FuKun W. Hoffmann

The selenoproteome exhibits widely varying, tissue-specific dependence on selenoprotein P for selenium supply

Selenoprotein P (Sel P) is a selenium-rich glycoprotein believed to play a key role in selenium (Se) transport throughout the body. Development of a Sel P knockout mouse model has supported this notion and initial studies have indicated that selenium supply to various tissues is differentially affected by genetic deletion of Sel P. Se in the form of the amino acid, selenocysteine, is incorporated into selenoproteins at UGA codons. Thus, Se availability affects not only selenoprotein levels, but also the turnover of selenoprotein mRNAs via the nonsense-mediated decay pathway. We investigated how genetic deletion of Sel P in mice affected levels of the mRNAs encoding all known members of the murine selenoprotein family, as well as three non-selenoprotein factors involved in their synthesis, selenophosphate synthetase 1 (SPS1), SECIS-binding protein 2 (SBP2) and SECp43. Our findings present a comprehensive description of selenoprotein mRNA expression in the following murine tissues: brain, heart, intestine, kidney, liver, lung, spleen and testes. We also describe how abundance of selenoproteins and selenoprotein-synthesis factors are affected by genetic deletion of Sel P in some of these tissues, providing insight into how the presence of this selenoprotein influences selenoprotein mRNA levels, and thus, the selenoproteome.

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.

Selenoprotein K Knockout Mice Exhibit Deficient Calcium Flux in Immune Cells and Impaired Immune Responses

Selenoprotein K (Sel K) is a selenium-containing protein for which no function has been identified. We found that Sel K is an endoplasmic reticulum transmembrane protein expressed at relatively high levels in immune cells and is regulated by dietary selenium. Sel K−/− mice were generated and found to be similar to wild-type controls regarding growth and fertility. Immune system development was not affected by Sel K deletion, but specific immune cell defects were found in Sel K−/− mice. Receptor-mediated Ca2+ flux was decreased in T cells, neutrophils, and macrophages from Sel K−/− mice compared with controls. Ca2+-dependent functions including T cell proliferation, T cell and neutrophil migration, and Fcγ receptor-mediated oxidative burst in macrophages were decreased in cells from Sel K−/− mice compared with that in cells from controls. West Nile virus infections were performed, and Sel K−/− mice exhibited decreased viral clearance in the periphery and increased viral titers in brain. Furthermore, West Nile virus-infected Sel K−/− mice demonstrated significantly lower survival (2 of 23; 8.7%) compared with that of wild-type controls (10 of 26; 38.5%). These results establish Sel K as an endoplasmic reticulum-membrane protein important for promoting effective Ca2+ flux during immune cell activation and provide insight into molecular mechanisms by which dietary selenium enhances immune responses.

A role for dietary selenium and selenoproteins in allergic airway inflammation.

Asthma is driven by allergic airway inflammation and involves increased levels of oxidative stress. This has led to speculation that antioxidants like selenium (Se) may play important roles in preventing or treating asthma. We fed diets containing low (0.08 parts per million), medium (0.25 parts per million), or high (2.7 parts per million) Se to female C57BL/6 mice and used an established OVA challenge protocol to determine the relationship between Se intake and the development of allergic airway inflammation. Results demonstrated that mice fed medium levels of Se had robust responses to OVA challenge in the lung as measured by lung cytokine levels, airway cellular infiltrate, eosinophilia, serum anti-OVA IgE, airway hyperreactivity, goblet cell hyperplasia, and phosphorylated STAT-6 levels in the lung. In contrast, responses to OVA challenge were less robust in mice fed low or high levels of Se. In particular, mice fed low Se chow showed significantly lower responses compared with mice fed medium Se chow for nearly all readouts. We also found that within the medium Se group the expression of lung glutathione peroxidase-1 and liver selenoprotein P were increased in OVA-challenged mice compared with PBS controls. These data suggest that Se intake and allergic airway inflammation are not related in a simple dose-response manner, which may explain the inconsistent results obtained from previous descriptive studies in humans. Also, our results suggest that certain selenoproteins may be induced in response to Ag challenges within the lung.

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

Significance The stimulation of certain surface receptors on immune cells triggers the release of calcium (Ca2+) stored in the endoplasmic reticulum (ER). This Ca2+ flux is required for efficient activation and function of immune cells, and involves the ER membrane Ca2+ channel, the inositol 1,4,5-triphosphate receptor (IP3R). We found that stable expression of IP3R requires the addition of a fatty acid through a process called palmitoylation catalyzed by an enzyme complex composed of DHHC6 (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) and selenoprotein K (Selk) proteins. These findings provide new mechanistic insight into the selenium-sensitive fine-tuning of immune cell activation through posttranslational modification of the IP3R Ca2+ channel. This study also reveals a novel DHHC6/Selk enzyme complex responsible for regulating stable expression of the IP3R. Calcium (Ca2+) is a secondary messenger in cells and Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.

Selenoprotein K is a novel target of m-calpain, and cleavage is regulated by Toll-like receptor-induced calpastatin in macrophages.

