Sentrin/sumo Specific Proteases As Novel Tissue-selective Modulators Of Vitamin D Receptor-mediated Signaling

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Security and trust in mobile interactions: a study of users’ perceptions and reasoning

This paper describes an ongoing investigation into the trust and security concerns of users who carry out interactions in mobile and ubiquitous computing environments. The study is motivated by future scenarios in which it is envisioned that people may be able to spontaneously make their personal, mobile devices interact with other devices in such environments—environments which may b

Depletion of endogenous SENP1 results in a diminished induction of <i>CYP24A</i> mRNA expression by 1,25D.

<p>Caco-2 cells were seeded and co-transfected with 100nM Dharmafect SENP1 siRNA or Non-Targeting (NT) control or as described in methods. Cells were then treated with vehicle or 1,25D (10⁻⁸M) for 24 hours before RNA/protein extraction and subsequent analysis through RT-PCR/qRT-PCR and immunoblotting. <b>A</b>. RT-PCR depicting gene specific PCR products obtained from Caco-2 cells transfected with non-targeting (NT) and SENP1-specific siRNAs and subsequently treated with 1,25D or vehicle control. The lower panels describe Q-PCR analysis of the impact of NT and SENP1-specific siRNAs upon the mRNA expression within 1,25D treated Caco-2 cells of (<b>B</b>) <i>SENP1</i>, (<b>C</b>) <i>CYP24A1</i> and (<b>D</b>) <i>TRPV6</i>. The experiment depicted in <b>A</b> is representative of three independent experiments while <b>B, C</b> and <b>D</b> describe the average of three independent experiments where each data point represents the means (± SD) of triplicate assays (<i>n = </i>3) and where <i>ns</i> p≥0.05, ** p = 0.001–0.01, **** p<0.0001.</p

SENPs facilitate deSUMOylation of VDR.

<p>Depicted are cell-based SUMOylation assays performed as described in materials and methods using the following experimental conditions: A. HEK-293 cells received expression constructs for V5-VDR, His-SUMO2 and UBC9 or parent control. Cell lysates from each treatment group were incubated with V5-agarose beads and the resulting precipitated material subjected to western blot analysis with antibodies specific for 6x His (upper panel) or V5 (lower panel) epitope tags. The arrowheads indicate unconjugated and SUMO2-modified versions of V5-VDR. B. Where indicated, cells were co-transfected with V5-VDR, His-SUMO2, UBC-9 in combination with FLAG-SENP-1 or FLAG-SENP-2 with (-) denoting inclusion of appropriate parent vector control. The upper panel describes detection of SUMO2-conjugated VDR within precipitated lysates with expression of V5-VDR and Flag-SENP within the appropriate treatment groups confirmed in the lower panel. C. Cells received the indicated combination of V5-VDR, His-SUMO2, UBC-9 along and FLAG-SENP-2 or parent vector control. Upper panel depicts detection of SUMO2 conjugated VDR and reversal of this modification by SENP2. Lower panels confirm the expression status of unconjugated SUMO2.</p

Transcriptional activities of VDR and RXRα in MCF-7 cells are differentially modulated by SENPs.

<p><b>A</b>. MCF-7 cells were seeded under conditions defined in methods and received the indicated SENP expression construct or corresponding parent vector control in combination with the pFLUC reporter and pCMVBD-VDRFL or pCMVBD- RXRαFL. <b>B</b>. pSG5-hVDR and pSG5-hRXRα were co-transfected into MCF-7 cells in combination with the pMCS-24OHase reporter and appropriate Flag-SENP expression plasmid or parent vector control. Cells were then dosed for 24 hours with the 1,25D (10⁻⁸M) or 9-<i>cis</i> RA (10⁻⁶M) cognate ligands or vehicle control where indicated. The fold-stimulation (ratio of activity in the presence:absence of ligand) is indicated above each set of bars. The results are presented as means (± SD) from three independent experiments with each data point measured in triplicate (<i>n</i> = 3) where <i>ns</i> p≥0.05, ** p = 0.001–0.01, **** p<0.0001.</p

SENP1 selectively potentiates the transcriptional activities of VDR and RXRα.

