Selenoprotein I is indispensable for ether lipid homeostasis and proper myelination.

Selenoprotein I (SELENOI) catalyzes the final reaction of the CDP-ethanolamine branch of the Kennedy pathway, generating the phospholipids phosphatidylethanolamine (PE) and plasmenyl-PE. Plasmenyl-PE is a key component of myelin and is characterized by a vinyl ether bond that preferentially reacts with oxidants, thus serves as a sacrificial antioxidant. In humans, multiple loss-of-function mutations in genes affecting plasmenyl-PE metabolism have been implicated in hereditary spastic paraplegia, including SELENOI. Herein, we developed a mouse model of nervous system-restricted SELENOI deficiency that circumvents embryonic lethality caused by constitutive deletion and recapitulates phenotypic features of hereditary spastic paraplegia. Resulting mice exhibited pronounced alterations in brain lipid composition, which coincided with motor deficits and neuropathology including hypomyelination, elevated reactive gliosis, and microcephaly. Further studies revealed increased lipid peroxidation in oligodendrocyte lineage cells and disrupted oligodendrocyte maturation both in vivo and in vitro. Altogether, these findings detail a critical role for SELENOI-derived plasmenyl-PE in myelination that is of paramount importance for neurodevelopment.

Upregulated selenoprotein I during lipopolysaccharide-induced B cell activation promotes lipidomic changes and is required for effective differentiation into IgM-secreting plasma B cells.

The mechanisms driving metabolic reprogramming during B cell activation are unclear, particularly roles for enzymatic pathways involved in lipid remodeling. We found that murine B cell activation with lipopolysaccharide (LPS) led to a 1.6-fold increase in total lipids that included higher levels of phosphatidylethanolamine (PE) and plasmenyl PE. Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase involved in the synthesis of both PE and plasmenyl PE, and SELENOI expression was also upregulated during activation. Selenoi knockout (KO) B cells exhibited decreased levels of plasmenyl PE, which plays an important antioxidant role. Lipid peroxidation was measured and found to increase ∼2-fold in KO vs. wild-type (WT) B cells. Cell death was not impacted by KO in LPS-treated B cells and proliferation was only slightly reduced, but differentiation into CD138 + Blimp-1+ plasma B cells was decreased ∼2-fold. This led to examination of B cell receptors important for differentiation that recognize the ligand B cell activating factor, and levels of TACI (transmembrane activator, calcium-modulator, and cytophilin ligand interactor) (CD267) were significantly decreased on KO B cells compared with WT control cells. Vaccination with ovalbumin/adjuvant led to decreased ovalbumin-specific immunoglobulin M (IgM) levels in sera of KO mice compared with WT mice. Real-time polymerase chain reaction analyses revealed a decreased switch from surface to secreted IgM in spleens of KO mice induced by vaccination or LP-BM5 retrovirus infection. Overall, these findings detail the lipidomic response of B cells to LPS activation and reveal the importance of upregulated SELENOI for promoting differentiation into IgM-secreting plasma B cells.

Selenoprotein I deficiency in T cells promotes differentiation into tolerant phenotypes while decreasing Th17 pathology.

Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase contributing to cellular metabolism and the synthesis of glycosylphosphatidylinositol (GPI) anchors. SELENOI knockout (KO) in T cells has been shown to impair metabolic reprogramming during T cell activation and reduce GPI-anchored Thy-1 levels, which are both crucial for Th17 differentiation. This suggests SELENOI may be important for Th17 differentiation, and we found that SELENOI was indeed up-regulated early during the activation of naïve CD4+ T cells in Th17 conditions. SELENOI KO reduced RORγt mRNA levels by decreasing SOX5 and STAT3 binding to promoter and enhancer regions in the RORC gene encoding this master regulator of Th17 cell differentiation. Differentiation of naïve CD4+ T cells into inflammatory versus tolerogenic Th cell subsets was analyzed and results showed that SELENOI deficiency skewed differentiation away from pathogenic Th17 cells (RORγt+ and IL-17A+ ) while promoting tolerogenic phenotypes (Foxp3+ and IL-10+ ). Wild-type and T cell-specific SELENOI KO mice were subjected to experimental autoimmune encephalitis (EAE), with KO mice exhibiting diminished clinical symptoms, reduced CNS pathology and decreased T cell infiltration. Flow cytometry showed that SELENOI T cell KO mice exhibited lower CD4+ RORγt+ and CD4+ IL-17A+ T cells and higher CD4+ CD25+ FoxP3+ T cells in CNS tissues of mice subjected to EAE. Thus, the metabolic enzyme SELENOI is up-regulated to promote RORγt transcription that drives Th17 differentiation, and SELENOI deficiency shifts differentiation toward tolerogenic phenotypes while protecting against pathogenic Th17 responses.

