Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction.

Emerging evidence suggests that protein acetylation is a broad-ranging regulatory mechanism. Here we utilize acetyl-peptide arrays and metabolomic analyses to identify substrates of mitochondrial deacetylase Sirt3. We identified ornithine transcarbamoylase (OTC) from the urea cycle, and enzymes involved in ß-oxidation. Metabolomic analyses of fasted mice lacking Sirt3 (sirt3(-/-)) revealed alterations in ß-oxidation and the urea cycle. Biochemical analysis demonstrated that Sirt3 directly deacetylates OTC and stimulates its activity. Mice under caloric restriction (CR) increased Sirt3 protein levels, leading to deacetylation and stimulation of OTC activity. In contrast, sirt3(-/-) mice failed to deacetylate OTC in response to CR. Inability to stimulate OTC under CR led to a failure to reduce orotic acid levels, a known outcome of OTC deficiency. Thus, Sirt3 directly regulates OTC activity and promotes the urea cycle during CR, and the results suggest that under low energy input, Sirt3 modulates mitochondria by promoting amino acid catabolism and ß-oxidation.

Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication.

Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared with other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3) enables the cells normally unsusceptible to RV-C infection to support both virus binding and replication. A coding single nucleotide polymorphism (rs6967330, C529Y) was previously linked to greater cell-surface expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared with wild-type CDHR3, cells transfected with the CDHR3-Y529 variant had about 10-fold increases in RV-C binding and progeny yields. We developed a transduced HeLa cell line (HeLa-E8) stably expressing CDHR3-Y529 that supports RV-C propagation in vitro. Modeling of CDHR3 structure identified potential binding sites that could impact the virus surface in regions that are highly conserved among all RV-C types. Our findings identify that the asthma susceptibility gene product CDHR3 mediates RV-C entry into host cells, and suggest that rs6967330 mutation could be a risk factor for RV-C wheezing illnesses.

Effect of exogenous interferons on rhinovirus replication and airway inflammatory responses.

BACKGROUND: Human rhinoviruses (HRVs) are the most common cause of asthma exacerbations. In airway epithelial cells, the primary site of HRV infection, decreased production of interferons (IFNs) may result in greater susceptibility to HRV and worsened symptoms. Thus, exogenous IFN could supplement the innate immune response and provide a treatment for virus-induced asthma exacerbations. Furthermore, the effects of exogenous IFN could be type specific in part because of the cellular distribution of type 1 and type 2 IFN receptors. OBJECTIVE: To investigate the effects of exogenous IFNs on HRV replication in bronchial epithelial cells. METHODS: Frozen stocks of primary human bronchial epithelial cells from healthy donors were cultured in monolayers; pretreated (24 hours) with 0.1-ng/mL, 1-ng/mL, or 10-ng/mL doses of IFN-α, -ß, -λ1, or -λ2; and infected with HRV-1A. Viral replication was quantified using real-time reverse transcription-polymerase chain reaction, and cytokine and chemokine secretion 24 hours after infection was measured by multiplex enzyme-linked immunosorbent assay. RESULTS: Compared with untreated samples, IFN-α, IFN-ß, IFN-λ1, and IFN-λ2 (0.1 ng/mL) significantly reduced HRV replication after high- (P < .02) and low-dose inoculation (P < .05). Similar effects were seen in 1-ng/mL and 10-ng/mL doses of IFN, where HRV replication was significantly decreased in both high- (P < .001) and low-dose inoculation (P < .001). Treatment with IFNs also enhanced HRV-induced IFN-γ-induced protein 10 secretion (P < .001). Finally, treatment with either IFN-λ1 or IFN-λ2 significantly increased HRV-induced secretion of RANTES (regulated on activation, normal T-expressed, and presumably secreted) (P < .05) but not IL-1ß or vascular endothelial growth factor. CONCLUSION: These findings suggest that exogenous IFNs, IFN-λ1 in particular, warrant further study as a potential therapy for virus-induced asthma exacerbations.

Innate immune responses to rhinovirus are reduced by the high-affinity IgE receptor in allergic asthmatic children.

