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

Actin cytoskeleton Xin repeat proteins

available at www.sciencedirect.com www.elsevier.com/locate/yexcr Research Article Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VAS

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

Spatial and temporal characterization of <i>mmp17b</i> expression.

<p>A is RT-PCR showing temporal expression of <i>mmp17b</i> and <i>mmp17a</i> compared to β-actin. <i>Mmp17b</i> expression commences at around 18 hpf while <i>mmp17a</i> is expressed as early as 5 hpf. B-E are whole mount in situ hybridization (WISH) panels of <i>mmp17b</i> at different embryonic stages (B and B’, 17 hpf; C and C’, 20 hpf; D and D’, 26 hpf). Earlier in development <i>mmp17b</i> is expressed more dorsally and then moves more posterior and ventral as development continues. Panel E shows <i>mmp17b</i> fluorescent in situ hybridization (FISH) in red and immunofluorescence (IF) for Flk-GFP in a 26 hpf embryo, which illustrates lack of <i>mmp17b</i> expression in the vasculature. Panels F, G-H, I-J and K are three color WISH staining performed as described in materials and methods with different probes as indicated in each panel. In panel F, <i>mmp17b</i> (purple), lower left is 4D9 staining (green) the muscle pioneer cells, lower right is Flk-GFP marking (red) the endothelial cells, and upper right is a merge. In this panel you can observe that 4D9 staining is located in the same region as <i>mmp17b</i>. G-H panel shows three color ISH image of 26 hpf zebrafish trunk and plexus with <i>mmp17b</i> (blue), Flk-GFP (red), and <i>crestin</i> (green). In the trunk image, <i>crestin</i> and <i>mmp17b</i> are overlapping in expression suggesting co-expression of these two genes in the same cell. The expression of both <i>crestin</i> and <i>mmp17b</i> are more dorsal in posterior regions of the embryo. Panels I-J represent high-powered image of the panel G. J is an optical section of panel G. Panel K is a three color image of 26 hpf zebrafish trunk with <i>mmp17a</i> (green), <i>mmp17b</i> (blue), and Flk-GFP (red). This image illustrates that <i>mmp17a</i> and <i>mmp17b</i> are expressed in different regions of the developing embryo. B-D are lateral views, B’-D’ are dorsal views. D” is a close-up of the panel D. Panels L-N are single plane confocal images of a 26 hpf embryo stained for <i>mmp17b</i> using FISH (panel L, green), and immunostaining for sox10 cells labelled with RFP (panel M, red). Panel N is the merged images showing co-localization of <i>mmp17b</i> and sox10 as indicated by yellow color (arrowhead).</p

Mmp17b Is Essential for Proper Neural Crest Cell Migration <i>In Vivo</i>

<div><p>The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish <i>mmp17b. Mmp17b</i> is expressed post-gastrulation in the developing NC. Morpholino inactivation of <i>mmp17b</i> function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, <u>Re</u>version-inducing-<u>C</u>ysteine-rich protein with <u>K</u>azal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing <i>mmp17b</i>, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for <i>mmp17b</i> show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish <i>sensory deprived</i> (<i>sdp</i>). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.</p> </div

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