Microsomal triglyceride transfer protein transfers and determines plasma concentrations of ceramide and sphingomyelin, but not glycosylceramide [Metabolism]

September 8th, 2015 by

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin, but normal hexosylceramide, lactosylceramide and different sphingosines compared with unaffected controls. Further, similar differential effects on plasma sphingolipids were seen in liver and intestine specific MTP knockout (L,I-Mttp-/-) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis, but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion, but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.
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Systematic analysis of mycobacterial acylation reveals first example of acylation-mediated regulation of enzyme activity of a bacterial phosphatase [Signal Transduction]

September 8th, 2015 by

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins is not studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys224. This residue is situated in a lid region that covers enzyme′s active site. Consequently, acetylation and succinylation negatively regulates the activity of PtpB.
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G0/G1 switch gene-2 regulates cardiac lipolysis [Metabolism]

September 8th, 2015 by

The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids (FAs) to prevent lipotoxicity, while TAG hydrolysis (lipolysis) remobilizes FAs from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G0/G1 switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild type and mutant mice we show that (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2-overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL-deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.

Na-H exchanger isoform-2 (NHE2) mediates butyrate-dependent Na+ absorption in dextran sulfate sodium (DSS)-induced colitis [Cell Biology]

September 8th, 2015 by Rajendran, V. M., Nanda Kumar, N. S., Tse, C. M., Binder, H. J.

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na+ absorption. Although colonic Na+ absorption is mediated by both epithelial Na+ channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na+ absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na+ absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na+ fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO3--dependent and butyrate-dependent Na+ absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO3--dependent Na+ absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO3--dependent and butyrate-dependent Na+ absorption, while DSS- induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO3--dependent) Na+ absorption. In in vivo loop studies HCO3--Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, while in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO3--Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-medated Na+ absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea.

Sph3 is a glycoside hydrolase required for the biosynthesis of galactosaminogalactan in Aspergillus fumigates [Enzymology]

September 4th, 2015 by

Aspergillus fumigatus is the most virulent species within the Aspergillus genus, and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, D166, E167, and E222, which are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of D166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GHXXX.

A novel peroxisome proliferator-activated receptor (PPAR){gamma} agonist 2-hydroxyethyl 5-chloro-4,5-didehydroȷasmonate exerts anti-inflammatory effects in colitis [Signal Transduction]

September 4th, 2015 by

Inflammatory bowel disease (IBD) is a chronic inflammatory disease with increasing incidence and prevalence worldwide. Here we investigated the newly synthesized jasmonate analogue 2-hydroxyethyl 5-chloro-4,5-didehydrojasmonate (J11-Cl) for its anti-inflammatory effects on intestinal inflammation. First of all, to test whether J11-Cl can activate peroxisome proliferator-activated receptors (PPARs), we performed docking simulation because J11-Cl has structural similarity with anti-inflammatory 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of endogenous ligands of PPARγ. J11-Cl bound to the ligand binding domain of PPARγ in the same manner as 15d-PGJ2 and rosiglitazone, and significantly increased transcriptional activity of PPARγ. In animal experiments, colitis was significantly reduced in mice with J11-Cl treatment, determined by analyses of survival rate, body weight changes, clinical symptoms and histological evaluation. Moreover, J11-Cl decreased production of pro-inflammatory cytokines including IL-6, IL-8 and G-CSF as well as chemokines including chemokine (C-C motif) ligand (CCL)20, chemokine (C-X-C motif) ligand (CXCL)2, CXCL3 and chemokine (C-X3-C motif) ligand 1 (CX3CL1) in colon tissues, and LPS or TNF-α-stimulated macrophages and epithelial cells. In contrast, production of anti-inflammatory cytokines including IL-2 and IL-4 as well as the proliferative factor, GM-CSF, was increased by J11-Cl. Furthermore, inhibition of MAPKs and NF-κB activation by J11-Cl was also observed. J11-Cl reduced intestinal inflammation by increasing transcriptional activity of PPARγ and modulating inflammatory signaling pathways. Therefore, our study suggests that J11-Cl may serve as a novel therapeutic agent against IBD.
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Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/IGF-1 Signalling Pathway [Metabolism]

September 4th, 2015 by

The insulin/IGF-1 signalling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output of for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation and Akt signalling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterised by marked disruption of both metabolism and growth factor signalling.

The dNTP triphosphohydrolase activity of SAMHD1 contributes to the mitochondrial DNA depletion associated with genetic deficiency of deoxyguanosine kinase [Molecular Bases of Disease]

September 4th, 2015 by Franzolin, E., Salata, C., Bianchi, V., Rampazzo, C.

The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Downregulation of SAMHD1 by siRNA expands all four dNTP pools with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in wt cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus.
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Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein [Gene Regulation]

September 4th, 2015 by Katsuma, S., Sugano, Y., Kiuchi, T., Shimada, T.

We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH-tandem zinc finger (ZF) protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two ZF domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, 301-Cys and 304-Cys, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.

Mapping key residues of ISD11 critical for NFS1-ISD11 subcomplex stability: Implications in the development of mitochondrial disorder, COXPD19 [Protein Structure and Folding]

September 4th, 2015 by Saha, P. P., Srivastava, S., Kumar S. K., P., Sinha, D., D'Silva, P.

Biogenesis of iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of Fe-S cluster assembly process is assisted by NFS1-ISD11 complex, which delivers sulfur to the scaffold protein ISCU during Fe-S cluster synthesis. Although, ISD11 is an essential protein but its cellular role in the Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (F40), helix 3 (L63, R68, Q69, I72, Y76) and C-terminal segment (L81, E84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of mitochondrial genetic disorder, COXPD19. Our findings highlight that, ISD11 R68A/L mutation display reduced affinity to form a stable subcomplex with NFS1, thereby fails to prevent NFS1 aggregation resulting impairment of Fe-S cluster biogenesis. The prime affected machinery is the ETC complex which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and Reactive Oxygen Species (ROS), leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.
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