Nucleoside diphosphate kinases 1 and 2 regulate a protective liver response to a high-fat diet.

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

Variation in Lipid Species Profiles among Leukemic Cells Significantly Impacts Their Sensitivity to the Drug Targeting of Lipid Metabolism and the Prognosis of AML Patients.

Several studies have linked bad prognoses of acute myeloid leukemia (AML) to the ability of leukemic cells to reprogram their metabolism and, in particular, their lipid metabolism. In this context, we performed "in-depth" characterization of fatty acids (FAs) and lipid species in leukemic cell lines and in plasma from AML patients. We firstly showed that leukemic cell lines harbored significant differences in their lipid profiles at steady state, and that under nutrient stress, they developed common mechanisms of protection that led to variation in the same lipid species; this highlights that the remodeling of lipid species is a major and shared mechanism of adaptation to stress in leukemic cells. We also showed that sensitivity to etomoxir, which blocks fatty acid oxidation (FAO), was dependent on the initial lipid profile of cell lines, suggesting that only a particular "lipidic phenotype" is sensitive to the drug targeting of FAO. We then showed that the lipid profiles of plasma samples from AML patients were significantly correlated with the prognosis of patients. In particular, we highlighted the impact of phosphocholine and phosphatidyl-choline metabolism on patients' survival. In conclusion, our data show that balance between lipid species is a phenotypic marker of the diversity of leukemic cells that significantly influences their proliferation and resistance to stress, and thereby, the prognosis of AML patients.

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca2+ signaling and motility in Toxoplasma gondii.

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca2+] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca2+ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K+] to activate cytosolic [Ca2+] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca2+ signaling and motility in Toxoplasma.

Identification of a Membrane Protein Required for Lipomannan Maturation and Lipoarabinomannan Synthesis in Corynebacterineae.

Mycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760 t-LM comprised a series of short LM species (8-27 residues long) containing an α1-6-linked mannose backbone with greatly reduced α1-2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane α1-6-mannosyltransferase involved in extending the α1-6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.

ALA10, a Phospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana.

The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature.

The lipoprotein LpqW is essential for the mannosylation of periplasmic glycolipids in Corynebacteria.

Phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) are essential components of the cell wall and plasma membrane of mycobacteria, including the human pathogen Mycobacterium tuberculosis, as well as the related Corynebacterineae. We have previously shown that the lipoprotein, LpqW, regulates PIM and LM/LAM biosynthesis in mycobacteria. Here, we provide direct evidence that LpqW regulates the activity of key mannosyltransferases in the periplasmic leaflet of the cell membrane. Inactivation of the Corynebacterium glutamicum lpqW ortholog, NCgl1054, resulted in a slow growth phenotype and a global defect in lipoglycan biosynthesis. The NCgl1054 mutant lacked LAMs and was defective in the elongation of the major PIM species, AcPIM2, as well as a second glycolipid, termed Gl-X (mannose-α1-4-glucuronic acid-α1-diacylglycerol), which function as membrane anchors for LM-A and LM-B, respectively. Elongation of AcPIM2 and Gl-X was found to be dependent on expression of polyprenol phosphomannose (ppMan) synthase. However, the ΔNCgl1054 mutant synthesized normal levels of ppMan, indicating that LpqW is not required for synthesis of this donor. A spontaneous suppressor strain was isolated in which lipoglycan synthesis in the ΔNCgl1054 mutant was partially restored. Genome-wide sequencing indicated that a single amino acid substitution within the ppMan-dependent mannosyltransferase MptB could bypass the need for LpqW. Further evidence of an interaction is provided by the observation that MptB activity in cell-free extracts was significantly reduced in the absence of LpqW. Collectively, our results suggest that LpqW may directly activate MptB, highlighting the role of lipoproteins in regulating key cell wall biosynthetic pathways in these bacteria.

Inositol lipid metabolism in mycobacteria: biosynthesis and regulatory mechanisms.

BACKGROUND: The genus Mycobacterium includes a number of medically important pathogens. The cell walls of these bacteria have many unique features, including the abundance of various inositol lipids, such as phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). The biosynthesis of these lipids is believed to be prime drug targets, and has been clarified in detail over the past several years. SCOPE OF REVIEW: Here we summarize our current understanding of the inositol lipid metabolism in mycobacteria. We will highlight unsolved issues and future directions especially in the context of metabolic regulation. MAJOR CONCLUSIONS: Inositol is a building block of phosphatidylinositol (PI), which is further elaborated to become PIMs, LM and LAM. d-myo-inositol 3-phosphate is an intermediate of the de novo inositol synthesis, but it is also the starting substrate for mycothiol synthesis. Controlling the level of d-myo-inositol 3-phosphate appears to be important for maintaining the steady state levels of mycothiol and inositol lipids. Several additional control mechanisms must exist to control the complex biosynthetic pathways of PI, PIMs, LM and LAM. These may include regulatory proteins such as a lipoprotein LpqW, and spatial separation of enzymes, such as the amphipathic PimA mannosyltransferase and later enzymes in the PIMs/LM biosynthetic pathway. Finally, we discuss mechanisms that underlie control of LM/LAM glycan polymer elongation. GENERAL SIGNIFICANCE: Mycobacteria have evolved a complex network of inositol metabolism. Clarifying its metabolism will not only provide better understanding of bacterial pathogenesis, but also understanding of the evolution and general functions of inositol lipids in nature.

Stress-induced synthesis of phosphatidylinositol 3-phosphate in mycobacteria.

Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. Here we show that Mycobacterium smegmatis and other Actinomycetes bacteria can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P). This lipid was transiently labeled with [(3)H]inositol. Sensitivity of the purified lipid to alkaline phosphatase, headgroup analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. This is the first example of de novo synthesis of PI3P in bacteria, and the transient synthesis in response to environmental stimuli suggests that some bacteria may have evolved similar lipid-mediated signaling pathways to those observed in eukaryotic cells.