Loss of SIRT3 Provides Growth Advantage for B Cell Malignancies [Metabolism]

December 2nd, 2015 by

B cell malignancies comprise a diverse group of cancers that proliferate in lymph nodes, bone marrow, and peripheral blood. Sirtuin 3 (SIRT3) is the major deacetylase within the mitochondrial matrix that promotes aerobic metabolism and controls reactive oxygen species (ROS) by deacetylating and activating isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2). There is controversy as to whether SIRT3 acts as an oncogene or a tumor suppressor, and here we investigated its role in B cell malignancies. Using mantle cell lymphoma (MCL), we found that lower SIRT3 protein expression was associated with worse overall survival. Further, SIRT3 protein expression was reduced in chronic lymphocytic leukemia (CLL) primary samples and malignant B cell lines compared to primary B cells from healthy donors. This lower level of expression correlated with hyperacetylation of IDH2 and SOD2 mitochondrial proteins, lowered enzymatic activities and higher ROS levels. Overexpression of SIRT3 decreased proliferation and diminished the Warburg-like phenotype in SIRT3-deficient cell lines, and this effect is largely dependent on deacetylation of IDH2 and SOD2. Lastly, depletion of SIRT3 from malignant B cell lines resulted in greater susceptibility to treatment with an ROS scavenger but did not result in greater susceptibility to inhibition of the HIF-1alpha pathway, suggesting that loss of SIRT3 increases proliferation via ROS-dependent but HIF-1alpha-independent mechanisms. Our study suggests that SIRT3 acts as a tumor suppressor in B cell malignancies, and activating the SIRT3 pathway might represent a novel therapeutic approach for treating B cell malignancies.

Purinergic Receptor P2Y2 Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP-1 Transcription Factor Site and Negative Regulator [Signal Transduction]

December 2nd, 2015 by Liu, Y., Zhang, L., Wang, C., Roy, S., Shen, J.

We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here we report the role of a newly identified AP-1 consensus sequence in TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at -1363 bp of human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2 and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2, but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation while Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y GPCR control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.
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The Prodomain-Bound Form of Bone Morphogenetic Protein 10 is Biologically Active on Endothelial Cells [Cell Biology]

December 2nd, 2015 by

BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality due to impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling.

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis [Protein Structure and Folding]

December 2nd, 2015 by

Enterococcus faecalis is a gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8°C, which is much higher than that of Escherichia coli ACP (67.2°C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (3-17), helix II (39-53), helix III (60-64), and helix IV (68-78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between F45 in helix II with F18 in the α1α2 loop and a hydrogen bonding between S15 in helix I and I20 in the α1α2 loop, resulting in its high thermal stability. F45-mediated hydrophobic packing may block acyl chain binding sub-pocket II entry. Furthermore, S58 in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.
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{beta}-subunit Binding is Sufficient for Ligands to open the Integrin {alpha}IIb{beta}3 Headpiece [Cell Biology]

December 2nd, 2015 by Lin, F.-Y., Zhu, J., Eng, E. T., Hudson, N. E., Springer, T. A.

The platelet integrin αIIbβ3 binds to a KQAGDV motif at the fibrinogen γ-chain C-terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and β subunits; the basic Lys or Arg sidechain hydrogen bonds to the αIIb-subunit and the acidic Asp sidechain coordinates to a metal ion held by the β3-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket towards αIIb, with movement of the βI-domain β1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg sidechain and αIIb. Here we test whether ligand interactions with β3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbβ3 headpiece with affinity comparable to the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbβ3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the β3 subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required.

The C-terminal region and SUMOylation of Cockayne syndrome group B protein play critical roles in transcription-coupled nucleotide excision repair [DNA and Chromosomes]

November 30th, 2015 by Sin, Y., Tanaka, K., Saijo, M.

Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a sub-pathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30 amino acid residues were required for UV resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier-2/3 in a UV-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues; however, the substitution of lysine residues in this region to arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region to arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER.
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Transcriptional Regulation of Cystathionine-{gamma}-lyase in Endothelial cells by NADPH oxidase 4-dependent Signaling [Gene Regulation]

November 30th, 2015 by

The gasotransmitter, hydrogen sulfide (H2S) is recognised as an important mediator of endothelial cell homeostasis and function, which impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species (ROS), H2O2. However, the cellular source of H2O2, and the redox-dependent molecular signaling pathway which modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor (HRI) kinase /eIF2α /Activating Transcription Factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE, to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic (eNox4 Tg) mice, compared to wild-type littermate controls. Using wire myography we demonstrate that eNox4 Tg mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with the CSE inhibitor, Propargyl-glycine. We therefore conclude that positive transcriptional regulation of CSE by Nox4 in endothelial cells results in increased H2S production, and contributes to the regulation of vascular tone.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in vitro Reconstitution of Lipid II Synthesis Pathway [Enzymology]

November 30th, 2015 by

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been amongst the first drug targets, compounds directed against the membrane integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi and Escherichia coli as well as of Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B.subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY/nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane associated protein MurG As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin and tunicamycin.

Binding Affinities among DNA Helicase-Primase, DNA Polymerase and Replication Intermediates in the Replisome of Bacteriophage T7 [Enzymology]

November 30th, 2015 by Zhang, H., Tang, Y., Lee, S.-J., Wei, Z., Cao, J., Richardson, C. C.

The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed the loop is released and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7 the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase-DNA complex are much faster at a nick DNA than the release from an ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.
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Uracil-DNA glycosylase UNG promotes Tet-mediated DNA demethylation [Gene Regulation]

November 30th, 2015 by

In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in ESCs and iPSCs, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. In order to search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation.