Genome-wide Mechanosensitive MicroRNA (MechanomiR) Screen Uncovers Dysregulation of their Regulatory Networks in the mdm Mouse Model of Muscular Dystrophy [Gene Regulation]

August 13th, 2015 by Mohamed, J. S., Hajira, A., Lopez, M. L., Boriek, A. M.

Muscular dystrophies (MDs) are a heterogeneous group of genetic and neuromuscular disorders, which result in severe loss of motor ability and skeletal muscle mass and function. Aberrant mechanotransduction and dysregulated-microRNA pathways are often associated with the progression of MD. Here, we hypothesized that dysregulation of mechanosensitive microRNAs (mechanomiRs) in dystrophic skeletal muscle play major roles in the progression of MD. To test our hypothesis, for the first time, we performed a genome-wide expression profile of anisotropically-regulated mechanomiRs and bioinformatically analyzed their target gene networks, and we assessed their roles in the advancement of MD using diaphragm muscles from wild-type and mdm (muscular dystrophy with myositis) mouse, an animal model of human tibial MD (titinopathy). We show that ex-vivo anisotropic mechanical stretch significantly alters the miRNA expression profile in diaphragm from WT and mdm mice, and as a result, some of the genes associated with MDs are dysregulated in mdm mice due to differential regulation of a distinct set of mechanomiRs. Interestingly, we found a contrasting expression pattern of the highly expressed let-7 family mechanomiRs let-7e-5p and miR-98-5p, and their target genes associated with extracellular matrix (ECM) and transforming growth factor-β signaling (TGF-β) pathways, respectively between WT and mdm mice. Gain- and loss-of-function analysis of let-7e-5p in myocytes isolated from the diaphragms of WT and mdm mice confirmed Col1a1, Col1a2, Col3a1, Col24a1, Col27a1, Itga1, Itga4, Scd1 and Thbs1 as target genes of let-7e-5p. Furthermore, we found that miR-98 negatively regulates myoblast differentiation. Our study therefore introducesanother biological player in the regulation of skeletal muscle structure and function that may contribute to unexplained disorders of MD.
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Allosteric Activation of Bacterial Swi2/Snf2 Protein RapA by RNA Polymerase: Biochemical and Structural Studies [Protein Structure and Folding]

August 13th, 2015 by

Members of the Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. It is not known, however, how the RapA-RNAP interaction activates the enzyme. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAΔN). Here, we report the ATPase activity of RapA and RapAΔN, in the absence or presence of RNAP, and the solution structures of RapA and RapAΔN, either ligand-free or in complex with RNAP. Determined by small-angle X-ray scattering, the solution structures reveal a new conformation of RapA, define the binding mode and binding site of RapA on RNAP, and show that the binding sites of RapA and σ70 on the surface of RNAP largely overlap. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These and previous findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins in general and of RapA in particular. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription.
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Degenerate CP1 Domain from Human Mitochondrial Leucyl-tRNA Synthetase [RNA]

August 13th, 2015 by

The connective polypeptide 1 (CP1) editing domain of leucyl-tRNA synthetase (LeuRS) from various species either harbors a conserved active site to exclude tRNA mis-charging with non-cognate amino acids or is evolutionarily truncated or lost because there is no requirement for high translational fidelity. However, human mitochondrial LeuRS (hmtLeuRS) contains a full-length but degenerate CP1 domain which has mutations in some residues important for post-transfer editing. The significance of such an inactive CP1 domain and a translational accuracy mechanism with different non-cognate amino acids are not completely understood. Herein, we identified the essential role of evolutionarily divergent CP1 domain in facilitating hmtLeuRS's catalytic efficiency and endowing enzyme with resistance to AN2690, broad-spectrum drug acting on LeuRSs. In addition, the canonical core of hmtLeuRS is not stringent for non-cognate norvaline (Nva) and valine (Val). HmtLeuRS has a very weak tRNA-independent pre-transfer editing activity for Nva, which is insufficient to remove mis-activated Nva. Besides, hmtLeuRS chimeras fused with a functional CP1 domain from LeuRSs of other species, regardless of origin, showed restored post-transfer editing activity, and acquired fidelity during aminoacylation. The present work offers a novel perspective on the role of the CP1 domain in optimizing aminoacylation efficiency.

