IL-1 induces mitochondrial translocation of IRAK2 to suppress oxidative metabolism in adipocytes.

Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R-MyD88-IRAK2-PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity.

MFN2 couples glutamate excitotoxicity and mitochondrial dysfunction in motor neurons.

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca(2+)-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.

Cited2, a transcriptional modulator protein, regulates metabolism in murine embryonic stem cells.

CREB-binding protein (CBP)/p300 interacting transactivator with glutamic acid (Glu) and aspartic acid (Asp)-tail 2 (Cited2) was recently shown to be essential for gluconeogenesis in the adult mouse. The metabolic function of Cited2 in mouse embryonic stem cells (mESCs) remains elusive. In the current study, the metabolism of glucose was investigated in mESCs, which contained a deletion in the gene for Cited2 (Cited2(Δ/-)). Compared with its parental wild type counterpart, Cited2(Δ/-) ESCs have enhanced glycolysis, alternations in mitochondria morphology, reduced glucose oxidation, and decreased ATP content. Cited2 is recruited to the hexokinase 1 (HK1) gene promoter to regulate transcription of HK1, which coordinates glucose metabolism in wild type ESCs. Reduced glucose oxidation and enhanced glycolytic activity in Cited2(Δ/-) ESCs correlates with defective differentiation during hypoxia, which is reflected in an increased expression of pluripotency marker (Oct4) and epiblast marker (Fgf5) and decreased expression of lineage specification markers (T, Gata-6, and Cdx2). Knockdown of hypoxia inducible factor-1α in Cited2(Δ/-) ESCs re-initiates the expression of differentiation markers T and Gata-6. Taken together, a deletion of Cited2 in mESCs results in abnormal mitochondrial morphology and impaired glucose metabolism, which correlates with a defective cell fate decision.

Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis.

Copper levels are known to be elevated in inflamed and malignant tissues. But the mechanism underlying this selective enrichment has been elusive. In this study, we report a axis by which inflammatory cytokines, such as IL-17, drive cellular copper uptake via the induction of a metalloreductase, STEAP4. IL-17-induced elevated intracellular copper level leads to the activation of an E3-ligase, XIAP, which potentiates IL-17-induced NFκB activation and suppresses the caspase 3 activity. Importantly, this IL-17-induced STEAP4-dependent cellular copper uptake is critical for colon tumor formation in a murine model of colitis-associated tumorigenesis and STEAP4 expression correlates with IL-17 level and XIAP activation in human colon cancer. In summary, this study reveals a IL-17-STEAP4-XIAP axis through which the inflammatory response induces copper uptake, promoting colon tumorigenesis.

Epithelial-derived gasdermin D mediates nonlytic IL-1ß release during experimental colitis.

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1ß. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1ß-containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro-IL-1ß, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1ß colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63+ sEVs containing IL-1ß, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1ß-containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1ß sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.

Dynamin-like protein 1 cleavage by calpain in Alzheimer's disease.

Abnormal mitochondrial dynamics contributes to mitochondrial dysfunction in Alzheimer's disease (AD), yet the underlying mechanism remains elusive. In the current study, we reported that DLP1, the key mitochondrial fission GTPase, is a substrate of calpain which produced specific N-terminal DLP1 cleavage fragments. In addition, various AD-related insults such as exposure to glutamate, soluble amyloid-ß oligomers, or reagents inducing tau hyperphosphorylation (i.e., okadaic acid) led to calpain-dependent cleavage of DLP1 in primary cortical neurons. DLP1 cleavage fragments were found in cortical neurons of CRND8 APP transgenic mice which can be inhibited by calpeptin, a potent small molecule inhibitor of calpain. Importantly, these N-terminal DLP1 fragments were also present in the human brains, and the levels of both full-length and N-terminal fragments of DLP1 and the full-length and calpain-specific cleavage product of spectrin were significantly reduced in AD brains along with significantly increased calpain. These results suggest that calpain-dependent cleavage is at least one of the posttranscriptional mechanisms that contribute to the dysregulation of mitochondrial dynamics in AD.

