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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Pro-oncogenic roles of HLXB9 in insulinoma cells through interaction with Nono and downregulation of the c-Met inhibitor Cblb [Cell Biology]

September 4th, 2015 by

Pancreatic islet β-cells that lack the MEN1 encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the β-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting β-cell tumors (insulinomas). In this study, we investigated the binding-partners and target-genes of phospho-HLXB9 in mouse insulinoma MIN6 β-cells. Co-immunoprecipitation coupled with mass spectrometry showed a significant association of phospho-HLXB9 with the survival factor p54nrb/Nono (54 kDa nuclear RNA binding protein, non-POU-domain-containing octamer). Endogenous phospho-HLXB9 co-localized with endogenous Nono in the nucleus. Overexpression of HLXB9 decreased the level of overexpressed Nono but not endogenous Nono. Anti-phospho-HLXB9 chromatin-immunoprecipitation followed by deep sequencing (ChIP-Seq) identified the c-Met inhibitor, Cblb, as a direct phospho-HLXB9 target gene. Phospho-HLXB9 occupied the promoter of Cblb and reduced the expression of Cblb mRNA. Cblb overexpression or HLXB9 knockdown decreased c-Met protein and reduced cell migration. Also, increased phospho-HLXB9 coincided with reduced Cblb and increased c-Met in insulinomas of two mouse models of menin loss. These data provide mechanistic insights into the role of phospho-HLXB9 as a pro-oncogenic factor by interacting with a survival factor, and by promoting the oncogenic c-Met pathway. These mechanisms have therapeutic implications for reducing β-cell proliferation in insulinomas by inhibiting phospho-HLXB9 or its interaction with Nono, and modulating the expression of its direct (Cblb) or indirect (c-Met) targets. Our data also implicate the use of pro-oncogenic activities of phospho-HLXB9 in β-cell expansion strategies to alleviate β-cell loss in diabetes.

Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2 [Gene Regulation]

September 4th, 2015 by

Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis.

Binding of galectin-3, a {beta}-galactoside-binding lectin, to MUC1 protein enhances phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, promoting tumor cell malignancy [Signal Transduction]

September 4th, 2015 by

Both mucin 1 (MUC1) and galectin-3 are known to be overexpressed in various malignant tumors and associated with a poor prognosis. It has been extensively reported that MUC1 is involved in potentiation of growth factor-dependent signal transduction. Because some carbohydrate moieties carried on MUC1 change to preferable ones for binding of galectin-3 in cancer cells, we speculated that MUC1-mediated signaling may occur through direct binding of galectin-3. Immunochemical studies showed that the distribution of galectin-3 coincided with that of MUC1 in various human tumor tissues but not in human nonmalignant tissues, and the level of galectin-3 retained on the surface of various cancer cells paralleled that of MUC1. Treatment of MUC1-expressing cells with galectin-3 induced phosphorylation of ERK1/2 and Akt following enhanced phosphorylation of MUC1 C-terminal domain (MUC1-CD), consistently promoting tumor cell malignancy. It is also noted that this enhanced phosphorylation occurred independently of EGF receptor (EGFR)-mediated signaling in both EGFR and MUC1 expressing cells, and multivalency of galectin-3 was important for initiation of MUC1-mediated signaling. Expectedly, both silencing of endogenous galectin-3 and treatment with galectin-3 antagonists down-regulated cell proliferation of MUC1-expressing cells. These results suggest that the binding of galectin-3 to MUC1 plays a key role in MUC1-mediated signaling. Thus, constitutive activation of MUC1-mediated signaling in an autocrine/paracrine manner due to ligation of galectin-3 promotes uncontrolled tumor cell malignancy. This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated signaling pathway.
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Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins [Protein Structure and Folding]

September 4th, 2015 by Bondos, S. E., Swint-Kruse, L., Matthews, K. S.

To modulate transcription, a variety of input signals must be sensed by genetic regulatory proteins. In these proteins, flexibility and disorder are emerging as common themes. Prokaryotic regulators generally have short, flexible segments, whereas eukaryotic regulators have extended regions that lack predicted secondary structure (intrinsic disorder). Two examples illustrate the impact of flexibility and disorder on gene regulation: The prokaryotic LacI/GalR family, with detailed information from studies on LacI, and the eukaryotic family of Hox proteins, with specific insights from investigations of Ultrabithorax (Ubx). The widespread importance of structural disorder in gene regulatory proteins may derive from the need for flexibility in signal response and, particularly in eukaryotes, in protein partner selection.