<p>HEK-293 cells were transfected with the pFLUC reporter vector in combination with the appropriate pCMVBD-based expression vector for each Gal4-nuclear receptor (LBD) hybrid protein under evaluation. Where indicated, cells also received the SENP1 expression plasmid, pFLAG-CMV-SENP1 (200 ng), or an equivalent amount of parent vector (minus SENP1 insert) as control. The total amount of DNA in each transfection was kept at a constant value through inclusion of the appropriate amount of empty expression vector. Treated cells were dosed with the appropriate cognate ligand or vehicle control for a period of 24 hours before measurement of luciferase activity. All ligands were used at a concentration of 10⁻⁶M, except 1,25D (10⁻⁸M). After normalization for transfection efficiency based on the activity of the pRL-TK control, results were expressed as relative luciferase units per well. The fold-stimulation (ratio of activity in the presence:absence of ligand) is indicated above each set of bars. Data represents analysis of at least three independent experiments with each treatment run in triplicate (<i>n = </i>3) mean ± SD; where <i>ns</i> = p≥0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001 and **** p<0.0001.</p

SENPs potentiate transactivation of the full length VDR protein.

<p><b>A</b>. HEK-293 cells received pCMVBD-VDRFL that encodes Gal4DBD fused to full length human VDR, in combination with the pFR-LUC reporter and the indicated SENP expression construct or parent vector control. The lower panel depicts an immunoblot analysis in which combined cellular lysates from each treatment group were probed with the antibodies specific for VDR (9A7) and β-actin. <b>B</b>. pSG5-hVDR that expresses full length human VDR were co-transfected into HEK-293 cells in combination with the reporter construct pMCS-24OHase, pSG5-hRXRα and the appropriate Flag-SENP expression plasmid or parent vector control. Transfected cells were incubated with 1,25D (10⁻⁸M) or vehicle for 24 hours before measurement of luciferase activity. The lower panel depicts immunoblot analysis of cell lysates transfected with the pFlag-SENP1 or pFlag-SENP2 expression constructs and then probed with the Flag or β-actin specific antibodies, with WCE representing the untransfected whole cell lysate control. The fold stimulation are expressed as means (± SD) and results presented are the average of three independent experiments, where <i>n = 3</i> in each assay. <b>C</b>. Caco-2 cells were co-transfected with pCMVBD-VDRFL and pFR-LUC reporter in combination with the appropriate SENP expression constructs or parent vector control. <b>D</b>. Caco-2 cells received pSG5-hVDR+pSG5-hRXRα, the pMCS-24OHase reporter, together with the indicated SENP expression plasmid or parent vector control. Transfected cells were then incubated with 1,25D (10⁻⁸M) or vehicle control for 24 hours before measurement of luciferase activity. All depicted data represents an average of four independent experiments with values expressed as means (± SD) of triplicate assays (<i>n</i> = 3) where ** p = 0.001–0.01, *** p = 0.0001 - 0.001, **** p<0.0001.</p

SENP1 potentiates the hormone responsiveness of an endogenous vitamin D target gene.

<p>Caco-2 cells were plated as described in methods and co-transfected with pSG5hVDR and, where indicated, pFLAG-CMV-SENP1 or corresponding parent vector control. Following incubation for a period of 24 hours with 1,25D (10 nM) or vehicle control, total RNA was isolated from cells, converted to cDNA and real time PCR analysis performed. The fold-stimulation of <i>CYP24A1</i> mRNA expression achieved through the presence of 1,25D is indicated above the black bars. The depicted data represents an average of three independent experiments with each data point a means (± SD) of triplicate assays (<i>n = </i>3) and **** p<0.0001.</p