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

Huang, Zhi; Hoffmann, FuKun W.; Fay, Jeffrey; Hashimoto, Ann C.; Chapagain, Moti; Kaufusi, Pakieli H.; Hoffmann, Peter

doi:10.1074/jbc.m111.315598

Cited by 54 publications

(45 citation statements)

References 42 publications

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“…The addition of fatty acid residues to the cytosolic region of the IP3R subunits may facilitate assembly of the subunits or stabilize the assembled complex within the ER membrane. This study, to our knowledge, is the first to describe palmitoylation of the IP3R as well as the enzyme complex consisting of DHHC6 and Selk, which provides mechanistic data to explain previous reports of regulation of Ca 2+ flux by dietary selenium and Selk expression in immune cells (15,16,31). Whereas the list of palmitoylated proteins has rapidly increased, surprisingly limited information is available regarding the members of the palmitoyl acyl transferase (PAT) family that carry out this posttranslational modification in mammalian cells.…”

Section: Discussionmentioning

confidence: 75%

“…T cells isolated from WT or Selk KO mice using a Miltenyi untouched mouse T-cell kit were plated at 4 × 10 4 cells per well in 20 μL PBS in a 96-well plate precoated with anti-CD3 (1 μg/mL) and anti-CD28 (10 μg/mL). For BMDMs, the same density of cells was stimulated with 20 μL of BSA/anti-BSA complexes as previously described (31). After 10 min of stimulation, the cells were lysed with 10 μL of 0.2 N perchloric acid and IP3 measured per manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

“…Purification of splenic T cells, B cells, and macrophages was performed using a Miltenyi magnetic separator and purity of cells was determined by evaluation of each cell-type marker via flow cytometry on a FACScaliber (BD Biosciences) and found to be >90%. Bone marrow-derived macrophages (BMDMs) were cultured as previously described (31).…”

Section: Methodsmentioning

confidence: 99%

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

Fredericks¹,

Hoffmann²,

Rose³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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.I mmune cell activation relies on receptor-mediated increases in cellular calcium (Ca 2+ ) concentrations, which is an indispensable step in proliferation, differentiation, migration, and effector functions during immune responses (1, 2). A rapid influx of Ca 2+ has been shown to be important during the activation of lymphocytes through the T-and B-cell receptors, macrophages through Fcγ receptors, and mast cells through Fce receptors and stimulation of various immune cells through chemokine receptors (3). Engagement of these receptors at the plasma membrane leads to the activation of phosphoinositide-specific phospholipase C, which catalyzes the degradation of phosphatidylinositol-4,5-bisphosphate to generate inositol 1,4,5-triphosphate (IP3) and diacylglycerol (4). IP3 binds to the IP3 receptor (IP3R) in the endoplasmic reticulum (ER) membrane leading to Ca 2+ mobilization from the ER, which is the main Ca 2+ store in immune cells (5). The release of Ca 2+ from the ER lumen to the cytosol causes structural changes and oligomerization of the ER transmembrane protein, stromal interaction molecule 1 (STIM1) (6, 7). These changes allow the cytosolic domain of STIM1 to directly inte...

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Section: Discussionmentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

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

Fredericks¹,

Hoffmann²,

Rose³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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134

123

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“…Selk is localized to the ER membrane in T cells, neutrophils, and macrophages, and its expression is required for optimal calcium flux triggered during engagement of the TCR, Fc␥Rs, chemokines, and TLRs. Several functions are particularly impaired in Selk Ϫ/Ϫ macrophages, including chemokineinduced migration, proinflammatory cytokine secretion in response to TLR ligands LPS and polyinosinic:polycytidylic acid, and the phagocytosis of IgG-coated particles [12,14].…”

Section: Introductionmentioning

confidence: 99%

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

Meiler

Baumer

Huang

et al. 2013

Journal of Leukocyte Biology

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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.

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“…Interestingly, calpain regulation of SelK was only found in innate immune cells such as macrophages, but not in T or B cells. The Ca 2 + flux mediated by SelK was found to be crucial for ERK activation and nitric oxide production required for phagocytosis of IgG-opsonized particles by macrophages (16). In addition to IgG-opsonized particles, oxidized lowdensity lipoprotein (LDL) uptake was impaired in SelK -/-macrophages, and this led to reduced foam cell formation and atherosclerosis (25).…”

Section: Introductionmentioning

confidence: 99%

Selenoprotein K and Protein Palmitoylation

Fredericks

Hoffmann

2015

Antioxidants & Redox Signaling

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Significance: Selenoprotein K (SelK) is an endoplasmic reticulum (ER) membrane protein, and its expression is sensitive to dietary selenium levels. A recently described role for SelK as a cofactor in catalyzing protein palmitoylation reactions provides an important link between low dietary selenium intake and suboptimal cellular functions that depend on this selenoprotein for palmitoylation. Recent Advances: A recent breakthrough provided insight into the contribution of SelK to calcium (Ca

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

Cited by 54 publications

References 42 publications

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

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

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

Selenoprotein K and Protein Palmitoylation

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