RNA‐binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral activity

The type I interferon (IFN‐I, IFN‐α/β)‐mediated immune response is the first line of host defense against invading viruses. IFN‐α/β binds to IFN‐α/β receptors (IFNARs) and triggers the expression of IFN‐stimulated genes (ISGs). Thus, stabilization of IFNARs is important for prolonging antiviral activity. Here, we report the induction of an RNA‐binding motif‐containing protein, RBM47, upon viral infection or interferon stimulation. Using multiple virus infection models, we demonstrate that RBM47 has broad‐spectrum antiviral activity in vitro and in vivo. RBM47 has no noticeable impact on IFN production, but significantly activates the IFN‐stimulated response element (ISRE) and enhances the expression of interferon‐stimulated genes (ISGs). Mechanistically, RBM47 binds to the 3''UTR of IFNAR1 mRNA, increases mRNA stability, and retards the degradation of IFNAR1. In summary, this study suggests that RBM47 is an interferon‐inducible RNA‐binding protein that plays an essential role in enhancing host IFN downstream signaling.     

Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4

Nuclear IκBα preferentially binds the acetylated N‐terminal tail of histone H4 in vivo, specifically in the skin and intestine stem cell compartments. N‐terminal cleavage of histone H4 facilitates IκBα dissociation and cellular differentiation.     

SARS‐CoV‐2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage

SARS‐CoV‐2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS‐CoV‐2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS‐CoV‐2 viral proteins. Here, we show that the nucleocapsid of SARS‐CoV‐2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS‐CoV‐2‐infected monocytes show enhanced cellular interleukin‐1β (IL‐1β) expression, but reduced IL‐1β secretion. While SARS‐CoV‐2 infection promotes activation of the NLRP3 inflammasome and caspase‐1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS‐CoV‐2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase‐1. These insights into how SARS‐CoV‐2 antagonizes cellular inflammatory responses may open new avenues for treating COVID‐19 in the future.     

Glycogen synthase kinase 3β inhibition enhanced proliferation,migration and functional re-endothelialization of endothelial progenitor cells in hypercholesterolemia microenvironment

Hypercholesterolemia impairs the quantity and function of endothelial progenitor cell. We hypothesized that glycogen synthase kinase 3β activity is involved in regulating biological function of endothelial progenitor cells in hypercholesterolemia microenvironment. For study, endothelial progenitor cells derived from apolipoprotein E-deficient mice fed with high-fat diet were used. Glycogen synthase kinase 3β activity was interfered with glycogen synthase kinase 3β inhibitor lithium chloride or transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β (GSK3β-KM). Functions of endothelial progenitor cells, proliferation, migration, secretion and network formation of endothelial progenitor cells were assessed in vitro. The expression of phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 in endothelial progenitor cells was detected by Western blot. The in vivo function re-endothelialization and vasodilation were also analyzed by artery injury model transplanted with glycogen synthase kinase 3β-inhibited endothelial progenitor cells. We demonstrated that while the proliferation, migration, network formation as well as VEGF and NO secretion were impaired in apolipoprotein E-deficient endothelial progenitor cells, glycogen synthase kinase 3β inhibition significantly improved all these functions. Apolipoprotein E-deficient endothelial progenitor cells showed decreased phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 expression, whereas these signals were enhanced by glycogen synthase kinase 3β inhibition and accompanied with β-catenin nuclear translocation. Our in vivo model showed that glycogen synthase kinase 3β inhibition remarkably increased re-endothelial and vasodilation. Taken together, our data suggest that inhibition of glycogen synthase kinase 3β is associated with endothelial progenitor cell biological functions both in vitro and in vivo. It might be an important interference target in hypercholesterolemia microenvironment.     

PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells

Type I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide– and natriuretic peptide–induced cGMP generation. However, some reports have shown that PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu²⁺ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGIα Cys⁴³, Cys¹¹⁸, and Cys¹⁹⁶ play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys⁴³ crosslinking in response to H₂O₂ (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo.     

LRP5 promotes cancer stem cell traits and chemoresistance in colorectal cancer

The overactivation of canonical Wnt/β‐catenin pathway and the maintenance of cancer stem cells (CSCs) are essential for the onset and malignant progression of most human cancers. However, their regulatory mechanism in colorectal cancer (CRC) has not yet been well demonstrated. Low‐density lipoprotein receptor‐related protein 5 (LRP5) has been identified as an indispensable co‐receptor with frizzled family members for the canonical Wnt/β‐catenin signal transduction. Herein, we show that activation of LRP5 gene promotes CSCs‐like phenotypes, including tumorigenicity and drug resistance in CRC cells, through activating the canonical Wnt/β‐catenin and IL‐6/STAT3 signalling pathways. Clinically, the expression of LRP5 is upregulated in human CRC tissues and closely associated with clinical stages of patients with CRC. Further analysis showed silencing of endogenous LRP5 gene is sufficient to suppress the CSCs‐like phenotypes of CRC through inhibiting these two pathways. In conclusion, our findings not only reveal a regulatory cross‐talk between canonical Wnt/β‐catenin signalling pathway, IL‐6/STAT3 signalling pathway and CD133‐related stemness that promote the malignant behaviour of CRC, but also provide a valuable target for the diagnosis and treatment of CRC.     

