Dynamic Regulation of ME1 Phosphorylation and Acetylation Affects Lipid Metabolism and Colorectal Tumorigenesis.

PGAM5 is a mitochondrial serine/threonine phosphatase that regulates multiple metabolic pathways and contributes to tumorigenesis in a poorly understood manner. We show here that PGAM5 inhibition attenuates lipid metabolism and colorectal tumorigenesis in mice. PGAM5-mediated dephosphorylation of malic enzyme 1 (ME1) at S336 allows increased ACAT1-mediated K337 acetylation, leading to ME1 dimerization and activation, both of which are reversed by NEK1 kinase-mediated S336 phosphorylation. SIRT6 deacetylase antagonizes ACAT1 function in a manner that involves mutually exclusive ME1 S336 phosphorylation and K337 acetylation. ME1 also promotes nicotinamide adenine dinucleotide phosphate (NADPH) production, lipogenesis, and colorectal cancers in which ME1 transcripts are upregulated and ME1 protein is hypophosphorylated at S336 and hyperacetylated at K337. PGAM5 and ME1 upregulation occur via direct transcriptional activation mediated by ß-catenin/TCF1. Thus, the balance between PGAM5-mediated dephosphorylation of ME1 S336 and ACAT1-mediated acetylation of K337 strongly influences NADPH generation, lipid metabolism, and the susceptibility to colorectal tumorigenesis.

An extended conformation of SARS-CoV-2 main protease reveals allosteric targets.

SignificanceThe coronavirus main protease (Mpro) is required for viral replication. Here, we obtained the extended conformation of the native monomer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro by trapping it with nanobodies and found that the catalytic domain and the helix domain dissociate, revealing allosteric targets. Another monomeric state is termed compact conformation and is similar to one protomer of the dimeric form. We designed a Nanoluc Binary Techonology (NanoBiT)-based high-throughput allosteric inhibitor assay based on structural conformational change. Our results provide insight into the maturation, dimerization, and catalysis of the coronavirus Mpro and pave a way to develop an anticoronaviral drug through targeting the maturation process to inhibit the autocleavage of Mpro.

Deacetylation of Notch1 by SIRT1 contributes to HBsAg- and HBeAg-mediated M2 macrophage polarization.

Hepatic macrophages are involved in both pathogen clearance and immunopathogenesis. Emerging evidence demonstrates that macrophage polarization plays a critical role in hepatitis B virus (HBV)-induced immune impairment and liver pathology. However, it remains largely unknown as to how HBV infection facilitates M2 macrophage polarization. Here, a mouse HBV infection model was established by hydrodynamic injection with a vector containing 1.3-fold overlength HBV genome via the tail vein. Coculture experiments with HBV-producing HepG2.2.15 cells and macrophages were established in vitro. We found that HBV-inhibited M1 while enhancing M2 markers, which was accompanied by decreased proinflammatory tumor necrosis factor-α (TNF-α) and augmented anti-inflammatory IL-10 expression. Furthermore, both hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secretion contributed to HBV-triggered macrophage polarization from M1 toward M2 phenotype. Mechanistically, HBsAg and HBeAg could upregulate the sirtuins 1 (SIRT1) deacetylase expression, which in turn promote deacetylation of the Notch1 intracellular domain (NICD), leading to increased Akt phosphorylation and decreased NF-κB nuclear translocation in macrophages. Our findings suggest that NICD deacetylation by SIRT1 contributes to HBsAg- and HBeAg-mediated M2 macrophage polarization, raising the possibility of targeting SIRT1/Notch1 pathway in macrophages to treat HBV immune evasion and chronic HBV infection.NEW & NOTEWORTHY This study identified a previously unrecognized molecular mechanism of HBV-mediated suppression of innate immune responses. We demonstrate that deacetylation of NICD by SIRT1 contributes to HBsAg- and HBeAg-mediated M2 macrophage polarization, which may aid in the development of new macrophage-based immunotherapy for chronic HBV infection and related diseases.

Repurposing old drugs as novel inhibitors of human MIF from structural and functional analysis.