T-cell activation decreases miRNA-15a/16 levels to promote MEK1-ERK1/2-Elk1 signaling and proliferative capacity.

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.

Upregulated ethanolamine phospholipid synthesis via selenoprotein I is required for effective metabolic reprogramming during T cell activation.

OBJECTIVE: T cell activation triggers metabolic reprogramming to meet increased demands for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cells and cancer cells, which led us to investigate its potential role during T cell activation. METHODS: As selenoprotein I (SELENOI) is an enzyme participating in two metabolic pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI-deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation. RESULTS: SELENOI knockout (KO) in mouse T cells led to reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor AMPK. AMPK was inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment CONCLUSIONS: T cell activation upregulates SELENOI-dependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.

Selenoprotein I is essential for murine embryogenesis.

Selenoprotein I (SELENOI) is an ethanolamine phosphotransferase that catalyzes the third reaction of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Since the role of SELENOI in murine embryogenesis has not been investigated, SELENOI-/+ mating pairs were used to generate global KO offspring. Of 323 weanling pups, no homozygous KO genotypes were found. E6.5-E18.5 embryos (165 total) were genotyped, and only two E18.5 KO embryos were detected with no discernable anatomical defects. To screen embryos prior to uterine implantation that occurs ~ E6, blastocyst embryos (E3.5-E4.4) were flushed from uteruses of pregnant females and analyzed for morphology and genotype. KO embryos were detected in 5 of 6 pregnant females, and 7 of the 32 genotyped embryos were found to be SELENOI KO that exhibited no overt pathological features. Overall, these results demonstrate that, except for rare cases (2/490 = 0.4%), global SELENOI deletion leads to early embryonic lethality.

Targeting the C-terminus of galectin-9 induces mesothelioma apoptosis and M2 macrophage depletion.

Galectin-9 has emerged as a promising biological target for cancer immunotherapy due to its role as a regulator of macrophage and T-cell differentiation. In addition, its expression in tumor cells modulates tumor cell adhesion, metastasis, and apoptosis. Malignant mesothelioma (MM) is an aggressive neoplasm of the mesothelial cells lining the pleural and peritoneal cavities, and in this study, we found that both human MM tissues and mouse MM cells express high levels of galectin-9. Using a novel monoclonal antibody (mAb) (Clone P4D2) that binds the C-terminal carbohydrate recognition domain (CRD) of galectin-9, we demonstrate unique agonistic properties resulting in MM cell apoptosis. Furthermore, the P4D2 mAb reduced tumor-associated macrophages differentiation toward a protumor phenotype. Importantly, these effects exerted by the P4D2 mAb were observed in both human and mouse in vitro experiments and not observed with another antigalectin-9 specific mAb (clone P1D9) that engages the N-terminus CRD of galectin-9. In syngeneic murine models of MM, P4D2 mAb treatment inhibited tumor growth and improved survival, with tumors from P4D2-treated mice exhibited reduced infiltration of tumor-associated M2 macrophages. This was consistent with an increased production of inducible nitric oxide synthase, which is a major enzyme-regulating macrophage inflammatory response to cancer. These data suggest that using an antigalectin 9 mAb with agonistic properties similar to those exerted by galectin-9 may provide a novel multitargeted strategy for the treatment of mesothelioma and possibly other galectin-9 expressing tumors.

Multi-antigen Vaccination With Simultaneous Engagement of the OX40 Receptor Delays Malignant Mesothelioma Growth and Increases Survival in Animal Models.