The selection pressure analysis of classical swine fever virus envelope protein genes Erns and E2.

Classical swine fever virus, one member of the family Flaviviridae, is the pathogen of CSF, an economically important and highly contagious disease of pigs. Knowledge of virus genes under positive selection pressure can help identify molecular determinants of virulence or pathogenesis without prior knowledge of the mechanisms governing virulence and pathogenesis and clarify the driving force of classical swine fever virus evolution. The positive selection pressure acting on envelope protein genes E(rns), E1 and E2 of classical swine fever virus were assessed and a site-by-site analysis of the d(N)/d(S) ratio was performed, to identify specific codons undergoing diversifying positive selection. Whilst no significant evidence for positive selection was observed in E1, four positively selected sites (208 in E(rns) and 72, 75, and 200 in E2) were identified. The positively selected site (208) of E(rns) corresponds to one of the amino acid substitutions (Ser to Arg) found in an HS-binding CSFV variant. The mutant at the positively selected site (75) is located within an O-glycosylation motif and altered the predicted glycosylation pattern. In addition, Thr at the positively selective site 200 are directly involved with mAb WH308 with which CS vaccine strain does not react, unlike most of the virulent CSFV strains.

Act1 is a negative regulator in T and B cells via direct inhibition of STAT3.

Although Act1 (adaptor for IL-17 receptors) is necessary for IL-17-mediated inflammatory responses, Act1- (but not Il17ra-, Il17rc-, or Il17rb-) deficient mice develop spontaneous SLE- and Sjögren's-like diseases. Here, we show that Act1 functions as a negative regulator in T and B cells via direct inhibition of STAT3. Mass spectrometry analysis detected an Act1-STAT3 complex, deficiency of Act1 (but not Il17ra-, Il17rc-, or Il17rb) results in hyper IL-23- and IL-21-induced STAT3 activation in T and B cells, respectively. IL-23R deletion or blockade of IL-21 ameliorates SLE- and Sjögren's-like diseases in Act1-/- mice. Act1 deficiency results in hyperactivated follicular Th17 cells with elevated IL-21 expression, which promotes T-B cell interaction for B cell expansion and antibody production. Moreover, anti-IL-21 ameliorates the SLE- and Sjögren's-like diseases in Act1-deficient mice. Thus, IL-21 blocking antibody might be an effective therapy for treating SLE- and Sjögren's-like syndrome in patients containing Act1 mutation.

Cited2 is required for the maintenance of glycolytic metabolism in adult hematopoietic stem cells.

Mammalian adult hematopoietic stem cells (HSCs) reside in the hypoxic bone marrow microenvironment and display a distinct metabolic phenotype compared with their progenitors. It has been proposed that HSCs generate energy mainly through anaerobic glycolysis in a pyruvate dehydrogenase kinase (Pdk)-dependent manner. Cited2 is an essential regulator for HSC quiescence, apoptosis, and function. Herein, we show that conditional deletion of Cited2 in murine HSCs results in elevated levels of reactive oxygen species, decreased cellular glutathione content, increased mitochondrial activity, and decreased glycolysis. At the molecular level, Cited2 deficiency significantly reduced the expression of genes involved in metabolism, such as Pdk2, Pdk4, and lactate dehydrogenases B and D (LDHB and LDHD). Cited2-deficient HSCs also exhibited increased Akt signaling, concomitant with elevated mTORC1 activity and phosphorylation of FoxOs. Further, inhibition of PI3/Akt, but not mTORC1, partially rescued the repression of Pdk4 caused by deletion of Cited2. Altogether, our results suggest that Cited2 is required for the maintenance of adult HSC glycolytic metabolism likely through regulating Pdk2, Pdk4, LDHB, LDHD, and Akt activity.

GFI1 is repressed by p53 and inhibits DNA damage-induced apoptosis.

GFI1 is a transcriptional repressor that plays a critical role in hematopoiesis and has also been implicated in lymphomagenesis. It is still poorly understood how GFI1 expression is regulated in the hematopoietic system. We show here that GFI1 transcription was repressed by the tumor suppressor p53 in hematopoietic cells. Knockdown of p53 resulted in increased GFI1 expression and abolished DNA damage-induced GFI1 downregulation. In contrast, GFI1 expression was reduced and its downregulation in response to DNA damage was rescued upon restoration of p53 function in p53-deficient cells. In luciferase reporter assays, wild type p53, but not a DNA binding-defective p53 mutant, repressed the GFI1 promoter. Chromatin immunoprecipitation (ChIP) assays demonstrated that p53 bound to the proximal region of the GFI1 promoter. Detailed mapping of the GFI1 promoter indicated that GFI1 core promoter region spanning from -33 to +6 bp is sufficient for p53-mediated repression. This core promoter region contains a putative p53 repressive response element, mutation of which abolished p53 binding to and repression of GFI1 promoter. Significantly, apoptosis induced by DNA damage was inhibited upon Gfi1 overexpression, but augmented following GFI1 knockdown. Our data establish for the first time that GFI1 is repressed by p53 and add to our understanding of the roles of GFI1 in normal hematopoiesis and lymphomagenesis.

Analysis of synonymous codon usage in classical swine fever virus.

Using the complete genome sequences of 35 classical swine fever viruses (CSFV) representing all three genotypes and all three kinds of virulence, we analyzed synonymous codon usage and the relative dinucleotide abundance in CSFV. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in CSFV. Furthermore, we observed that the relative abundance of dinucleotides in CSFV is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as the subgenotypes and aromaticity, also influence the codon usage variation among the genomes of CSFV. This study represents the most comprehensive analysis to date of CSFV codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.

12-nt insertion in 3' untranslated region leads to attenuation of classic swine fever virus and protects host against lethal challenge.

We report here the discovery of an attenuation mechanism of classic swine fever virus (CSFV) induced by introduction of a continuous 12-nt (CUUUUUUCUUUU) insertion in viral 3' UTR. The 12-nt insertion sequence was first found in one attenuated vaccine strain HCLV (Hog Cholera Lapinized Virus) which did not exist in other CSFV strains. To address the function of the 12-nt insertion in viral replication and attenuation, we constructed and analyzed two chimeras stemmed from a highly virulent strain Shimen either with introduction of the 12-nt insertion in 3' UTR or the replacement of viral 3' UTR by the 3' UTR of HCLV. We found that the two chimeras' maximum titers declined approximately 100-fold than their parental strain Shimen in PK15 cells. An animal experiment showed that the two chimeras were both dramatically attenuated in pigs. All the chimera-infected pigs survived infection and remained clinically normal with the exception of a transient fever while the 100% mortality was observed for the Shimen-infected pigs. In addition, the two chimeras can induce neutralization antibody to completely protect the pigs against lethal challenge with highly virulent CSFV, which was similar to the vaccine strain HCLV. These data demonstrate that the 12-nt insertion in 3' UTR is sufficient for the attenuation of CSFV. Taken together, a novel attenuation mechanism of CSFV is found and may pave a way to further research for new attenuated vaccine.

Evidence for positive selection on the E2 gene of bovine viral diarrhoea virus type 1.

Despite the growing interest in the molecular epidemiology of pestivirus, there have been few attempts to determine which regions of the pestivirus genome are subject to positive selection, although this may be a key indicator of the nature of the interaction between host and virus. By using likelihood-based methods for phylogenetic inference, the positive selection pressure of BVDV-1 E2 gene were assessed and a site-by-site analysis of the dN/dS ratio was performed, to identify specific codons undergoing diversifying positive selection. The overall omega was 0.20, indicating that most sites were subject to strong purifying selection and five positively selected sites (886, 888, 905, 944, and 946) were identified. It is surprising to find that all the potential positively selected sites fall within the C-terminal of E2, and out of the N-terminal of E2 which is thought to be surface-exposed and therefore prime targets for host antibody response. In conclusion, these results suggest that selection favoring avoidance of antibody recognition has not been a major factor in the history of BVDV-1. Further analysis is necessary to see if amino acid substitutions in the BVDV-1 positively selected sites can lead to change of host tropism or\and escape from epitope-specific CD8 T-cell response.