Human macrophage migration inhibitory factor (MIF) is an important pro-inflammatory cytokine that plays multiple pleiotropic functions. It is considered as a promising therapeutic target for the infectious, autoimmune, and cardiovascular diseases and cancers. The development of MIF inhibitors has not been translated into clinical success despite decades of research. Given the time and cost of developing new drugs, existing drugs with clarified safety and pharmacokinetics are explored for their potential as novel MIF inhibitors. This study identified five known drugs that could inhibit MIF's tautomerase activity and MIF-mediated cell chemotaxis in RAW264.7 cells. It was found that compounds D2 (histamine), D5 (metaraminol), and D8 (nebivolol) exhibited micromolar-range inhibition potency close to the positive control ISO-1. Kinetics and the mechanism for inhibition were subsequently determined. Moreover, the detailed inhibitor-binding patterns were investigated by X-ray crystallography, computational molecular docking, and structure-based analysis. Therefore, this study elucidates the molecular mechanism of repurposed drugs acting on MIF and provides a structural foundation for lead optimization to promote the clinical development of MIF-targeted drugs.

The bacterial deubiquitinase Ceg23 regulates the association of Lys-63-linked polyubiquitin molecules on the Legionella phagosome.

Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

Hepatitis B e Antigen Inhibits NF-κB Activity by Interrupting K63-Linked Ubiquitination of NEMO.

Viruses have adopted diverse strategies to suppress antiviral responses. Hepatitis B virus (HBV), a virus that is prevalent worldwide, manipulates the host's innate immune system to evade scavenging. It is reported that the hepatitis B e antigen (HBeAg) can interfere with NF-κB activity, which then leads to high viral loads, while HBV with the G1896A mutation remains infectious without the production of HBeAg but can induce more severe proinflammatory response and liver damage. The aim of current work was to study the molecular mechanism by which HBeAg suppresses interleukin-1ß (IL-1ß)-stimulated NF-κB activity, which leads to the suppression of the innate immune responses to HBV infection. Our study revealed that HBeAg could interact with NEMO, a regulatory subunit associated with IκB kinase, which regulates the activation of NF-κB. HBeAg suppressed the IL-1ß-induced tumor necrosis factor (TNF)-associated factor 6 (TRAF6)-dependent K63-linked ubiquitination of NEMO, thereby downregulating NF-κB activity and promoting virus replication. We further demonstrated the inhibitory effect of HBeAg on the NF-κB signaling pathway using primary human hepatocytes, HBV-infected HepG2-NTCP cells, and clinical liver samples. Our study reveals a molecular mechanism whereby HBeAg suppresses IL-1ß-induced NF-κB activation by decreasing the TRAF6-dependent K63-linked ubiquitination of NEMO, which may thereby enhance HBV replication and promote a persistent infection.IMPORTANCE The role of HBeAg in inflammatory responses during the infection of hepatitis B virus (HBV) is not fully understood, and several previous reports with regard to the NF-κB pathway are controversial. In this study, we showed that HBeAg could suppress both Toll-like receptor 2 (TLR2)- and IL-1ß-induced activation of NF-κB in cells and clinical samples, and we further revealed novel molecular mechanisms. We found that HBeAg can associate with NEMO, the regulatory subunit for IκB kinase (IKK) that controls the NF-κB signaling pathway, and thereby inhibits TRAF6-mediated K63-linked ubiquitination of NEMO, resulting in downregulation of NF-κB activity and promotion of virus replication. In contrast, the HBeAg-negative HBV mutant can induce higher levels of NF-κB activity. These results are important for understanding the HBV-induced pathogenesis of chronic hepatitis and indicate that different clinical measures should be considered to treat HBeAg-positive and HBeAg-negative infections. Our findings represent a conceptual advance in HBV-related suppression of NF-κB signaling.

Female-specific activation of pregnane X receptor mediates sex difference in fetal hepatotoxicity by prenatal monocrotaline exposure.

Pyrrolizidine alkaloids (PAs) are a group of hepatic toxicant widely present in plants. Cytochrome P450 (CYP) 3A plays a key role in metabolic activation of PAs to generate electrophilic metabolites, which is the main cause of hepatotoxicity. We have previously demonstrated the sex difference in developmental toxicity and hepatotoxicity in fetal rats exposed to monocrotaline (MCT), a representative toxic PA. The aim of this study was to explore the underlying mechanism. 20 mg·kg-1·d-1 MCT was intragastrically given to pregnant Wistar rats from gestation day 9 to 20. CYP3As expression and pregnane X receptor (PXR) activation were specifically enhanced in female fetal liver. After MCT treatment, we also observed a significant increase of CYP3As expression in LO2 cells (high PXR level) or hPXR-transfected HepG2 cells (low PXR level). Employing hPXR and CYP3A4 dual-luciferase reporter gene assay, we confirmed the agonism effect of MCT on PXR-dependent transcriptional activity of CYP3A4. Agonism and antagonism of the androgen receptor (AR) either induced or blocked MCT-induced PXR activation, respectively. This study was the first report identifying that MCT served as PXR agonist to induce CYP3A expression. CYP3A induction may increase self-metabolic activation of MCT and subsequently lead to more severe hepatotoxicity in female fetus. While in male, during the intrauterine period, activated AR by testosterone secretion from developing testes represses MCT-induced PXR activation and CYP3A induction, which may partially protect male fetus from MCT-induced hepatotoxicity.

Identification and Characterization of a Ribose 2'-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales.

UNLABELLED: The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2'-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2'-O-MTase activity, although we could not detect 2'-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2'-O-MTase, the roniviral 2'-O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2'-O-MTases and can target only the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with the coronavirus protein, roniviral 2'-O-MTase does not require a protein cofactor for stimulation of its activity and differs in its preference for several biochemical parameters, such as reaction temperature and pH. Furthermore, the ronivirus 2'-O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family. IMPORTANCE: Methylation of the 5'-cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as a 2'-O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales In this study, we identified a 2'-O-methyltransferase encoded by ronivirus that shows common and unique features in comparison with that of coronaviruses. Ronivirus 2'-O-MTase does not need a protein cofactor for MTase activity, whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2'-O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional links between roniviruses and coronaviruses.

Hepatitis B e antigen and its precursors promote the progress of hepatocellular carcinoma by interacting with NUMB and decreasing p53 activity.

UNLABELLED: Hepatitis B viral infection is one of the leading causes of hepatocellular carcinoma (HCC) worldwide. Although several viral factors have been identified that may increase the risk for HCC development, the molecular mechanisms leading to the transformation of normal hepatocytes into cancer cells remain elusive. In this study, we demonstrated that the intracellular hepatitis B e antigen (HBeAg) and its precore precursors, but not their homologous core protein, could associate with NUMB and thereby impair the stability and transcriptional activity of tumor suppressor p53. HBeAg and its precursors could disrupt p53-NUMB and HDM2-NUMB interactions and tricomplex p53-HDM2-NUMB formation, inhibit the acetylation and translocation of p53 from cytosol to the nucleus, promote HDM2-mediated ubiquitination and degradation of p53, and suppress p53-dependent apoptosis. A xenograft tumorigenicity assay showed that expression of HBeAg and its precursors promoted carcinogenesis in a mouse model. Immunohistochemical analysis of the bioptic liver samples of HCC patients revealed that HBeAg positivity was associated with reduced transcriptional activity of p53. Taken together, the results suggest a role of intracellular HBeAg and its precursors in HCC development. CONCLUSION: HBeAg and its precursors promote HDM2-mediated degradation and impair transcriptional activity of p53 by interacting with NUMB, consequently contributing to HCC development. (Hepatology 2016;64:390-404).

MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic concentrations.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. In addition to its known receptor-mediated biological activities, MIF possesses a catalytic site of unknown function between subunits of a homotrimer. Each subunit contributes three ß-strands to adjacent subunits to form a core seven-stranded ß-sheet for each monomer. MIF monomers, dimers, or trimers have been reported, but the active form that binds and activates the MIF receptor (CD74) is still a matter of debate. A cysteine mutant (N110C) that covalently locks MIF into a trimer by forming a disulfide with Cys-80 of an adjacent subunit is used to study this issue. Partial catalytic activity and receptor binding to CD74 are retained by N110C (locked trimer), but there is no cellular signaling. Wild-type MIF-induced cellular signaling, in vivo lung neutrophil accumulation, and alveolar permeability are inhibited with a fivefold excess of N110C. NMR and size-exclusion chromatography with light scattering reveal that N110C can form a higher-order oligomer in equilibrium with a single locked trimer. The X-ray structure confirms a local conformational change that disrupts the subunit interface and results in global changes responsible for the oligomeric form. The structure also confirms these changes are consistent for the partial catalytic and receptor binding activities. The absence of any potential monomer and the retention of partial catalytic and receptor binding activities despite changes in conformation (and dynamics) in the mutant support an endogenous MIF trimer that binds and activates CD74 at nanomolar concentrations. This conclusion has implications for therapeutic development.

Structural insight into the evolution of a new chemokine family from zebrafish.

The mammalian chemokine family is segregated into four families - CC, CXC, CX3C, and XC-based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N-terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur-SAD phasing to determine the three-dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin-binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family.

A novel finding for enterovirus virulence from the capsid protein VP1 of EV71 circulating in mainland China.

Enterovirus 71 (EV71) is a neurotropic virus that causes various clinical manifestations in young children, ranging from asymptomatic to fatal. Different pathotypes of EV71 notably differ in virulence. Several virulence determinants of EV71 have been predicted. However, these reported virulence determinants could not be used to identify the EV71 strains of subgenotype C4, which mainly circulate in China. In this study, VP1 sequences of 37 EV71 strains from severe cases (SC-EV71) and 192 EV71 strains from mild cases (MC-EV71) in mainland China were analyzed to determine the potential virulence determinants in the capsid protein VP1 of EV71. Although most SC-EV71 strains belonged to subgenotype C4a, no specific genetic lineages in C4a were correlated with EV71 virulence. Interestingly, amino acid substitutions at nine positions (H22Q, P27S, N31S/D, E98K, E145G/Q, D164E, T240A/S, V249I, and A289T) were detected by aligning the VP1 sequences of the SC-EV71 and MC-EV71 strains. Moreover, both the constituent ratios of the conservative or mutated residues in the MC-EV71 and SC-EV71 strains and the changes in the VP1 3D structure resulting from these mutations confirmed that the conservative residues (22H, 249V, and 289A) and the mutated residues (27S, 31S/D, 98K, 145G/Q, 164E, and 240A/S) might be potential virulence determinants in VP1 of EV71. Furthermore, these results led to the hypothesis that VP1 acts as a sandwich switch for viral particle stabilization and cellular receptors attachment, and specific mutations in this protein can convert mild cases into severe cases. These findings highlight new opportunities for diagnostic and therapeutic interventions.

The effects of weather factors on road traffic casualties: Analysis on provincial panel data of China from 2006 to 2021.

Introduction: Road traffic injuries stands as a major concern globally, as they result in significant loss of life, economic impact, and erode trust in government and societal safety. The influence of weather on road traffic safety is undeniable, impacting road conditions, individuals, and vehicles. However, the specific influence of weather on road traffic casualties has seldom been explored. Method: This study assesses the effect of weather factors on road traffic casualties in China from 2006 to 2021. Vector error correction models (VECM) were utilized to determine the Granger causality between weather factors and covariates. Furthermore, panel autoregressive distribution lag models (ARDL) were applied to quantify the association between weather factors and road traffic casualties. Results: The findings indicate that rainfall and temperature exert a short-term negative impact on casualty risk, which intriguingly becomes positive in the long term. A standout discovery is the significant role of health investments, which are shown to reduce casualty numbers in both the short and long-terms. In the long run, the gross domestic product significantly enhances casualties, while expressway mileage notably decreases them. Conclusions: These results demonstrate the significant influence of weather on road traffic casualties and highlight the critical roles played by factors such as gross domestic product, health investment, and expressway mileage. The evidence presented in the study underscores the urgent need for more effective strategies to mitigate road traffic casualties. Thus, some effective measures are proposed to reduce road traffic casualties. This study is conducive to the improvement of traffic in severe weather in China and provides guidance for traffic management departments.