Malignant Mesothelioma (MM) is a rare and highly aggressive cancer that develops from mesothelial cells lining the pleura and other internal cavities, and is often associated with asbestos exposure. To date, no effective treatments have been made available for this pathology. Herein, we propose a novel immunotherapeutic approach based on a unique vaccine targeting a series of antigens that we found expressed in different MM tumors, but largely undetectable in normal tissues. This vaccine, that we term p-Tvax, is comprised of a series of immunogenic peptides presented by both MHC-I and -II to generate robust immune responses. The peptides were designed using in silico algorithms that discriminate between highly immunogenic T cell epitopes and other harmful epitopes, such as suppressive regulatory T cell epitopes and autoimmune epitopes. Vaccination of mice with p-Tvax led to antigen-specific immune responses that involved both CD8+ and CD4+ T cells, which exhibited cytolytic activity against MM cells in vitro. In mice carrying MM tumors, p-Tvax increased tumor infiltration of CD4+ T cells. Moreover, combining p-Tvax with an OX40 agonist led to decreased tumor growth and increased survival. Mice treated with this combination immunotherapy displayed higher numbers of tumor-infiltrating CD8+ and CD4+ T cells and reduced T regulatory cells in tumors. Collectively, these data suggest that the combination of p-Tvax with an OX40 agonist could be an effective strategy for MM treatment.

Correction: Selenoprotein K deficiency inhibits melanoma by reducing calcium flux required for tumor growth and metastasis.

[This corrects the article DOI: 10.18632/oncotarget.24388.].

Selenoprotein K deficiency inhibits melanoma by reducing calcium flux required for tumor growth and metastasis.

Interest has emerged in the therapeutic potential of inhibiting store operated calcium (Ca2+) entry (SOCE) for melanoma and other cancers because malignant cells exhibit a strong dependence on Ca2+ flux for disease progression. We investigated the effects of deleting Selenoprotein K (SELENOK) in melanoma since previous work in immune cells showed SELENOK was required for efficient Ca2+ flux through the endoplasmic reticulum Ca2+ channel protein, inositol 1,4,5-trisphosphate receptor (IP3R), which is due to the role SELENOK plays in palmitoylating and stabilizing the expression of IP3R. CRISPR/Cas9 was used to generate SELENOK-deficiency in human melanoma cells and this led to reduced Ca2+ flux and impaired IP3R function, which inhibited cell proliferation, invasion, and migration. Ca2+-dependent signaling through calcineurin was inhibited with SELENOK-deficiency, and gene array analyses together with evaluation of transcript and protein levels showed altered transcriptional programs that ultimately disrupted stemness and pro-growth properties. In vivo investigations were conducted using the Grm1-Tg transgenic mouse strain that develops spontaneous metastatic melanoma, which was crossed with SELENOK-/- mice to generate the following littermates: Grm1-Tg/SELENOK-/-, Grm1-Tg/SELENOK-/+, Grm1-Tg/SELENOK+/+. SELENOK-deficiency in Grm1-Tg/SELENOK-/- male and female mice inhibited primary tumor growth on tails and ears and reduced metastasis to draining lymph nodes down to levels equivalent to non-tumor control mice. Cancer stem cell pools were also decreased in Grm1-Tg/SELENOK-/- mice compared to littermates. These results suggest that melanoma requires SELENOK expression for IP3R dependent maintenance of stemness, tumor growth and metastasic potential, thus revealing a new potential therapeutic target for treating melanoma and possibly other cancers.

Calpain-2 inhibitor treatment preferentially reduces tumor progression for human colon cancer cells expressing highest levels of this enzyme.

Calpain-2 levels are higher in colorectal tumors resistant to chemotherapy and previous work showed calpain-2 inhibitor therapy reduced inflammation-driven colorectal cancer, but direct effects of the inhibitor on colon cancer cells themselves were not demonstrated. In the present study, five human colon cancer cell lines were directly treated with a calpain-2 inhibitor and results showed increased cell death in 4 of 5 cell lines and decreased anchorage-independent growth for all cell five lines. When tested for levels of calpain-2, three cell lines exhibited increasing levels of this enzyme: HCT15 (low), HCC2998 (medium), and HCT116 (significantly higher). This was consistent with gel shift assays showing that calpain-2 inhibitor reduced of NF-κB nuclear translocation most effectively in HCT116 cells. Ability of calpain-2 inhibitor to impede tumor progression in vivo was evaluated using intrarectal transplant of luciferase-expressing cells for these three cell lines. Results showed that calpain-2 inhibitor therapy reduced tumor growth and increased survival only in mice injected with HCT116 cells. These data suggest calpain-2 inhibitor treatment may be most effective on colorectal tumors expressing highest levels of calpain-2.

