genes failed to be up-regulated across the diauxic shift in a manner similar to the Δisc1 strain. Isc1p- and mitochondrial function-dependent genes significantly overlapped with Adr1p-, Snf1p-, and Cat8p-dependent genes, suggesting some functional link among these factors. However, the retrograde response was not activated in Δisc1, suggesting that the response of Δisc1 cannot be simply attributed to mitochondrial dysfunction. These results suggest a novel role for Isc1p in allowing the reprogramming of gene expression during the transition from anaerobic to aerobic metabolism.

Inositolsphingolipid phospholipase C (Isc1p) is the Saccharomyces cerevisiae member of the extended family of neutral sphingomyelinases that regulates the generation of bioactive ceramides. Recently, we reported that Isc1p is post-translationally activated in the post-diauxic phase of growth and that it localizes to mitochondria (Vaena de Avalos, S., Okamoto, Y., and Hannun, Y. A. (2004) J. Biol. Chem. 279, 11537-11545). In this study the in vivo mechanisms of activation and function of Isc1p were investigated. Deletion of ISC1 resulted in markedly lower growth in non-fermentable carbon sources. Interestingly, the growth defect of isc1Delta strains resembled that of pgs1Delta strains, lacking the committed step in the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL), which were shown to activate Isc1p in vitro. Therefore, the role of Pgs1p in activation of Isc1p in vivo was investigated. The results showed that in the pgs1Delta strain, the growth-dependent activation of Isc1p was impaired as was the ISC1-dependent increase in the levels of phytoceramide during the post-diauxic phase, demonstrating that the activation of Isc1p in vivo is dependent on PGS1 and on the mitochondrial phospholipids PG/CL. Mechanistically, loss of Isc1p resulted in lower levels of mitochondrial cytochrome c oxidase subunits cox3p and cox4p, previously established targets of both PG and CL (Ostrander, D. B., Zhang, M., Mileykovskaya, E., Rho, M., and Dowhan, W. (2001) J. Biol. Chem. 276, 25262-25272), thus suggesting that Isc1p mediates at least some functions downstream of PG/CL. This study provides the first evidence for the mechanism of in vivo activation and function of Isc1p. A model with endogenous PG/CL as the in vivo activator of Isc1p is proposed.

We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p [Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798]. The present study was conducted to determine specific domains in Isc1p required for catalysis. Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1. Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues. Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins. In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain". G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis. D163A and K168A lost activity completely. Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity. The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p. Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding. Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain. This study provides novel insight into a new domain found in Isc1p and related enzymes.

In the present paper, we report that an inositolphosphoglycan (IPG), derived from a Trypanosoma cruzi glycoinositolphosphoceramide (LPPG), is able to inhibit ACTH-mediated accumulation of a glucocorticoid, cortisol, in calf adrenocortical cells. This IPG is also able to inhibit the stimulation by ACTH of the production of the main glucocorticoid, corticosterone and the main mineralocorticoid, aldosterone, in rat adrenocortical cells. Nitrous acid deamination confirmed that IPG is responsible for this inhibition. In order to study the involvement of glycosylphosphatidylinositol (GPI) in ACTH response in rat adrenal cortex, the activation of a phospholipase that hydrolyzes GPI (GPI-PLC) was evaluated. It was found that the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium is increased in rat adrenocortical cells by ACTH treatment. In addition, ACTH stimulates the release of ceramide from the glycoinositolphosphoceramide purified from T. cruzi. These data suggest that ACTH activates a GPI-PLC in rat adrenal cortex, which is in agreement with our previous data in calf adrenocortical cells; thus, the hydrolysis of GPI provoked by ACTH takes place in different mammals and the IPG released could inhibit ACTH-mediated synthesis of aldosterone, corticosterone and cortisol.

The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly purified mitochondria, and western blots using highly purified mitochondrial membrane fractions demonstrated that epitope-tagged Isc1p localized to the outer mitochondrial membrane as an integral membrane protein. Next, LC/MS was employed to determine the sphingolipid composition of highly purified mitochondria which were found to be significantly enriched in alpha-hydroxylated phytoceramides (21.7 fold) relative to the whole cell. Mitochondria, on the other hand, were significantly depleted in sphingoid bases. Compared to the parental strain, mitochondria from isc1Delta in the post-diauxic phase showed drastic reduction in the levels of alpha-hydroxylated phytoceramide (93.1% loss compared to WT mitochondria with only 2.58 fold enrichment in mitochondria compared to whole cell). Functionally, isc1Delta showed a higher rate of respiratory-deficient cells after incubation at high temperature and was more sensitive to hydrogen peroxide and ethidium bromide, indicating that isc1Delta exhibits defects related to mitochondrial function. These results suggest that Isc1p generates ceramide in mitochondria, and the generated ceramide contributes to the normal function of mitochondria. This study provides a first insight into the specific composition of ceramides in mitochondria.