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  • Long-term bio-power of ceramic Microbial Fuel Cells in individual and stacked configurations
    Bioelectrochemistry (IF 4.474) Pub Date : 2020-01-17
    Iwona Gajda; Oluwatosin Obata; Maria Jose Salar-Garcia; John Greenman; Ioannis A. Ieropoulos
    更新日期:2020-01-17
  • Zinc oxide-gold nanocomposite as a proper platform for label-free DNA biosensor
    Bioelectrochemistry (IF 4.474) Pub Date : 2020-01-16
    Zahra Hatami; Elham Ragheb; Fahimeh Jalali; Mahmoud Amouzadeh Tabrizi; Mojtaba Shamsipur

    In this study, a simple and cost-effective electrochemical DNA biosensor was developed for sensitive detection of mycobacterium tuberculosis (TB). Nanocomposite of zinc oxide (ZnO) and gold nanoparticles (AuNPs) was used as a platform for immobilizing thiolated TB DNA (probe DNA). ZnO was electrodeposited on a glassy carbon electrode by potentiostat electrolysis of Zn (NO3)2 solution at -1.0 V (vs. Ag/AgCl), then AuNPs were loaded as the second layer at -0.4 V from HAuCl4 solution. Thiolated probe DNA was then covalently attached to AuNPs. Anodic peak current of Fe (CN)6 3-/4- was followed in hybridization experiments and a linear calibration curve was obtained in concentration range of 2.5–250 pM and limit of detection (LOD) of 1.8 pM for target DNA. The label-free TB biosensor exhibited high selectivity, suitable stability, and reproducibility.

    更新日期:2020-01-17
  • Stable Isotopes for Tracing Mammalian-Cell Metabolism In Vivo
    Trends Biochem. Sci. (IF 16.889) Pub Date : 2020-01-17
    Juan Fernández-García; Patricia Altea-Manzano; Erica Pranzini; Sarah-Maria Fendt

    Metabolism is at the cornerstone of all cellular functions and mounting evidence of its deregulation in different diseases emphasizes the importance of a comprehensive understanding of metabolic regulation at the whole-organism level. Stable-isotope measurements are a powerful tool for probing cellular metabolism and, as a result, are increasingly used to study metabolism in in vivo settings. The additional complexity of in vivo metabolic measurements requires paying special attention to experimental design and data interpretation. Here, we review recent work where in vivo stable-isotope measurements have been used to address relevant biological questions within an in vivo context, summarize different experimental and data interpretation approaches and their limitations, and discuss future opportunities in the field.

    更新日期:2020-01-17
  • Response of Isovalerate-Degrading Methanogenic Microbial Community to Inhibitors
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-17
    Jie Li, Hui-Zhong Wang, Yue Yi, Min Gou, Masaru Konishi Nobu, Ya-Ting Chen, Yue-Qin Tang

    Isovalerate is one of the key intermediates during anaerobic digestion treating protein-containing waste/wastewater. Investigating the effect of different kinds of inhibitors on isovalerate-degrading microbial community is necessary to develop measures for improving the effectiveness of the treatment plants. In the present study, dynamic changes in the isovalerate-degrading microbial community in presence of inhibitors (ammonium, sulfide, mixed ammonium and sulfide, and chlortetracycline (CTC)) were investigated using high-throughput sequencing of 16S rRNA gene. Our observations showed that the isovalerate-degrading microbial community responded differently to different inhibitors and that the isovalerate degradation and gas production were strongly repressed by each inhibitor. We found that sulfide inhibited both isovalerate oxidation followed by methanogenesis, while ammonium, mixed ammonium and sulfide, and CTC mainly inhibited isovalerate oxidation. Genera classified into Proteobacteria and Chloroflexi were less sensitive to inhibitors. The two dominant genera, which are potential syntrophic isovalerate oxidizers, exhibited different responses to inhibitors that the unclassified_Peptococcaceae_3 was more sensitive to inhibitors than the unclassified_Syntrophaceae. Upon comparison to acetoclastic methanogen Methanosaeta, hydrogenotrophic methanogens Methanoculleus and Methanobacterium were less sensitive to inhibitors.