Selenoprotein K Increases Efficiency of DHHC6 Catalyzed Protein Palmitoylation by Stabilizing the Acyl-DHHC6 Intermediate.

Selenoprotein K (SELENOK) is a selenocysteine (Sec)-containing protein localized in the endoplasmic reticulum (ER) membrane where it interacts with the DHHC6 (where single letter symbols represent Asp-His-His-Cys amino acids) enzyme to promote protein acyl transferase (PAT) reactions. PAT reactions involve the DHHC enzymatic capture of palmitate via a thioester bond to cysteine (Cys) residues that form an unstable palmitoyl-DHHC intermediate, followed by transfer of palmitate to Cys residues of target proteins. How SELENOK facilitates this reaction has not been determined. Splenocyte microsomal preparations from wild-type mice versus SELENOK knockout mice were used to establish PAT assays and showed decreased PAT activity (~50%) under conditions of SELENOK deficiency. Using recombinant, soluble versions of DHHC6 along with SELENOK containing Sec92, Cys92, or alanine (Ala92), we evaluated the stability of the acyl-DHHC6 intermediate and its capacity to transfer the palmitate residue to Cys residues on target peptides. Versions of SELENOK containing either Ala or Cys residues in place of Sec were equivalently less effective than Sec at stabilizing the acyl-DHHC6 intermediate or promoting PAT activity. These data suggest that Sec92 in SELENOK serves to stabilize the palmitoyl-DHHC6 intermediate by reducing hydrolyzation of the thioester bond until transfer of the palmitoyl group to the Cys residue on the target protein can occur.

Selenoprotein K regulation of palmitoylation and calpain cleavage of ASAP2 is required for efficient FcγR-mediated phagocytosis.

Effective activation of macrophages through phagocytic Fcγ receptors (FcγR) has been shown to require selenoprotein K (Selk). We set out to determine whether the FcγR-mediated uptake process itself also requires Selk and potential underlying mechanisms. Macrophages from Selk knockout (KO) mice were less efficient compared with wild-type (WT) controls in engulfing IgG-coated fluorescent beads. Using LC-MS/MS to screen for Selk-binding partners involved in FcγR-mediated phagocytosis, we identified Arf-GAP with SH3 domain, ANK repeat, and PH domain-containing protein 2 (ASAP2). Coimmunoprecipitation assays confirmed interactions between Selk and ASAP2. Selk was required for ASAP2 to be cleaved by calpain-2 within the Bin/Amphiphysin/Rvs (BAR) domain of ASAP2. BAR domains promote membrane association, which was consistent with our data showing that Selk deficiency led to retention of ASAP2 within the phagocytic cup. Because Selk was recently identified as a cofactor for the palmitoylation of certain proteins, we investigated whether ASAP2 was palmitoylated and whether this was related to its cleavage by calpain-2. Acyl/biotin exchange assays and MALDI-TOF analysis showed that cysteine-86 in ASAP2 was palmitoylated in WT, but to a much lesser extent in KO, mouse macrophages. Inhibitors of either palmitoylation or calpain-2 cleavage and rescue experiments with different versions of Selk demonstrated that Selk-dependent palmitoylation of ASAP2 leads to cleavage by calpain-2 within the BAR domain, which releases this protein from the maturing phagocytic cup. Overall, these findings identify ASAP2 as a new target of Selk-dependent palmitoylation and reveal a new mechanism regulating the efficiency of FcγR-mediated phagocytosis.

Calpain-2 Inhibitor Therapy Reduces Murine Colitis and Colitis-associated Cancer.

