<i>Shigella</i> infection increases PML-NB number.

<p>HeLa cells were not infected (UI), infected with a non-invasive strain (BS176), or infected with an invasive wild-type strain (M90T) as indicated, both strains contained a plasmid expressing the <i>afaE</i> adhesin (A). Cells were incubated for 3 hours, fixed and immunostained for PML (red). M90T-infected cells with normal and high levels of PML nuclear bodies are shown as indicated. DNA was visualized with DAPI (blue) and white arrow heads indicate PML NB “doublets”. Scale bars = 10μm. The average number of PML bodies per cell for each treatment condition was quantified (B) and the <i>p</i>-value of Student’s t-test was calculated (n.s. = not significant). The percentage of cells containing either 0, 1–5, 6–10 or 11+ PML bodies was also determined (C) and the <i>p</i>-value of Student’s t-test was calculated. Values in B and C represent the mean +/- SE, n = 3. Total PML protein levels for each treatment condition was determined by Western blot analysis (D).</p

A decrease in SUMO-conjugated proteins accompanies <i>Shigella</i> infection.

<p>A comparison of SUMO-conjugated proteins from HeLa cells infected with wild type <i>Shigella</i> (M90T), a non-invasive strain (BS176), or uninfected cells for 1, 2, and 3 hours, both strains contained a plasmid expressing the <i>afaE</i> adhesin. Whole cell lysates were prepared from infected cells, separated on 7% polyacrylamide gels and immunoblotted using antibodies specific for SUMO1 (A) or SUMO2/3 (B). In (C) whole cell lysates from HeLa cells infected with wild type <i>Shigella</i> (M90T), a non-invasive mutant defective in the T3SS (<i>mxiD</i>), or an invasive mutant that does not produce a number of effectors (<i>mxiE</i>), was analyzed by immunoblotting using SUMO1-specific antibodies. Immunoblotting using GAPDH served as a loading control.</p

<i>Shigella</i> Infection Interferes with SUMOylation and Increases PML-NB Number

<div><p>Shigellosis is a severe diarrheal disease that affects hundreds of thousands of individuals resulting in significant morbidity and mortality worldwide. Shigellosis is caused by <i>Shigella</i> spp., a gram-negative bacterium that uses a Type 3 Secretion System (T3SS) to deliver effector proteins into the cytosol of infected human cells. <i>Shigella</i> infection triggers multiple signaling programs that result in a robust host transcriptional response that includes the induction of multiple proinflammatory cytokines. PML nuclear bodies (PML-NBs) are dynamic subnuclear structures that coordinate immune signaling programs and have a demonstrated role in controlling viral infection. We show that PML-NB number increases upon <i>Shigella</i> infection. We examined the effects of <i>Shigella</i> infection on SUMOylation and found that upon <i>Shigella</i> infection the localization of SUMOylated proteins is altered and the level of SUMOylated proteins decreases. Although <i>Shigella</i> infection does not alter the abundance of SUMO activating enzymes SAE1 or SAE2, it dramatically decreases the level of the SUMO conjugating enzyme Ubc9. All <i>Shigella</i>-induced alterations to the SUMOylation system are dependent upon a T3SS. Thus, we demonstrate that <i>Shigella</i> uses one or more T3SS effectors to influence both PML-NB number and the SUMOylation machinery in human cells.</p></div

<i>Shigella</i> targets SUMO conjugating enzyme Ubc9.

<p>In (A-C) whole cell lysates prepared from HeLa cells infected for 1, 2 or 3 hours with the indicated strains of <i>Shigella</i> bearing a plasmid expressing the <i>afaE</i> adhesin were analyzed by immunoblotting using antibodies specific for SAE1 (A), SAE2 (B), or Ubc9 (C). In (D) HeLa cells were treated with the proteasome inhibitor MG132 prior to infection, or not, as indicated prior to infection and preparation of lysates. Lysates were analyzed for Ubc9 by immunoblotting. In (D) HeLa cells were infected with the indicated strains and lysates were analyzed by immunoblotting for UbcH5.</p

PRP4K is a HER2-regulated modifier of taxane sensitivity

<p>The taxanes are used alone or in combination with anthracyclines or platinum drugs to treat breast and ovarian cancer, respectively. Taxanes target microtubules in cancer cells and modifiers of taxane sensitivity have been identified <i>in vitro</i>, including drug efflux and mitotic checkpoint proteins. Human epidermal growth factor receptor 2 (HER2/ERBB2) gene amplification is associated with benefit from taxane therapy in breast cancer yet high HER2 expression also correlates with poor survival in both breast and ovarian cancer. The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), which we identified as a component of the U5 snRNP also plays a role in regulating the spindle assembly checkpoint (SAC) in response to microtubule-targeting drugs. In this study, we found a positive correlation between PRP4K expression and HER2 status in breast and ovarian cancer patient tumors, which we determined was a direct result of PRP4K regulation by HER2 signaling. Knock-down of PRP4K expression reduced the sensitivity of breast and ovarian cancer cell lines to taxanes, and low PRP4K levels correlated with in vitro-derived and patient acquired taxane resistance in breast and ovarian cancer. Patients with high-grade serous ovarian cancer and high HER2 levels had poor overall survival; however, better survival in the low HER2 patient subgroup treated with platinum/taxane-based therapy correlated positively with PRP4K expression (HR = 0.37 [95% CI 0.15-0.88]; p = 0.03). Thus, PRP4K functions as a HER2-regulated modifier of taxane sensitivity that may have prognostic value as a marker of better overall survival in taxane-treated ovarian cancer patients.</p

DRiP78 interaction with CCR5 homodimers.

<p>a) CXCR4 and CCR5 sequences corresponding to the potential interaction site of DRiP78, starting at the beginning of the carboxy-terminal tail of the receptor. b) Co-immunoprecipitation of DRiP78 with CCR5 WT or with a mutation of the potential interaction site (phenylalanine residues mutated to alanine residues) and with CXCR4-v1/CCR5-v2. c) Interaction of CXCR4 with DRiP78. d) CCR5 c-tail interaction with DRiP78. The GST-CCR5 c-tails (wild type or F/A mutant) were incubated HEK293A cell lysates expressing DRiP78 to detect the interaction.</p

Effect of DRiP78 on CCR5 localization.

<p>Cells were plated on coverslips for 24 hours and then transfected with HA-CCR5 (WT (a) or F/A mutant (b)) and a cell surface marker, the CB1 receptor. c) expression of WT CCR5 with DRiP78 and d) Expression of mutated CCR5 with DRiP78. Green shows HA expression, red shows CB1 receptor or FLAG expression while the overlay column shows merged images of the previous two as acquired via fluorescence microscopy. DRiP78 is an ER chaperone, and therefore serves as an ER marker.</p

DRiP78 regulates CCR5 interaction with G protein subunits during assembly.

<p>a) HEK293 cells were co-transfected with cDNAs encoding DRiP78 WT, HA-CCR5 WT or F/A, and various G protein subunits (Gαi-<i>R</i>luc, Gβ1-<i>R</i>luc in presence of Gγ2. Cells were then lysed and immunoprecipitations using an antibody directed against HA were performed. An immunoblot was then performed using an antibody directed against <i>R</i>luc to reveal the G protein subunits. b) Histogram representation of the results obtained by immunoblotting. c) and d) BRET experiment showing WT CCR5-GFP₁₀ or CCR5 F/A-eGFP interaction with <i>R</i>luc-tagged Gαi, Gαs and Gβ1 subunits. * = <i>p</i><0.05; ** = <i>p</i><0.01 compared with negative controls. Results are representative of 3 independent experiments.</p

DRiP78 levels in cells affect function.

<p>a) Increasing amounts of DRiP78 cDNA were transfected in HEK293 cells and expression levels of chemokine receptor dimers were measured. b) Cells expressing chemokine receptor dimers were transfected for 48 hours with various amounts of cDNA encoding DRiP78 and fluorescence levels were then measured. Fluorescence appears only when receptors dimerize therefore any variation in fluorescence level would be attributed to a change in the capacity of those receptors to associate. c) Quantification of <i>R</i>luc levels in cells, when expressed along with constructs mentioned in b). <i>R</i>luc levels do not change, so fluorescence levels are not attributed to changes in the cell capacity to produce the proteins.</p

Interaction of DRiP78 with chemokine receptor dimmers.

<p>HEK293 cells were co-transfected for 48 hours with various contructs harboring non-functional parts of a YFP variant, venus. Upon interaction between two receptors, the venus parts can associate together and regenerate a functional fluorescent signal. Receptor pairs were expressed with DRiP78-<i>R</i>luc and bioluminescence resonance energy transfer was measured. * = <i>p</i><0.05 compared with controls.</p