The stiffness‐controlled release of interleukin‐6 by cardiac fibroblasts is dependent on integrin α2β1

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin‐6 (IL‐6), interleukin‐11 (IL‐11) and soluble receptor of IL‐6 (sIL‐6R). It also examines the roles of integrin α2β1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2β1 integrin count and released higher levels of IL‐6 and sIL‐6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL‐11 content. The silencing of the α2 integrin subunit decreased the release of IL‐6. Similar effects were induced by TC‐I 15 (an α2β1 integrin inhibitor). The IL‐6 levels in the serum and heart were markedly lower in α2 integrin‐deficient mice B6.Cg‐Itga2^{tm1.1Tkun/tm1.1Tkun} than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL‐6 level. sIL‐6R secretion is not dependent on α2β1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL‐6 by cardiac fibroblasts, and this effect is dependent on α2β1 integrin and kinase Src activation.     

Nur77 Prevents Osteoporosis by Inhibiting the NF‐κB Signalling Pathway and Osteoclast Differentiation

Inflammation is a major risk factor for osteoporosis, and reducing inflammatory levels is important for the prevention of osteoporosis. Although nuclear receptor 77 (Nur77) protects against inflammation in a variety of diseases, its role in osteoporosis is unknown. Therefore, the main purpose of this study was to investigate the osteoprotective and anti‐inflammatory effects of Nur77. The microCT and haematoxylin and eosin staining results indicated that knockout of Nur77 accelerated femoral bone loss in mice. The enzyme‐linked immunosorbent assay (ELISA) results showed that knockout of Nur77 increased the serum levels of hsCRP and IL‐6. The expression levels of NF‐κB, IL‐6, TNF‐α and osteoclastogenesis factors (TRAP, NFATC1, Car2, Ctsk) in the femurs of Nur77 knockout mice were increased significantly. Furthermore, in vitro, shNur77 promoted the differentiation of RAW264.7 cells into osteoclasts by activating NF‐κB, which was confirmed by PDTC treatment. Mechanistically, Nur77 inhibited osteoclast differentiation by inducing IκB‐α and suppressing IKK‐β. In RAW264.7 cells, overexpression of Nur77 alleviated inflammation induced by siIκB‐α, while siIKK‐β alleviated inflammation induced by shNur77. Consistent with the in vivo studies, we found that compared with control group, older adults with high serum hsCRP levels were more likely to suffer from osteoporosis (OR = 1.76, p < 0.001). Our data suggest that Nur77 suppresses osteoclast differentiation by inhibiting the NF‐κB signalling pathway, strongly supporting the notion that Nur77 has the potential to prevent and treat osteoporosis.     

Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM.     

TIFA has dual functions in Helicobacter pylori‐induced classical and alternative NF‐κB pathways

Helicobacter pylori infection constitutes one of the major risk factors for the development of gastric diseases including gastric cancer. The activation of nuclear factor‐kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) via classical and alternative pathways is a hallmark of H. pylori infection leading to inflammation in gastric epithelial cells. Tumor necrosis factor receptor‐associated factor (TRAF)‐interacting protein with forkhead‐associated domain (TIFA) was previously suggested to trigger classical NF‐κB activation, but its role in alternative NF‐κB activation remains unexplored. Here, we identify TRAF6 and TRAF2 as binding partners of TIFA, contributing to the formation of TIFAsomes upon H. pylori infection. Importantly, the TIFA/TRAF6 interaction enables binding of TGFβ‐activated kinase 1 (TAK1), leading to the activation of classical NF‐κB signaling, while the TIFA/TRAF2 interaction causes the transient displacement of cellular inhibitor of apoptosis 1 (cIAP1) from TRAF2, and proteasomal degradation of cIAP1, to facilitate the activation of the alternative NF‐κB pathway. Our findings therefore establish a dual function of TIFA in the activation of classical and alternative NF‐κB signaling in H. pylori‐infected gastric epithelial cells.     

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKβ, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the “NEMO-binding domain” at the C terminus of IKK2/IKKβ. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKβ through this secondary interaction in vitro and for full activation of IKK2/IKKβ in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKβ within its scaffold-dimerization domain proximal to the kinase domain–Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture–based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKβ for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.     

PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

Variants identified in genome‐wide association studies have implicated immune pathways in the development of Alzheimer’s disease (AD). Here, we investigated the mechanistic basis for protection from AD associated with PLCγ2 R522, a rare coding variant of the PLCG2 gene. We studied the variant''s role in macrophages and microglia of newly generated PLCG2‐R522‐expressing human induced pluripotent cell lines (hiPSC) and knockin mice, which exhibit normal endogenous PLCG2 expression. In all models, cells expressing the R522 mutation show a consistent non‐redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This manifests as enhanced release of cellular calcium ion stores in response to physiologically relevant stimuli like Fc‐receptor ligation or exposure to Aβ oligomers. Expression of the PLCγ2‐R522 variant resulted in increased stimulus‐dependent PIP₂ depletion and reduced basal PIP₂ levels in vivo. Furthermore, it was associated with impaired phagocytosis and enhanced endocytosis. PLCγ2 acts downstream of other AD‐related factors, such as TREM2 and CSF1R, and alterations in its activity directly impact cell function. The inherent druggability of enzymes such as PLCγ2 raises the prospect of PLCγ2 manipulation as a future therapeutic approach in AD.     