Natural evidence of coronaviral 2'-O-methyltransferase activity affecting viral pathogenesis via improved substrate RNA binding.

Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune evasion. However, the K-D-K-E catalytic motif of 2'-O-MTase is highly conserved across numerous viruses, including flaviviruses, vaccinia viruses, coronaviruses, and extends even to mammals. Here, we observed a stronger 2'-O-MTase activity in SARS-CoV-2 compared to SARS-CoV, despite the presence of a consistently active catalytic center. We further identified critical residues (Leu-36, Asn-138 and Ile-153) which served as determinants of discrepancy in 2'-O-MTase activity between SARS-CoV-2 and SARS-CoV. These residues significantly enhanced the RNA binding affinity of 2'-O-MTase and boosted its versatility toward RNA substrates. Of interest, a triple substitution (Leu36 → Ile36, Asn138 → His138, Ile153 → Leu153, from SARS-CoV-2 to SARS-CoV) within nsp16 resulted in a proportional reduction in viral 2'-O-methylation and impaired viral replication. Furthermore, it led to a significant upregulation of type I interferon (IFN-I) and proinflammatory cytokines both in vitro and vivo, relying on the cooperative sensing of melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). In conclusion, our findings demonstrated that alterations in residues other than K-D-K-E of 2'-O-MTase may affect viral replication and subsequently influence pathogenesis. Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.

Inhibition of paclitaxel-induced decreases in calcium signaling.

Peripheral neuropathy is one of the most severe and irreversible side effects caused by treatment from several chemotherapeutic drugs, including paclitaxel (Taxol®) and vincristine. Strategies are needed that inhibit this unwanted side effect without altering the chemotherapeutic action of these drugs. We previously identified two proteins in the cellular pathway that lead to Taxol-induced peripheral neuropathy, neuronal calcium sensor-1 (NCS-1) and calpain. Prolonged treatment with Taxol induces activation of calpain, degradation of NCS-1, and loss of intracellular calcium signaling. This paper has focused on understanding the molecular basis for prevention of peripheral neuropathy by testing the effects of addition of two candidate compounds to the existing chemotherapeutic drug regime: lithium and ibudilast. We found that the co-administration of either lithium or ibudilast to neuroblastoma cells that were treated with Taxol or vincristine inhibited activation of calpain and the reductions in NCS-1 levels and calcium signaling associated with these chemotherapeutic drugs. The ability of Taxol to alter microtubule formation was unchanged by the addition of either candidate compound. These results allow us to suggest that it is possible to prevent the unnecessary and irreversible damage caused by chemotherapeutic drugs while still maintaining therapeutic efficacy. Specifically, the addition of either lithium or ibudilast to existing chemotherapy treatment protocols has the potential to prevent chemotherapy-induced peripheral neuropathy.

Discovery and Development of 4-Hydroxyphenylpyruvate Dioxygenase as a Novel Crop Fungicide Target.

Plant pathogenic fungi pose a significant threat to crop yields and quality, and the emergence of fungicide resistance has further exacerbated the problem in agriculture. Therefore, there is an urgent need for efficient and environmentally friendly fungicides. In this study, we investigated the antifungal activity of (+)-Usnic acid and its inhibitory effect on crop pathogenic fungal 4-hydroxyphenylpyruvate dioxygenases (HPPDs) and determined the structure of Zymoseptoria tritici HPPD (ZtHPPD)-(+)-Usnic acid complex. Thus, the antifungal target of (+)-Usnic acid and its inhibitory basis toward HPPD were uncovered. Additionally, we discovered a potential lead fungicide possessing a novel scaffold that displayed remarkable antifungal activities. Furthermore, our molecular docking analysis revealed the unique binding mode of this compound with ZtHPPD, explaining its high inhibitory effect. We concluded that HPPD represents a promising target for the control of phytopathogenic fungi, and the new compound serves as a novel starting point for the development of fungicides and dual-purpose pesticides.

