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.

Selenoprotein K and protein palmitoylation.

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(2+)) flux in immune cells. In particular, SelK is required for palmitoylation of the Ca(2+) channel protein, inositol-1,4,5-triphosphate receptor (IP3R) in the ER membrane. Without this post-translational modification, expression and function of the IP3R is impaired. SelK is required for palmitoylation of another transmembrane protein, CD36, and very likely other proteins. SelK serves as a cofactor during protein palmitoylation by binding to the protein acyltransferase, DHHC6, thereby facilitating addition of the palmitate via a thioester bond to the sulfhydryl group of cysteine residues of target proteins. CRITICAL ISSUES: The association of DHHC6 and SelK is clearly important for immune cell functions and possibly other cell types. The step in the DHHC6 catalyzed S-acylation reaction on which SelK acts remains unclear and possible mechanisms of how the kinetics of the reaction are impacted by SelK binding to DHHC6 are presented here. FUTURE DIRECTIONS: Uncovering the specific role of SelK in promoting DHHC6 catalyzed protein palmitoylation may open a new line of inquiry into other selenoproteins playing similar roles as cofactors for different enzymatic processes.

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.

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.

Characterization of the cortisol stress response to sedation and anesthesia in children.

CONTEXT: The cortisol stress response to sedation and anesthesia in children is not well characterized. It is not clear whether it is necessary to give stress doses of corticosteroids to children with adrenal insufficiency undergoing sedated procedures. OBJECTIVE: Our objective was to describe the cortisol stress response to sedation and anesthesia in normal children. DESIGN, SETTING, AND PATIENTS: This was a prospective cohort study of 149 children ages 1 month to 17 yr who presented for routine sedated procedures. Salivary cortisol was measured at baseline, every 30 min during procedures, at completion, and at recovery. MAIN OUTCOME MEASURES: We evaluated relative change in salivary cortisol from baseline for level of sedation achieved and type of procedure performed. RESULTS: In total, 117 patients had adequate samples collected, and 110 were included in the main analysis. Twenty-five percent of patients showed an increase in salivary cortisol greater than four times baseline, consistent with a stress response. Mean salivary cortisol increased more than 3-fold from baseline (3.7±0.4, P<0.001) for all patients in the study. There was no difference for change in cortisol when comparing by level of sedation achieved or by type of procedure performed. The majority of patients with a stress response had their highest levels in the recovery phase, after their procedure was completed. CONCLUSION: Sedation and anesthesia can induce a significant rise in cortisol in children. Additional studies should be performed to validate our results and to determine whether stress dosing of corticosteroids may be needed for children with adrenal insufficiency undergoing sedated procedures.

A pilot project for improving paediatric diabetes outcomes using a website: the Pediatric Diabetes Education Portal.

We created a website for patients and families that allowed them to review clinic test results, review educational materials related to these results and post questions to their diabetes educator. Fingerstick haemoglobin A(1c) (HbA(1c)) testing and periodic use of a continuous glucose monitoring system (CGMS) were offered to all patients. The HbA(1c) and CGMS results were posted to the website after each clinic visit. A total of 52 patients with type 1 diabetes were enrolled in the study. There were 16 patients with HbA(1c) values within ADA guidelines and 16 with HbA(1c) values above guidelines; 20 patients were excluded for various reasons. Users of the website were defined as families who logged in four or more times over the six-month study period. For patients whose HbA(1c) started above ADA guidelines, the mean HbA(1c) for website users decreased from 10.5% (SD 2.2) at baseline to 9.1% (SD 1.2) after six months. In the non-users, the mean HbA(1c) increased from 9.5% (SD 1.5) at baseline to 10.4% (SD 2.5). However, these changes were not significant. A between groups comparison (users versus non-users) showed a significant improvement in HbA(1c) for website users (P = 0.03). This change in HbA(1c) was clinically relevant. Further studies with more patients are needed to see if these improvements can be sustained over a longer period.

RNA interference of transforming growth factor-ß 2 in human respiratory fibroblasts.

OBJECTIVE: Transforming growth factor-ß2 (TGF-ß2) is a principal cytokine of interest in the pathogenesis of scar formation and is a potential target for future molecular-based adjunctive therapies. The authors hypothesize that interfering RNA (RNAi) can be used to modulate TGF-ß2 production in cultured human respiratory fibroblasts. STUDY DESIGN: Basic science. Setting. Laboratory. SUBJECTS AND METHODS: RNAi constructs targeted to the TGF-ß2 transcript were complexed with microsomal lipids and applied to human fibroblasts in cell culture. Transfection efficiency and cell viability were measured by fluorescence microscopy. Messenger RNA (mRNA) for TGF-ß2 was measured 48 hours posttransfection using real-time quantitative PCR. The quantity of TGF-ß2 protein produced with increasing concentrations of RNAi was measured using enzyme-linked immunosorbent assay. The function of RNAi-treated fibroblasts was tested using a wound-healing assay. RESULTS: Transfection efficiency of more than 80% was achieved with minimal induced cell death. Treated cells showed selective knockdown of 80% of TGF-ß2 mRNA, which was confirmed with negative controls. As the concentration of RNAi was increased, an incremental decrease in TGF-ß2 protein was measured. CONCLUSION: RNAi technology is an effective means of localized and transient gene silencing in cultured human fibroblasts. Transfection can be achieved using microsome complexed RNAi with minimal induced cell death. This preliminary result shows promise for future in vitro studies.

Total and acylated ghrelin levels in children with poor growth.

Ghrelin, an enteric hormone with potent appetite stimulating effects, also stimulates growth hormone release. We hypothesized that altered levels of total ghrelin (TG) or acylated ghrelin (AG) could affect growth by altering growth hormone secretion, subsequently affecting insulin-like growth factor-1 (IGF-1) generation or by altering appetite and food intake. After institutional review board approval, 52 children presenting for evaluation of chronic gastrointestinal symptoms (group 1), poor weight gain (group 2), or poor linear growth (group 3) were evaluated for fasting TG and AG levels in addition to their regular evaluation. Serum ghrelin, IGF-1, and prealbumin were compared between groups. No difference was observed for mean fasting TG between groups. However, mean fasting AG was highest in patients in group 2 (465 ± 128 pg/mL) versus group 1 (176 ± 37 pg/mL) and group 3 (190 ± 34 pg/mL). IGF-1 was lowest in patients in group 2 despite similar prealbumin levels among the three groups. We conclude that serum AG levels are highest in children with isolated poor weight gain compared with children with short stature or chronic gastrointestinal symptoms, suggesting the possibility of resistance to AG in underweight children. Additional studies are needed to further clarify ghrelin's role in growth and appetite.