Characterization of theThreshold for NAD(P)H:quinone Oxidoreductase Activity in Intact Sulforaphane-treated Pulmonary Arterial Endothelial Cells

Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5 μM, 24 h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP+ in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP+ ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the Km for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold

Protein Expression, Characterization and Activity Comparisons of Wild Type and Mutant DUSP5 Proteins

Background The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies

Sucrose Nonfermenting-Related Kinase Enzyme-Mediated Rho-Associated Kinase Signaling is Responsible for Cardiac Function.

BACKGROUND: Cardiac metabolism is critical for the functioning of the heart, and disturbance in this homeostasis is likely to influence cardiac disorders or cardiomyopathy. Our laboratory has previously shown that SNRK (sucrose nonfermenting related kinase) enzyme, which belongs to the AMPK (adenosine monophosphate-activated kinase) family, was essential for cardiac metabolism in mammals. Snrk global homozygous knockout (KO) mice die at postnatal day 0, and conditional deletion of Snrk in cardiomyocytes (Snrk cmcKO) leads to cardiac failure and death by 8 to 10 months. METHODS AND RESULTS: We performed additional cardiac functional studies using echocardiography and identified further cardiac functional deficits in Snrk cmcKO mice. Nuclear magnetic resonance-based metabolomics analysis identified key metabolic pathway deficits in SNRK knockdown cardiomyocytes in vitro. Specifically, metabolites involved in lipid metabolism and oxidative phosphorylation are altered, and perturbations in these pathways can result in cardiac function deficits and heart failure. A phosphopeptide-based proteomic screen identified ROCK (Rho-associated kinase) as a putative substrate for SNRK, and mass spec-based fragment analysis confirmed key amino acid residues on ROCK that are phosphorylated by SNRK. Western blot analysis on heart lysates from Snrk cmcKO adult mice and SNRK knockdown cardiomyocytes showed increased ROCK activity. In addition, in vivo inhibition of ROCK partially rescued the in vivo Snrk cmcKO cardiac function deficits. CONCLUSIONS: Collectively, our data suggest that SNRK in cardiomyocytes is responsible for maintaining cardiac metabolic homeostasis, which is mediated in part by ROCK, and alteration of this homeostasis influences cardiac function in the adult heart

Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets

Background: Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. We present a p-nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5. Methods: pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.6 ± 0.4 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual-phosphorylated ERK2 substrate tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate. Results: The most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 μM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD3 _2320) with IC50 of 33 μM that is similar to NTS and does not aggregate. Conclusions: The new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs. Keywords: DUSP5, Phosphatase, Drug discovery, Docking, Suramin, Vascular anomalie

RECK blocks MMP17 activity <i>in vitro</i>.

<p>Elavl1 staining of control MO (cMO) (A) or <i>mmp17b</i> MO1 (B) injected 72 hpf embryos. Asterisks show the location of DRGs (dorsal root ganglia). <i>Mmp17b</i> knockdown embryos lack proper DRG development compared to controls. Quantitation of number of DRGs and migrated DRGs are indicated in panel C. Panel D is immunoprecipitation of myc-Mmp17b and RFP-Reck in Cos7 cells. Left panels indicate input of the two proteins in the sample, and right panels are RFP antibody immunoprecipitated samples followed by western blot. Arrow shows the pull down Mmp17b protein. Panel E is immunoprecipitation of myc-MMP17 and RFP-Reck in Cos7 cells. Left panel indicates input of MMP17 proteins in the lysate and the right panels indicate RFP antibody immunoprecipitated samples followed by western for myc epitope. Panel F is western blot of myc-MMP17 unactivated and activated with 4-aminophenylmercuric acetate (APMA) using MMP17 antibody. Activated MMP lanes show multiple bands, which are absent in samples co-incubated with RECK protein. Panel G is FRET-based fluorescence readout for MMP activity using the samples run on western blots in panel F.</p

Bioinformatic and biochemical analysis of Mmp17b.

<p>Panel A depicts amino acid alignment of human and mouse MMP17 and zebrafish Mmp17a and Mmp17b. Red color indicates conserved amino acids and blue color indicates less conserved regions. B is a cartoon of Mmp17b protein. The predicted domains include a zinc catalytic domain, hemopexin-like domains, and a GPI-anchor. C and D are myc and MMP17 western blots of HEK293T cell lysates respectively. Supernatant (S) and pellet (P) fractions were generated as described in Methods S1. Mmp17b protein is observed only in the P fraction. MMP17 and MMP25 proteins are more robustly expressed, and were observed in both S and P fractions. D depicts bands at the proper size for Mmp17b and MMP17 with some cross reactivity to MMP25. Bands of higher molecular weight are also observed. UT = Untransfected, S = supernatant, P = pellet, + = positive control, E = empty vector control. E-J are myc tagged <i>MMP17</i> and myc-HIS tagged <i>MMP17b</i> cDNAs transiently expressed in HEK293T cells. The enriched metalloproteinase fusion proteins were detected using human specific MMP17 antibody (E & H; shown in green) and a caveolin mAB antibody (F & I; shown in red). The overlay images (G & J) show co-localization of caveolin with MMPs in positively transfected cells. Image micrograph depicting nucleus stained with DAPI not shown. Scale bars are 10 micron.</p

Mmp17b is involved in neural crest patterning.

<p>A-C and D-F are WISH staining for <i>crestin</i> in control MO (cMO) (A), MO1 (B), and MO2 (C) injected 26 hpf or DMSO (D), Marimastat (E) and ONO-4817 (F) treated 26 hpf embryos. A’-C’ are high powered images of the trunk regions of A-C. Arrowheads indicate <i>crestin</i>^<i>+</i> cells misplaced in the trunk. There is a mis-patterning of <i>crestin</i> in the trunk of the MO1 and MO2 injected embryos compared to control. There is also an accumulation of <i>crestin</i>⁺ cells in the posterior of the embryo (white bracket) compared to control. This is quantitated in panel G. N=25 for cMO; n=19 for MO1; n=18 for MO2. In panels D-F, WISH staining for <i>crestin</i> in MMP inhibitor treated 26 hpf embryos shows mis-patterning similar to <i>mmp17b</i> KD embryos (A-C). An accumulation of <i>crestin</i>⁺ cells in the posterior of the MMP inhibitor treated embryos is also observed (red brackets). This is quantitated in panel H. N=12 for DMSO; n=8 for ONO 4817; n=10 for Marimastat. I-K shows melanocyte quantitation done on 72 hpf fish. Dorsal images were taken of 72 hpf fish injected with either control MO (J) or <i>mmp17b</i> MO1 (I). The number of medial (M, red arrow) and lateral (L, red arrow) melanocytes is counted between the two vertical bars illustrated in panels I and J for 10 fish in each category. The results were quantitated in panel K. The medial cells were not statistically different but the lateral cells were at a p-value of less than 0.05.</p