HOIL‐1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.     

Fibronectin type III domain‐containing 5 improves aging‐related cardiac dysfunction in mice

Aging is an important risk factor for cardiovascular diseases, and aging‐related cardiac dysfunction serves as a major determinant of morbidity and mortality in elderly populations. Our previous study has identified fibronectin type III domain‐containing 5 (FNDC5) and its cleaved form, irisin, as the cardioprotectant against doxorubicin‐induced cardiomyopathy. Herein, aging or matched young mice were overexpressed with FNDC5 by adeno‐associated virus serotype 9 (AAV9) vectors, or subcutaneously infused with irisin to uncover the role of FNDC5 in aging‐related cardiac dysfunction. To verify the involvement of nucleotide‐binding oligomerization domain‐like receptor with a pyrin domain 3 (NLRP3) and AMP‐activated protein kinase α (AMPKα), Nlrp3 or Ampkα2 global knockout mice were used. Besides, young mice were injected with AAV9‐FNDC5 and maintained for 12 months to determine the preventive effect of FNDC5. Moreover, neonatal rat cardiomyocytes were stimulated with tumor necrosis factor‐α (TNF‐α) to examine the role of FNDC5 in vitro. We found that FNDC5 was downregulated in aging hearts. Cardiac‐specific overexpression of FNDC5 or irisin infusion significantly suppressed NLRP3 inflammasome and cardiac inflammation, thereby attenuating aging‐related cardiac remodeling and dysfunction. In addition, irisin treatment also inhibited cellular senescence in TNF‐α‐stimulated cardiomyocytes in vitro. Mechanistically, FNDC5 activated AMPKα through blocking the lysosomal degradation of glucagon‐like peptide‐1 receptor. More importantly, FNDC5 gene transfer in early life could delay the onset of cardiac dysfunction during aging process. We prove that FNDC5 improves aging‐related cardiac dysfunction by activating AMPKα, and it might be a promising therapeutic target to support cardiovascular health in elderly populations.     

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-β polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a K_D of 15–47 μM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC₅₀ values in the range of 15–164 μM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.     

Induction of γδT cells from HSC‐enriched BMCs co‐cultured with iPSC‐derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co‐culturing HSC‐enriched bone marrow cells (HSC‐eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC‐eBMCs and iTECs cultured with IL‐2 + IL‐7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T‐cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia‐bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.     

Karyopherin-Centric Control of Nuclear Pores Based on Molecular Occupancy and Kinetic Analysis of Multivalent Binding with FG Nucleoporins

Intrinsically disordered Phe-Gly nucleoporins (FG Nups) within nuclear pore complexes exert multivalent interactions with transport receptors (Karyopherins (Kaps)) that orchestrate nucleocytoplasmic transport. Current FG-centric views reason that selective Kap translocation is promoted by alterations in the barrier-like FG Nup conformations. However, the strong binding of Kaps with the FG Nups due to avidity contradicts rapid Kap translocation in vivo. Here, using surface plasmon resonance, we innovate a means to correlate in situ mechanistic (molecular occupancy and conformational changes) with equilibrium (binding affinity) and kinetic (multivalent binding kinetics) aspects of Karyopherinβ1 (Kapβ1) binding to four different FG Nups. A general feature of the FxFG domains of Nup214, Nup62, and Nup153 is their capacity to extend and accommodate large numbers of Kapβ1 molecules at physiological Kapβ1 concentrations. A notable exception is the GLFG domain of Nup98, which forms a partially penetrable cohesive layer. Interestingly, we find that a slowly exchanging Kapβ1 phase forms an integral constituent within the FG Nups that coexists with a fast phase, which dominates transport kinetics due to limited binding with the pre-occupied FG Nups at physiological Kapβ1 concentrations. Altogether, our data reveal an emergent Kap-centric barrier mechanism that may underlie mechanistic and kinetic control in the nuclear pore complex.     

Human DND1‐RRM2 forms a non‐canonical domain swapped dimer

RNA recognition motif (RRM) being the most abundant RNA binding domain in eukaryotes, is a major player in cellular regulation. Several variations in the canonical βαββαβ topology have been observed. We have determined the 2.3 Å crystal structure of the human DND1‐RRM2 domain. The structure revealed an interesting non‐canonical RRM fold, which is maintained by the formation of a 3D domain swapped dimer between β₁ and β₄ strands across protomers. We have delineated the structural basis of the stable domain swapped dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our structural and dynamics studies substantiate major determinants and molecular basis for domain swapped dimerization observed in the RRM domain.     

SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice

Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide‑binding oligomerization domain, leucine rich repeat, and pyrin domain‑containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.