Background: Selenoprotein K is important for calcium-dependent activation of immune cells. Results: Selenoprotein K is cleaved by m-calpain in resting macrophages, but Toll-like receptor activation induces calpastatin generating full-length, functional selenoprotein K. Conclusion: Proteolytic modulation of selenoprotein K is important for macrophage activation. Significance: New roles are defined for the calpain/calpastatin system and selenoprotein K during macrophage activation and inflammation. Calpains are proteolytic enzymes that modulate cellular function through cleavage of targets, thereby modifying their actions. An important role is emerging for calpains in regulating inflammation and immune responses, although specific mechanisms by which this occurs have not been clearly defined. In this study, we identify a novel target of calpain, selenoprotein K (SelK), which is an endoplasmic reticulum transmembrane protein important for Ca2+ flux in immune cells. Calpain-mediated cleavage of SelK was detected in myeloid cells (macrophages, neutrophils, and dendritic cells) but not in lymphoid cells (B and T cells). Both m- and μ-calpain were capable of cleaving immunoprecipitated SelK, but m-calpain was the predominant isoform expressed in mouse immune cells. Consistent with these results, specific inhibitors were used to show that only m-calpain cleaved SelK in macrophages. The cleavage site in SelK was identified between Arg81 and Gly82 and the resulting truncated SelK was shown to lack selenocysteine, the amino acid that defines selenoproteins. Resting macrophages predominantly expressed cleaved SelK and, when activated through different Toll-like receptors (TLRs), SelK cleavage was inhibited. We found that decreased calpain cleavage was due to TLR-induced up-regulation of the endogenous inhibitor, calpastatin. TLR-induced calpastatin expression not only inhibited SelK cleavage, but cleavage of another calpain target, talin. Moreover, the expression of the calpain isoforms and calpastatin in macrophages were different from T and B cells. Overall, our findings identify SelK as a novel calpain target and reveal dynamic changes in the calpain/calpastatin system during TLR-induced activation of macrophages.

Stimulation of Unprimed Macrophages with Immune Complexes Triggers a Low Output of Nitric Oxide by Calcium-dependent Neuronal Nitric-oxide Synthase

Background: Nitric oxide production by macrophages is conventionally attributed to inducible nitric-oxide synthase activity. Results: Low levels of nitric oxide are generated by neuronal nitric-oxide synthase during engagement of Fcγ-receptors on unprimed macrophages. Conclusion: Immune complexes trigger low output nitric oxide that promotes autocrine/paracrine phagocytosis. Significance: A new role is identified for neuronal nitric-oxide synthase in macrophages. Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca2+) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca2+ flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca2+-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca2+ flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca2+-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.

Selenoprotein K is required for palmitoylation of CD36 in macrophages: implications in foam cell formation and atherogenesis

Selk is an ER transmembrane protein important for calcium flux and macrophage activation, but its role in foam cell formation and atherosclerosis has not been evaluated. BMDMs from Selk−/− mice exhibited decreased uptake of modLDL and foam cell formation compared with WT controls, and the differences were eliminated with anti‐CD36 blocking antibody. CD36 expression was decreased in TNF‐α‐stimulated Selk−/− BMDMs compared with WT controls. Fluorescence microscopy revealed TNF‐α‐induced clustering of CD36 in WT BMDMs indicative of lipid raft localization, which was absent in Selk−/− BMDMs. Fractionation revealed lower levels of CD36 reaching lipid rafts in TNF‐α‐stimulated Selk−/− BMDMs. Immunoprecipitation showed that Selk−/− BMDMs have decreased CD36 palmitoylation, which occurs at the ER membrane and is crucial for stabilizing CD36 expression and directing its localization to lipid rafts. To assess if this phenomenon had a role in atherogenesis, a HFD was fed to irradiated Ldlr−/− mice reconstituted with BM from Selk−/− or WT mice. Selk was detected in aortic plaques of controls, particularly in macrophages. Selk−/− in immune cells led to reduction in atherosclerotic lesion formation without affecting leukocyte migration into the arterial wall. These findings suggest that Selk is important for stable, localized expression of CD36 in macrophages during inflammation, thereby contributing to foam cell formation and atherogenesis.

Specific antioxidant selenoproteins are induced in the heart during hypertrophy.

Selenium (Se) is thought to confer cardioprotective effects through the actions of antioxidant selenoprotein enzymes that directly limit levels of ROS such as hydrogen peroxide (H(2)O(2)) or that reverse oxidative damage to lipids and proteins. To determine how the selenoproteome responds to myocardial hypertrophy, two mouse models were employed: triidothyronine (T3)- or isoproterenol (ISO)-treatment. After 7days of T3- and ISO-treatment, cardiac stress was demonstrated by increased H(2)O(2) and caspase-3 activity. Neither treatment produced significant increases in phospholipid peroxidation or TUNEL-positive cells, suggesting that antioxidant systems were protecting the cardiomyocytes from damage. Many selenoprotein mRNAs were induced by T3- and ISO-treatment, with levels of methionine sulfoxide reductase 1 (MsrB1, also called SelR) mRNA showing the largest increases. MsrB enzymatic activity was also elevated in both models of cardiac stress, while glutathione peroxidase (GPx) activity and thioredoxin reductase (Trxrd) activity were moderately and nonsignificantly increased, respectively. Western blot assays revealed a marked increase in MsrB1 and moderate increases in GPx3, GPx4, and Trxrd1, particularly in T3-treated hearts. Thus, the main response of the selenoproteome during hypertrophy does not involve increased GPx1, but increased GPx3 for reducing extracellular H(2)O(2) and increased GPx4, Trxrd1, and MsrB1 for minimizing intracellular oxidative damage.

Calpastatin Prevents NF-κB–Mediated Hyperactivation of Macrophages and Attenuates Colitis

Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated, reconstituted with either CAST-knockout (KO) or wild-type (WT) bone marrow, and subjected to dextran sulfate sodium–induced colitis. CAST-KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared with WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNF-α, and IFN-γ mRNA levels compared with WT controls. Mechanistic investigations using small interfering RNA and KO bone marrow to generate CAST-deficient macrophages showed that CAST deficiency during activation with bacterial pathogen associated molecular patterns, including heat-killed Enterococcus faecalis or CpG DNA, led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNF-α secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.