BACKGROUND: Children with allergic asthma have more frequent and severe human rhinovirus (HRV)-induced wheezing and asthma exacerbations through unclear mechanisms. OBJECTIVE: We sought to determine whether increased high-affinity IgE receptor (FcεRI) expression and cross-linking impairs innate immune responses to HRV, particularly in allergic asthmatic children. METHODS: PBMCs were obtained from 44 children, and surface expression of FcεRI on plasmacytoid dendritic cells (pDCs), myeloid dendritic cells, monocytes, and basophils was assessed by using flow cytometry. Cells were also incubated with rabbit anti-human IgE to cross-link FcεRI, followed by stimulation with HRV-16, and IFN-α and IFN-λ1 production was measured by Luminex. The relationships among FcεRI expression and cross-linking, HRV-induced IFN-α and IFN-λ1 production, and childhood allergy and asthma were subsequently analyzed. RESULTS: FcεRIα expression on pDCs was inversely associated with HRV-induced IFN-α and IFN-λ1 production. Cross-linking FcεRI before HRV stimulation further reduced PBMC IFN-α (47% relative reduction; 95% CI, 32% to 62%; P< .0001) and IFN-λ1 (81% relative reduction; 95% CI, 69% to 93%; P< .0001) secretion. Allergic asthmatic children had higher surface expression of FcεRIα on pDCs and myeloid dendritic cells when compared with that seen in nonallergic nonasthmatic children. Furthermore, after FcεRI cross-linking, allergic asthmatic children had significantly lower HRV-induced IFN responses than allergic nonasthmatic children (IFN-α, P= .004; IFN-λ1, P= .02) and nonallergic nonasthmatic children (IFN-α, P= .002; IFN-λ1, P= .01). CONCLUSIONS: Allergic asthmatic children have impaired innate immune responses to HRV that correlate with increased FcεRI expression on pDCs and are reduced by FcεRI cross-linking. These effects likely increase susceptibility to HRV-induced wheezing and asthma exacerbations.

Recent Clinical Isolates of Enterovirus D68 Have Increased Replication and Induce Enhanced Epithelial Immune Response Compared to the Prototype Fermon Strain.

In 2014, enterovirus D68 (EV-D68), previously associated primarily with mild respiratory illness, caused a large outbreak of severe respiratory illness and, in rare instances, paralysis. We compared the viral binding and replication of eight recent EV-D68 clinical isolates collected both before and during the 2014 outbreak and the prototype Fermon strain from 1962 in cultured HeLa cells and differentiated human primary bronchial epithelial cells (BEC) to understand the possible reasons for the change in virus pathogenicity. We selected pairs of closely related isolates from the same phylogenetic clade that were associated with severe vs. asymptomatic infections. We found no significant differences in binding or replication in HeLa cell cultures between the recent clinical isolates. However, in HeLa cells, Fermon had significantly greater binding (2-3 logs) and virus progeny yields (2-4 logs) but a similar level of replication (1.5-2 log increase in viral RNA from 2 h to 24 h post infection) compared to recent isolates. In differentiated BECs, Fermon and the recent EV-D68 isolates had similar levels of binding; however, the recent isolates produced 1.5-2-log higher virus progeny yields than Fermon due to increased replication. Interestingly, no significant differences in replication were identified between the pairs of genetically close recent EV-D68 clinical isolates despite the observed differences in associated disease severity. We then utilized RNA-seq to define the transcriptional responses in BECs infected with four recent EV-D68 isolates, representing major phylogenetic clades, and the Fermon strain. All the tested clinical isolates induced similar responses in BECs; however, numerous upregulated genes in antiviral and pro-inflammatory response pathways were identified when comparing the response to clinical isolates versus Fermon. These results indicate that the recent emergence in severe EV-D68 cases could be explained by an increased replication efficiency and enhanced inflammatory response induced by newly emerged clinical isolates; however, host factors are likely the main determinants of illness severity.

Structure and biochemical functions of SIRT6.

SIRT6 is a member of the evolutionarily conserved sirtuin family of NAD(+)-dependent protein deacetylases and functions in genomic stability and transcriptional control of glucose metabolism. Early reports suggested that SIRT6 performs ADP-ribosylation, whereas more recent studies have suggested that SIRT6 functions mainly as a histone deacetylase. Thus, the molecular functions of SIRT6 remain uncertain. Here, we perform biochemical, kinetic, and structural studies to provide new mechanistic insight into the functions of SIRT6. Utilizing three different assays, we provide biochemical and kinetic evidence that SIRT6-dependent histone deacetylation produces O-acetyl-ADP-ribose but at a rate ∼1,000 times slower than other highly active sirtuins. To understand the molecular basis for such low deacetylase activity, we solved the first crystal structures of this class IV sirtuin in complex with ADP-ribose and the non-hydrolyzable analog of O-acetyl-ADP-ribose, 2'-N-acetyl-ADP-ribose. The structures revealed unique features of human SIRT6, including a splayed zinc-binding domain and the absence of a helix bundle that in other sirtuin structures connects the zinc-binding motif and Rossmann fold domain. SIRT6 also lacks the conserved, highly flexible, NAD(+)-binding loop and instead contains a stable single helix. These differences led us to hypothesize that SIRT6, unlike all other studied sirtuins, would be able to bind NAD(+) in the absence of an acetylated substrate. Indeed, we found that SIRT6 binds NAD(+) with relatively high affinity (K(d) = 27 ± 1 µM) in the absence of an acetylated substrate. Isothermal titration calorimetry and tryptophan fluorescence binding assays suggested that ADP-ribose and NAD(+) induce different structural perturbations and that NADH does not bind to SIRT6. Collectively, these new insights imply a unique activating mechanism and/or the possibility that SIRT6 could act as an NAD(+) metabolite sensor.