Capsid virus-like particle display improves recombinant influenza neuraminidase antigen stability and immunogenicity in mice.

Supplementing influenza vaccines with additional protective antigens such as neuraminidase (NA) is a promising strategy for increasing the breadth of the immune response. Here, we improved the immunogenicity and stability of secreted recombinant NA (rNA) tetramers by covalently conjugating them onto the surface of AP205 capsid virus-like particles (cVLPs) using a Tag/Catcher ligation system. cVLP display increased the induction of IgG2a subclass anti-NA antibodies, which exhibited cross-reactivity with an antigenically distant homologous NA. It also reduced the single dose rNA amounts needed for protection against viral challenge in mice, demonstrating a dose-sparing effect. Moreover, effective cVLP-display was achieved across different NA subtypes, even when the conjugation was performed shortly before administration. Notably, the rNA-cVLP immunogenicity was retained upon mixing or co-administering with commercial vaccines. These results highlight the potential of this approach for bolstering the protective immune responses elicited by influenza vaccines.

Synthetic Sialosides Terminated with 8-N-Substituted Sialic Acid as Selective Substrates for Sialidases from Bacteria and Influenza Viruses.

Sialosides containing C8-modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid-interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para-nitrophenol-tagged α2-3- and α2-6-linked sialyl galactosides containing C8-acetamido, C8-azido, or C8-amino derivatized N-acetylneuraminic acid (Neu5Ac). High-throughput substrate specificity studies showed that the C8-modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8-azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8-acetamido-modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8-amino-modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8-amino or C8-acetamido-modified sialic acid can be used with other sialosides for diagnostic profiling of disease-causing sialidase-producing pathogens.

Isolation by multistep chromatography improves the consistency of secreted recombinant influenza neuraminidase antigens.

Recombinant protein-based approaches are ideally suited for producing vaccine antigens that are not overly abundant in viruses, such as influenza neuraminidase (NA). However, obtaining sufficient quantities of recombinant viral surface antigens remains challenging, often resulting in the use of chimeric proteins with affinity tags that can invariably impact the antigen's properties. Here, we developed multistep chromatography approaches for purifying secreted recombinant NA (rNA) antigens that are derived from recent H1N1 and H3N2 viruses and produced using insect cells. Analytical analyses showed that these isolation procedures yielded homogenous tetrameric rNA preparations with consistent specific activities that were not possible from a common immobilized metal affinity chromatography purification procedure. The use of classical chromatography improved the rNA tetramer homogeneity by removing the requirement of the N-terminal poly-histidine affinity tag that was shown to promote higher order rNA oligomer formation. In addition, these procedures reduced the specific activity variation by eliminating the exposure to Ni2+ ions and imidazole, with the latter showing pH and NA subtype dependent effects. Together, these results demonstrate that purification by multistep chromatography improves the homogeneity of secreted rNAs and eliminates the need for affinity tag-based approaches that can potentially alter the properties of these recombinant antigens.

Inactivated influenza virions are a flexible vaccine platform for eliciting protective antibody responses against neuraminidase.

Most seasonal influenza vaccines are produced using hemagglutinin (HA) surface antigens from inactivated virions. However, virions are thought to be a suboptimal source for the less abundant neuraminidase (NA) surface antigen, which is also protective against severe disease. Here, we demonstrate that inactivated influenza virions are compatible with two modern approaches for improving protective antibody responses against NA. Using a DBA/2J mouse model, we show that the strong infection-induced NA inhibitory (NAI) antibody responses are only achieved by high dose immunizations of inactivated virions, likely due to the low viral NA content. Based on this observation, we first produced virions with higher NA content by using reverse genetics to exchange the viral internal gene segments. Single immunizations with these inactivated virions showed enhanced NAI antibody responses and improved NA-based protection from a lethal viral challenge while also allowing for the development of natural immunity to the heterotypic challenge virus HA. Second, we combined inactivated virions with recombinant NA protein antigens. These combination vaccines increased NA-based protection following viral challenge and elicited stronger antibody responses against NA than either component alone, especially when the NAs possessed similar antigenicity. Together, these results indicate that inactivated virions are a flexible platform that can be easily combined with protein-based vaccines to improve protective antibody responses against influenza antigens.

Sialosides Containing 7-N-Acetyl Sialic Acid Are Selective Substrates for Neuraminidases from Influenza A Viruses.

Sialidases or neuraminidases are sialic-acid-cleaving enzymes that are expressed by a broad spectrum of organisms, including pathogens. In nature, sialic acids are monosaccharides with diverse structural variations, but the lack of novel probes has made it difficult to determine how sialic acid modifications impact the recognition by sialidases. Here, we used a chemoenzymatic synthon strategy to generate a set of α2-3- and α2-6-linked sialoside probes that contain 7-N-acetyl or 7,9-di-N-acetyl sialic acid as structure mimics for those containing the less stable naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl modifications. These probes were used to compare the substrate specificity of several sialidases from different origins. Our results show that 7-N-acetyl sialic acid was readily cleaved by neuraminidases from H1N1 and H3N2 influenza A viruses, but not by sialidases of human or bacterial origin, thereby indicating that the influenza enzymes possess a distinctive and more promiscuous substrate binding pocket.

VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease.

Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, thereby triggering the type Ι interferon (IFN) response. Mitochondrial outer membrane permeabilization, which is required for mtDNA release, has been extensively studied in apoptotic cells, but little is known about its role in live cells. We found that oxidatively stressed mitochondria release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane. Furthermore, the positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, inhibiting VDAC oligomerization is a potential therapeutic approach for diseases associated with mtDNA release.

Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins.

Sirt1 is an NAD+-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1. ATP suppresses Sirt1 activity by impairing the CTD's ability to bind to the deacetylase domain as well as its ability to function as the substrate recruitment site. ATP, but not NAD+, causes a conformational shift to a less compact structure. Mutations that prevent ATP binding increase Sirt1's ability to promote stress resistance and inhibit adipogenesis under high-ATP conditions. Interestingly, the CTD can be attached to other proteins, thereby converting them into energy-regulated proteins. These discoveries provide insight into how extreme energy deprivation can impact Sirt1 activity and underscore the complex nature of Sirt1 structure and regulation.

Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK.

The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK), an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE) in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

Inactivation of EWS reduces PGC-1α protein stability and mitochondrial homeostasis.

EWS (Ewing sarcoma) encodes an RNA/ssDNA binding protein that is frequently rearranged in a number of different cancers by chromosomal translocations. Physiologically, EWS has diverse and essential roles in various organ development and cellular processes. In this study, we uncovered a new role of EWS in mitochondrial homeostasis and energy metabolism. Loss of EWS leads to a significant decrease in mitochondria abundance and activity, which is caused by a rapid degradation of Peroxisome proliferator-activated receptor γ Coactivator (PGC-1α), a central regulator of mitochondria biogenesis, function, and cellular energy metabolism. EWS inactivation leads to increased ubiquitination and proteolysis of PGC-1α via proteasome pathway. Complementation of EWS in Ews-deficient cells restores PGC-1α and mitochondrial abundance. We found that expression of E3 ubiquitin ligase, FBXW7 (F-box/WD40 domain protein 7), is increased in the absence of Ews and depletion of Fbxw7 in Ews-null cells restores PGC-1α expression and mitochondrial density. Consistent with these findings, mitochondrial abundance and activity are significantly reduced in brown fat and skeletal muscles of Ews-deficient mice. Furthermore, expression of mitochondrial biogenesis, respiration and fatty acid ß-oxidation genes is significantly reduced in the liver of Ews-null mice. These results demonstrate a novel role of EWS in mitochondrial and cellular energy homeostasis by controlling PGC-1α protein stability, and further implicate altered mitochondrial and energy metabolism in cancers harboring the EWS translocation.

The multifunctional sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate p21-dependent cell-cycle arrest.

SIRT1 regulates the DNA damage response by deacetylating p53, thereby repressing p53 transcriptional output. Here, we demonstrate that the sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate the DNA damage response. PACS-2 knockdown cells failed to efficiently undergo p53-induced cell-cycle arrest in response to DNA damage. Accordingly, p53 acetylation was reduced both in PACS-2 knockdown cells and thymocytes from Pacs-2(-/-) mice, thereby blunting induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A). The SIRT1 inhibitor EX-527 or SIRT1 knockdown restored p53 acetylation and p21 induction as well as p21-dependent cell-cycle arrest in PACS-2 knockdown cells. Trafficking studies revealed that cytoplasmic PACS-2 shuttled to the nucleus, where it interacted with SIRT1 and repressed SIRT1-mediated p53 deacetylation. Correspondingly, in vitro assays demonstrated that PACS-2 directly inhibited SIRT1-catalyzed p53 deacetylation. Together, these findings identify PACS-2 as an in vivo mediator of the SIRT1-p53-p21 axis that modulates the DNA damage response.

Peptide switch is essential for Sirt1 deacetylase activity.

In mammals, the Sirtuins are composed of seven Sir2 orthologs (Sirt1-7) with a conserved deacetylase core that utilizes NAD(+) as a cofactor. Interestingly, the deacetylase core of Sirt1 by itself has no catalytic activity. We found within the C-terminal domain a 25 aa sequence that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an "on switch" for the deacetylase core. The endogenous Sirt1 inhibitor DBC1, which also binds to the deacetylase core, competes with and inhibits the ESA region from interacting with the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Therefore, the ESA region is a potential target for development of therapies to regulate Sirt1.

Promyelocytic leukemia inhibits adipogenesis, and loss of promyelocytic leukemia results in fat accumulation in mice.

The function of the tumor suppressor promyelocytic leukemia (PML) protein is disrupted in promyelocytic leukemia. PML has been reported to function as a negative regulator of mTOR (mammalian target of rapamycin) and nuclear Akt under some conditions. mTOR and Akt pathways regulate a diverse array of pathways, including those that control insulin signaling, energy metabolism, growth, cellular survival, and lifespan. Although the PML-mTOR/Akt link suggests that PML may have metabolic functions in the whole organism, very little is known about the metabolic functions of PML. Here we report that PML(-/-) mice did not show any significant metabolic defects. There was no impairment in the mTOR/Akt or AMPK signaling in white adipose tissue, liver, or muscle. However, despite having normal food intake and activity levels, PML(-/-) mice gained body weight faster and had more fat mass, particularly subcutaneous fat mass, in the diet-induced obesity model. Using in vitro adipogenesis models, we discovered that PML is a suppressor of adipogenesis. PML expression decreased during adipogenesis and was undetectable in fully differentiated adipocytes. Loss of PML increased expression of the adipogenic transcription factors CCAAT/enhancer binding protein-α and peroxisome proliferator-activated receptor-γ. We found that the Sirt1-NCor-SMRT corepressor complex, which represses pparg transcription, does not bind to the pparg promoter efficiently upon PML depletion. On the basis of these findings, we propose that PML is a negative regulator of the adipogenic transcription factors and that, in times of energy excess, PML may limit fat accumulation by suppressing the differentiation of preadipocytes into adipocytes.

AMP-activated protein kinase-deficient mice are resistant to the metabolic effects of resveratrol.

OBJECTIVE: Resveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis, and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5' AMP-activated protein kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPKalpha1 or -alpha2 to determine whether the metabolic effects of resveratrol are mediated by AMPK. RESEARCH DESIGN AND METHODS: Mice deficient in the catalytic subunit of AMPK (alpha1 or alpha2) and wild-type mice were fed a high-fat diet or high-fat diet supplemented with resveratrol for 13 weeks. Body weight was recorded biweekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro. RESULTS: Resveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPKalpha1(-/-) mice. In the absence of either AMPKalpha1 or -alpha2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis, and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD-to-NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly. CONCLUSIONS: We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.

CK2 is the regulator of SIRT1 substrate-binding affinity, deacetylase activity and cellular response to DNA-damage.

SIRT1, an NAD(+) (nicotinamide adenine dinucleotide)-dependent deacetylase, protects cells from stress-induced apoptosis, and its orthologues delay aging in lower eukaryotes. SIRT1 increases survival in response to stress such as DNA damage by deacetylating a number of substrates including pro-apoptotic protein p53. The molecular mechanism by which DNA-damage activates SIRT1 is not known. By screening a kinase inhibitor library, we identified CK2 as a SIRT1 kinase. CK2 is a pleiotropic kinase with more than 300 substrates and well-known anti-apoptotic and pro-growth activities. We find that CK2 is recruited to SIRT1 after ionizing radiation (IR) and phosphorylates conserved residues Ser 154, 649, 651 and 683 in the N- and C-terminal domains of mouse SIRT1. Phosphorylation of SIRT1 increases its deacetylation rate but not if the four Ser residues are mutated. In addition, phosphorylation of SIRT1 increases its substrate-binding affinity. CK2-mediated phosphorylation increases the ability of SIRT1 to deacetylate p53 and protect cells from apoptosis after DNA damage. Based on these findings, we propose that CK2 protects against IR-induced apoptosis partly by phosphorylating and activating SIRT1. Thus, this work suggests that SIRT1 is a component of the expansive anti-apoptotic network controlled by CK2. Since expression of both CK2 and SIRT1 is upregulated with tumorigenesis and downregulated with senescence, the CK2-SIRT1 link sheds new light on how CK2 may regulate cancer development and aging.

Activation of 5'-AMP-activated kinase with diabetes drug metformin induces casein kinase Iepsilon (CKIepsilon)-dependent degradation of clock protein mPer2.

Metformin is one of the most commonly used first line drugs for type II diabetes. Metformin lowers serum glucose levels by activating 5'-AMP-activated kinase (AMPK), which maintains energy homeostasis by directly sensing the AMP/ATP ratio. AMPK plays a central role in food intake and energy metabolism through its activities in central nervous system and peripheral tissues. Since food intake and energy metabolism is synchronized to the light-dark (LD) cycle of the environment, we investigated the possibility that AMPK may affect circadian rhythm. We discovered that the circadian period of Rat-1 fibroblasts treated with metformin was shortened by 1 h. One of the regulators of the period length is casein kinase Iepsilon (CKIepsilon), which by phosphorylating and inducing the degradation of the circadian clock component, mPer2, shortens the period length. AMPK phosphorylates Ser-389 of CKIepsilon, resulting in increased CKIepsilon activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK alpha2 knock-out mice. We conclude that metformin and AMPK have a previously unrecognized role in regulating the circadian rhythm.

ATR, PML, and CHK2 play a role in arsenic trioxide-induced apoptosis.

Arsenic trioxide (ATO) is a potent anti-leukemic chemotherapeutic agent for acute promyelocytic leukemia (APL) that results from a t (15, 17) chromosomal translocation that produces PML-RARalpha, a fusion protein between a tumor suppressor PML and the retinoic acid receptor RARalpha. APL patients are initially treated with retinoic acid, but most develop resistance and relapse. In contrast, ATO induces prolonged remissions even in the relapsed cases. However, the molecular mechanisms by which ATO kills the leukemic cells are not fully understood. We find that ATO induces apoptosis, at least in part, by activating proapoptotic kinase Chk2. ATO does this by stimulating ATR (ataxia telangiectasia mutated and Rad3-related) kinase, a Chk2-activating kinase. In conjunction, ATO degrades PML-RARalpha, resulting in the restoration of PML, which is required for autophosphorylation and full activation of Chk2. As a result, the p53-dependent apoptosis pathway is activated. Based on this, we propose that a pathway composed of ATR, PML, Chk2, and p53 plays a role in ATO-mediated apoptosis, a notion that is consistent with the observation that Chk2 is genetically intact and mutations in the p53 gene are extremely rare in APL.

BRG1 controls the activity of the retinoblastoma protein via regulation of p21CIP1/WAF1/SDI.

The ubiquitous mammalian chromatin-remodeling SWI/SNF-like BAF complexes play critical roles in tumorigenesis. It was suggested that the direct interaction of BRG1 with the retinoblastoma protein pRB is required for regulation of cell cycle progression by pRB. We present evidence that the BRG1-containing complexes regulate the expression of the cdk inhibitor p21(CIP1/WAF1/SDI). Furthermore, we show that the physical interaction between BRG1 and pRB is not required for induction of cell growth arrest and transcriptional repression of E2F target genes by pRB. Instead, BRG1 activates pRB by inducing its hypophosphorylation through up-regulation of the cdk inhibitor p21. The hypophosphorylation of pRB is reinforced by down-regulation of critical components, including cdk2, cyclin E, and cyclin D, in the pRB regulatory network. We demonstrate that up-regulation of p21 by BRG1 is necessary to induce formation of flat cells, growth arrest, and finally, cell senescence. Our results suggest that the BRG1-containing complexes control cellular proliferation and senescence by modulating the pRB pathway via multiple mechanisms.

Hydrogen peroxide mediates arsenite activation of p70(s6k) and extracellular signal-regulated kinase.

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70(s6k) and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H(2)O(2), because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H(2)O(2) by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70(s6k) and ERK1/2, indicating the possible role of H(2)O(2) in the arsenite activation of the p70(s6k) and the ERK1/2 signaling pathways. A specific inhibitor of p70(s6k), rapamycin, and calcium chelators significantly blocked the activation of p70(s6k) induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70(s6k), ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H(2)O(2) might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H(2)O(2) production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H(2)O(2) plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70(s6k) signaling pathways.

Maximal induction of a subset of interferon target genes requires the chromatin-remodeling activity of the BAF complex.

The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-alpha). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-alpha induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.