Structural interactions dictate the kinetics of macrophage migration inhibitory factor inhibition by different cancer-preventive isothiocyanates.

Regulation of cellular processes by dietary nutrients is known to affect the likelihood of cancer development. One class of cancer-preventive nutrients, isothiocyanates (ITCs), derived from the consumption of cruciferous vegetables, is known to have various effects on cellular biochemistry. One target of ITCs is macrophage migration inhibitory factor (MIF), a widely expressed protein with known inflammatory, pro-tumorigenic, pro-angiogenic, and anti-apoptotic properties. MIF is covalently inhibited by a variety of ITCs, which in part may explain how they exert their cancer-preventive effects. We report the crystallographic structures of human MIF bound to phenethylisothiocyanate and to l-sulforaphane (dietary isothiocyanates derived from watercress and broccoli, respectively) and correlate structural features of these two isothiocyanates with their second-order rate constants for MIF inactivation. We also characterize changes in the MIF structure using nuclear magnetic resonance heteronuclear single-quantum coherence spectra of these complexes and observe many changes at the subunit interface. While a number of chemical shifts do not change, many of those that change do not have features similar in magnitude or direction for the two isothiocyanates. The difference in the binding modes of these two ITCs provides a means of using structure-activity relationships to reveal insights into MIF biological interactions. The results of this study provide a framework for the development of therapeutics that target MIF.

Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition.

c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH--the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control.

Crystal Structure of an Intramolecular Mesaconyl-Coenzyme A Transferase From the 3-Hydroxypropionic Acid Cycle of Roseiflexus castenholzii.

Coenzyme A (CoA) transferases catalyze reversible transfer of CoA groups from CoA-thioesters to free acids, playing important roles in the metabolism of carboxylic acids in all organisms. An intramolecular CoA transferase, Mesaconyl-CoA C1-C4 CoA transferase (MCT) was identified in the autotrophic CO2 fixation pathway, 3-hydroxypropionic acid cycle of filamentous anoxygenic phototrophs (FAPs). Different from the well-known CoA transferases that catalyze CoA transfer between two distinct substrates, MCT specifically catalyzes the reversible transformation of mesaconyl-C1-CoA to mesaconyl-C4-CoA, a key reaction intermediate for carbon fixation. However, the molecular mechanism of MCT in employing one substrate is enigmatic. Here we determined the crystal structure of MCT from a chlorosome-less FAP Roseiflexus castenholzii at 2.5 Å resolution, and characterized the catalytic mechanisms through structural analyses and molecular dynamic simulations. The structure of R. castenholzii MCT consists of a Rossmann fold larger domain and a small domain that are connected by two linkers. Two MCT subunits are cross interlocked at the linker regions to form a functional dimer in solution, in which the substrate binding pockets are located at the interface of the Rossmann fold larger domain from one subunit and the small domain from the other subunit. In the simulated binding structures, both the substrate mesaconyl-C1-CoA and product mesaconyl-C4-CoA form extensive electrostatic and hydrogen bonding interactions with MCT. But some differences exist in the binding mode of these two CoA analogs, Arg314' from the second subunit of the dimer presenting dramatic conformational changes in binding with mesaconyl-C4-CoA. Together with Arg47 and one water molecule, a strictly conserved residue Asp165 are essential for catalyzing the reversible intramolecular CoA transfer reaction, through the electrostatic and hydrogen bonding interactions with the mesaconic tail of both the substrate and product. This study revealed a previously unrecognized mechanism for the uncommon intramolecular CoA transfer reaction, which will not only broaden the knowledge on the catalytic mechanisms of CoA transferases, but also contribute to enzyme engineering or biosynthetic applications of the 3-HP cycle for synthesis of fine chemicals and important metabolites.