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa is a 3-Mercaptopropionate Dioxygenase [Enzymology]

August 13th, 2015 by

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear-iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed p3MDO is expressed in low levels suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid (3-MPA) in vitro, with a marked preference for 3-MPA. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mossbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.

A novel GLP-1 receptor interacting protein ATP6ap2 regulates insulin secretion in pancreatic beta cells [Metabolism]

August 13th, 2015 by

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid (MYTH) assay to identify novel GLP1R interactors in both mouse and human islets. Among these, the V-type ATPase associated protein 6 (ATP6ap2), was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion, however, siRNA knockdown decreased both glucose stimulated- and GLP1-induced insulin secretion (GIIS). Decreases in GIIS were accompanied by attenuated GLP1 stimulated cAMP accumulation. Since ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knock-down but paradoxically increased proinsulin secretion. Importantly as GLP1R interactor, ATP6ap2 was required for GLP1 induced Ca2+ influx, in part explaining decreased insulin secretion in ATP6ap2 knock-down cells. Taken together our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signalling and insulin processing to affect insulin secretion.

Unliganded Fibroblast Growth Factor Receptor 1 Forms Density-Independent Dimers [Molecular Bases of Disease]

August 13th, 2015 by Comps-Agrar, L., Dunshee, D. R., Eaton, D. L., Sonoda, J.

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Forster resonance energy transfer (TR-FRET) combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full length FGFR1 at the cell-surface with or without the coreceptor βKlotho (hKLB). Using this approach, we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR), and was further increased by addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and KLB. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.

Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental and theoretical IR spectroscopy [Protein Structure and Folding]

August 13th, 2015 by

Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR-difference spectroscopy and QM/MM simulations we elucidate that the Mg2+ coordination by the phosphate groups, which varies largely among the X-ray structures, is the same for Ran and Ras. A new X-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg2+ in GTPases. The Mg2+ coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr39 side chain of Ran between the γ-phosphate and Gln69 prevents the optimal positioning of the attacking water molecule by the Gln69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr25 to the α-phosphate in Ran. By integration of X-ray structure analysis, experimental and theoretical IR spectroscopy the catalytic center of the X-ray structural models are further refined to sub-Ångstrom resolution, allowing an improved understanding of catalysis.
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A novel basal body protein and Polo-like kinase substrate is required for basal body segregation and flagellum adhesion in Trypanosoma brucei [Microbiology]

August 13th, 2015 by Hu, H., Zhou, Q., Li, Z.

The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confines TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of the bilobe resident TbCentrin2. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.
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Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content [Membrane Biology]

August 13th, 2015 by

Cancer cells emit extracellular vesicles (EVs) containing unique molecular signatures. Here we report that oncogenic EGF receptor (EGFR) and its inhibitors reprogram phosphoproteomes and cargo of tumor cell derived EVs. Thus, phosphorylated EGFR (P-EGFR) and several other receptor tyrosine kinases (RTKs) can be detected in EVs purified from plasma of tumor bearing mice and from conditioned media of cultured cancer cells. Treatment of EGFR-driven tumor cells with second generation EGFR kinase inhibitors (EKIs), including CI-1033 and PF-00299804, but not with anti-EGFR antibody (Cetuximab), or etoposide triggers a burst in emission of exosome-like EVs containing EGFR, P-EGFR and genomic DNA (exo-gDNA). The EV release can be attenuated by treatment with inhibitors of exosome biogenesis (GW4869) and caspase pathways (ZVAD). The content of P-EGFR isoforms (Y845, Y1068 and Y1173), ERK and AKT varies between cells and their corresponding EVs, and as a function of EKI treatment. Immunocapture experiments reveal the presence of EGFR and exo-gDNA within the same EV population following EKI treatment. These findings suggest that targeted agents may induce cancer cells to change the EV emission profiles reflective of drug-related therapeutic stress. We suggest that EV-based assays may serve as companion diagnostics for targeted anticancer agents.
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Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4{alpha} [Metabolism]

August 13th, 2015 by

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein (apo)B-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short-term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long-term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long-term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.