BACKGROUND: An important role has emerged for calpain enzymes in regulating inflammation with one isoform, calpain-2, particularly important for macrophage activation. The goal of this study was to determine the therapeutic potential of a synthetic calpain-2 inhibitor, zLLY-CH2F, for colitis and inflammation-associated colorectal cancer. METHODS: Mice were then subjected to the azoxymethane/dextran sulfate sodium model of colitis and colitis-associated cancer incorporating intervention with daily injections of 0.75 mg/kg calpain-2 inhibitor beginning after the first signs of colitis. RESULTS: Calpain-2 inhibitor treatment alleviated weight loss and bloody diarrhea, and reduced inflammatory infiltration into colon tissues and inflammatory cytokine mRNA. Calpain-2 inhibitor intervention also reduced total colitis-associated cancer tumor volume by up to 70% in vehicle control mice and decreased cancer pathology scores of blinded histological colon tissue analyses. Mechanistic investigations showed that calpain-2 inhibition during macrophage activation reduced inhibitor of kappa beta (IκB) degradation and nuclear factor kappa beta (NFκB) nuclear localization as well as secretion of specific inflammatory cytokines. In addition, calpain-2 inhibitor treatment of CT26.WT mouse and HT-29 human colorectal cancer cells decreased proliferation and reduced IκB degradation and NFκB translocation. CONCLUSIONS: Overall, these findings suggest that intervention with a calpain-2 inhibitor may reduce colitis and colitis-associated cancer through a two-hit process of limiting macrophage activation and inhibiting growth of the colorectal cancer cells themselves.

Preclinical development of HIvax: Human survivin highly immunogenic vaccines.

Our previous work involved the development of a recombinant fowlpox virus encoding survivin (FP-surv) vaccine that was evaluated for efficacy in mesothelioma mouse models. Results showed that FP-surv vaccination generated significant immune responses, which led to delayed tumor growth and improved animal survival. We have extended those previous findings in the current study, which involves the pre-clinical development of an optimized version of FP-surv designed for human immunization (HIvax). Survivin-derived peptides for the most common haplotypes in the human population were identified and their immunogenicity confirmed in co-culture experiments using dendritic cells and T cells isolated from healthy donors. Peptides confirmed to induce CD8(+) and CD4(+) T cells activation in humans were then included in 2 transgenes optimized for presentation of processed peptides on MHC-I (HIvax1) and MHC-II (HIvax2). Fowlpox vectors expressing the HIvax transgenes were then generated and their efficacy was evaluated with subsequent co-culture experiments to measure interferon-γ and granzyme B secretion. In these experiments, both antigen specific CD4(+) and CD8(+) T cells were activated by HIvax vaccines with resultant cytotoxic activity against survivin-overexpressing mesothelioma cancer cells. These results provide a rationale for clinical testing of HIvax1 and HIvax2 vaccines in patients with survivin-expressing cancers.

Adjuvants may reduce in vivo transfection levels for DNA vaccination in mice leading to reduced antigen-specific CD8+ T cell responses.

Adjuvants for DNA vaccination are designed to promote transformation of transgenes into target cells and increase inflammation in the site of injection, with resultant immune cell recruitment. Numerous studies indicated cationic liposomes as effective adjuvants for DNA vaccination due to their ability to promote in vivo transfection and innate immune system activation. Commercial reagents as Adjuplex and in vivo-JetPEI are also intended to facilitate DNA vaccination. Here, we evaluate the adjuvant properties of cationic liposomes, Adjuplex and in vivo-JetPEI compared to injection of DNA without adjuvant. In mice vaccinated with piggyBac pDNA vaccines, we assessed in vivo antigen expression, innate immune responses in draining lymph nodes, and antigen-specific T cell responses in spleens and blood. Surprisingly, vaccination with DNA in PBS emerged as the most efficient in promoting in vivo transfection and consequent antigen expression, while the addition of adjuvant reduced the amount of antigen expressed. On the other hand, we discovered higher numbers of innate immune cells and activated dendritic cells in the lymph nodes of mice injected with adjuvants than those vaccinated in PBS. The analysis of eGFP-specific immune responses revealed that all the different immunizations induced functional antigen-specific T cells in spleens, although only T cells generated by non-adjuvant vaccination and Adjuplex were identified in the blood of vaccinated mice. These results provide insight into the effects of these 3 adjuvants and may facilitate appropriate use off adjuvants by researchers using DNA vaccines in laboratory animals.

Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex.

Calcium (Ca(2+)) is a secondary messenger in cells and Ca(2+) flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca(2+) flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca(2+) flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially palmitoylated cysteine, and mutation of these three cysteines to alanines resulted in decreased IP3R palmitoylation and function. These findings reveal IP3R palmitoylation as a critical regulator of Ca(2+) flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.

Increasing dietary selenium elevates reducing capacity and ERK activation associated with accelerated progression of select mesothelioma tumors.

To study the effect of the micronutrient selenium on malignant mesothelioma (MM) progression, we cultured four different MM cell lines in media containing increasing amounts of sodium selenite (30, 50, and 80 nmol/L). Increasing selenium levels increased density-dependent proliferation and mobility for CRH5 and EKKH5 but not AB12 and AK7. Comparing these cell lines revealed that extracellular regulated kinase (ERK) phosphorylation was sensitive to a selenium increase in CRH5 and EKKH5 but not AB12 and AK7 cells. Stable expression of a dominant-negative mutant ERK eliminated the effects of increasing selenium. Because ERK is redox sensitive, we compared the MM cell lines in terms of glutathione levels and the capacity to reduce exogenous hydrogen peroxide. Increasing selenium levels led to higher glutathione and reducing capacity in CRH5 and EKKH5 but not AB12 and AK7. The reducing agent N-acetylcysteine eliminated the effects of selenium on ERK activation, proliferation, and mobility. Mice fed diets containing increasing levels of selenium (0.08, 0.25, and 1.0 ppm) showed increased tumor progression for CRH5 but not AB12, MM cells, and in vivo N-acetylcysteine treatment eliminated these effects. These data suggest that the effects of dietary selenium on MM tumor progression depend on the arising cancer cells' redox metabolism, and the tumors able to convert increased selenium into a stronger reducing capacity actually benefit from increased selenium intake.

Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8(+) T cell responses.

DNA vaccination with plasmid has conventionally involved vectors designed for transient expression of antigens in injected tissues. Next generation plasmids are being developed for site-directed integration of transgenes into safe sites in host genomes and may provide an innovative approach for stable and sustained expression of antigens for vaccination. The goal of this study was to evaluate in vivo antigen expression and the generation of cell mediated immunity in mice injected with a non-integrating plasmid compared to a plasmid with integrating potential. Hyperactive piggyBac transposase-based integrating vectors (pmhyGENIE-3) contained a transgene encoding either eGFP (pmhyGENIE-3-eGFP) or luciferase (pmhyGENIE-3-GL3), and were compared to transposase-deficient plasmids with the same transgene and DNA backbone. Both non-integrating and integrating plasmids were equivalent at day 1 for protein expression at the site of injection. While protein expression from the non-integrating plasmid was lost by day 14, the pmhyGENIE-3 was found to exhibit sustained protein expression up to 28 days post-injection. Vaccination with pmhyGENIE-3-eGFP resulted in a robust CD8(+) T cell response that was three-fold higher than that of non-integrating plasmid vaccinations. Additionally we observed in splenocyte restimulation experiments that only the vaccination with pmhyGENIE-3-eGFP was characterized by IFNγ producing CD8(+) T cells. Overall, these findings suggest that plasmids designed to direct integration of transgenes into the host genome are a promising approach for designing DNA vaccines. Robust cell mediated CD8(+) T cell responses generated using integrating plasmids may provide effective, sustained protection against intracellular pathogens or tumor antigens.

MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation.

Redox control of protein function involves oxidation and reduction of amino acid residues, but the mechanisms and regulators involved are insufficiently understood. Here, we report that in conjunction with Mical proteins, methionine-R-sulfoxide reductase B1 (MsrB1) regulates mammalian actin assembly via stereoselective methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin are specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by selenoprotein MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilize this redox control during cellular activation by stimulating MsrB1 expression and activity as a part of innate immunity. We identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study shows that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation.