Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

Background: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. Methods: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. Results: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD81 suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)–150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH2/2 or miRNA-1502/2 mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains

Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

<div><h3>Background</h3><p>Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.</p> <h3>Methods</h3><p>Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.</p> <h3>Results</h3><p>Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.</p> <h3>Conclusion</h3><p>Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.</p> </div

Adipose KLF15 Controls Lipid Handling to Adapt to Nutrient Availability

Adipose tissue stores energy in the form of triglycerides. The ability to regulate triglyceride synthesis and breakdown based on nutrient status (e.g., fed versus fasted) is critical for physiological homeostasis and dysregulation of this process can contribute to metabolic disease. Whereas much is known about hormonal control of this cycle, transcriptional regulation is not well understood. Here, we show that the transcription factor Kruppel-like factor 15 (KLF15) is critical for the control of adipocyte lipid turnover. Mice lacking Klf15 in adipose tissue (AK15KO) display decreased adiposity and are protected from diet-induced obesity. Mechanistic studies suggest that adipose KLF15 regulates key genes of triglyceride synthesis and inhibits lipolytic action, thereby promoting lipid storage in an insulin-dependent manner. Finally, AK15KO mice demonstrate accelerated lipolysis and altered systemic energetics (e.g., locomotion, ketogenesis) during fasting conditions. Our study identifies adipose KLF15 as an essential regulator of adipocyte lipid metabolism and systemic energy balance

EMMPRIN, GOLPH3 and eNOS are co-localized at the Golgi apparatus in endothelial cells.

<p>(<b>A</b>) Immunolabeling of EMMPRIN, eNOS, GOLPH3 and GM130 (a Golgi marker) in bovine aortic endothelial cells (BAECs). The upper panel shows EMMPRIN (red, left) and eNOS (green, center) and their merged image with DNA in blue (right). Arrows indicate where EMMPRIN and eNOS are located. The lower panel shows GOLPH3 (red, left), GM130 (green, center), and their merged image with nucleus in blue (right). Arrows indicate where GOLPH3 and GM130 are located. Scale bar; 10 µm. Images were taken using a Nikon E800 Microscope with a Plan-Fluorchromat 40×/0.75 objective (Nikon, Melville, NY). Shown are representative images from at least 3 independent experiments. (<b>B</b>) Immunolabeling of COS cells that were transfected with wild-type eNOS (blue, upper left panel), YFP-EMMPRIN (green, upper right panel) and HA-tagged GOLPH3 (red, lower left panel). The lower right panel shows their merged image. Scale bar; 10 µm. Arrows indicate eNOS-, EMMPRIN- and GOLPH3-rich areas. Scale bar; 10 µm. Shown are representative images from at least 3 independent transfection experiments. (<b>C</b>) Immunoprecipitation (IP) using anti-EMMPRIN (EMP) and total goat IgG from BAEC lysates. IP samples were blotted with eNOS and GOLPH3 antibodies. Input refers to eNOS levels in lysates before IP, indicating an equal amount of proteins in lysates used for IP. The blots are representative images from three independent experiments.</p

S-nitrosylation of EMMPRIN is increased in the aorta isolated from cirrhotic rats.

<p>Aorta samples were lysed in a lysis buffer. Three aorta samples were combined per group to obtain a sufficient amount of proteins for the biotin-switch assay. Lysates before the biotin-switch assay were blotted for EMMPRIN and a loading control, heat shock protein 90 (Hsp90) (input, left panel). Equal amounts of proteins (500 µg) in the lysates were used for the biotin-switch assay. Biotinylated proteins were captured by streptavidin agarose beads and blotted with an EMMPRIN antibody (right panel). The aorta from cirrhotic rats showed a higher level of S-nitrosylated EMMPRIN than that of normal rats. Interestingly, only those glycosylated EMMPRIN were S-nitrosylated in the aorta.</p

Proteins at the Golgi apparatus can be targets for S-nitrosylation.

<p>(<b>A</b>) Golgi membrane enrichment and its purity were assessed by Western blot with a Golgi marker (GM130), an endoplasmic reticulum (ER) marker (Calnexin) and a nuclear marker (Lamin A/C). Fraction III of the preparation showed a successful enrichment and purity of Golgi membranes and was used in this study. (<b>B</b>) S-nitrosylated Golgi membrane proteins were increased dose-dependently in response to the addition of an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). Golgi membranes were treated with indicated concentrations of SNAP <i>in vitro</i> at room temperature (RM) for 30 min. Protein S-nitrosylation was assessed by the biotin-switch assay. Asterisks indicate endogenous biotin-containing proteins, thus considered as non-specific bands. Dithiothreitol (DTT), which cleaves nitroso-cysteine bonds, serves as a negative control. Shown are representative blots from 3 independent experiments. (<b>C</b>) S-nitrosylated Golgi membrane proteins were increased time-dependently in response to 100 µM SNAP <i>in vitro</i> for indicated incubation times (0, 30 and 60 min). Arrows indicate those proteins increased in response to SNAP. Asterisks indicate endogenous biotin-containing proteins, thus considered as non-specific bands. Shown are representative images from 3 independent experiments. (<b>D</b>) The sample quality was verified before performing a proteomic analysis. Successful biotinylation (i.e., S-nitrosylation) by the biotin-switch assay was determined by Western blot. Golgi membranes were incubated with or without 10 µM S-nitrosocysteine (Cys-NO) for 15 min at 37°C. Then, the biotin-switch assay was performed in the presence or absence of 2 mM ascorbic acid (AA) (right panel). Proteins were separated by SDS-PAGE and an equal protein loading to each lane was confirmed by Ponceau staining (left panel). Subsequent Western blotting using an anti-biotin antibody detected biotinylated proteins (right panel). Arrows indicate unique bands of biotinylated proteins that appeared in the presence of AA. Those biotinylated proteins increased by Cys-NO treatment (the 4^th lane in the right panel) were identified by mass spectrometry.</p

Putative S-nitrosylated Golgi membrane proteins in rats.

<p>Putative S-nitrosylated Golgi membrane proteins in rats.</p