    更新日期:2020-01-17
  • Kinetics Analysis of the Inhibitory Effects of Alpha-Glucosidase and Identification of Compounds from Ganoderma lipsiense Mycelium
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-17
    Tania Maria Costa, Diego Alex Mayer, Diogo Alexandre Siebert, Gustavo Amadeu Micke, Michele Debiasi Alberton, Lorena Benathar Ballod Tavares, Débora de Oliveira

    Abstract The studies on natural compounds to diabetes mellitus treatment have been increasing in recent years. Research suggests that natural components can inhibit alpha-glucosidase activities, an important strategy in the management of blood glucose levels. In this work, for the first time in the literature, the compounds produced by Ganoderma lipsiense extracts were identified and evaluated on the inhibitory effect of these on alpha-glucosidase activity. Four phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to crude extract from G. lipsiense grown in red rice medium (RCE) and synthetic medium (SCE), being syringic acid identified in both extracts. Gas chromatography-mass spectrometry (GC-MS) analysis showed fatty acids and their derivatives, terpene, steroid, niacin, and nitrogen compounds to SCE, while RCE was rich in fatty acids and their derivatives. Both extracts demonstrated alpha-glucosidase inhibition (RCE IC50 = 0.269 ± 8.25 mg mL−1; SCE IC50 = 0.218 ± 9.67 mg mL−1), and the purified hexane fraction of RCE (RHEX) demonstrated the highest inhibition of enzyme (81.1%). Studies on kinetic inhibition showed competitive inhibition mode to RCE, while SCE showed uncompetitive inhibition mode. Although the inhibitory effects of RCE and SCE were satisfactory, the present findings identified some unpublished compounds to G. lipsiense in the literature with important therapeutic properties.

    更新日期:2020-01-17
  • Adipose Tissue–Derived Mesenchymal Stem Cells Protect Against Amiodarone-Induced Lung Injury in Rats
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-17
    Sara M. Radwan, Dalia Ghoneim, Manar Salem, Menna Saeed, Yara Saleh, Mohanad Elhamy, Kholoud Wael, Omnia Shokair, Sara A. Wahdan

    Pulmonary fibrosis (PF) is a progressive and irreversible lung disease, characterized by poor prognosis with limited treatment options. Mesenchymal stem cells (MSCs) are multi-potent cells having the ability to self-renew and differentiate into multiple tissues, thus considered a novel treatment option. The present study aimed to investigate the possible antifibrotic effect of undifferentiated adipose tissue–derived mesenchymal stem cell (AD-MSC) therapy on PF experimentally induced in rats using amiodarone (AMD). AMD (30 mg/kg) was given orally, once daily for 12 consecutive weeks to induce lung fibrosis. Following the confirmation of lung damage with histopathological examination, AD-MSCs (2 × 106 and 4 × 106 undifferentiated MSCs) were injected once intravenously, followed by 2 months for treatment. AMD induced focal fibroblastic cells proliferation in the peribronchiolar tissue, as well as in between the collapsed emphysematous alveoli. Also, AMD significantly increased serum and lung homogenate fibroblast growth factor-7 (FGF7), Clara cell protein-16 (CC16), and cytokeratin -19 (CK19) levels, as well as the expression of both iNOS and NFкB in the lung alveoli. Moreover, AMD caused excessive collagen deposition and increased alpha smooth muscle actin (α-SMA) expression. All findings significantly regressed on stem cell therapy in both doses, with superior effect of the high dose, providing evidence that adipose tissue–derived MSCs could be a promising approach for the treatment of PF.

    更新日期:2020-01-17
  • Extracellular Vesicles Derived from Epidural Fat-Mesenchymal Stem Cells Attenuate NLRP3 Inflammasome Activation and Improve Functional Recovery After Spinal Cord Injury
    Neurochem. Res. (IF 2.782) Pub Date : 2020-01-17
    Jiang-Hu Huang, Chun-Hui Fu, Yang Xu, Xiao-Ming Yin, Yong Cao, Fei-Yue Lin

    Abstract Spinal cord injury (SCI) is a devastating event which caused high mortality and morbidity. Recently, nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome has been showed to act a critical t role in the secondly injury phase of SCI. In current study, we aimed to investigate the effect and underlying molecular mechanisms of extracellular vesicles derived from epidural fat (EF)- mesenchymal stem cells (MSCs) for the treatment of SCI. Ninety-six Sprague–Dawley rats were used for current study and randomly divided into four groups: sham group, SCI group, SCI + Saline group, SCI + Extracellular vesicles group. Basso‐Beattie‐Bresnahan (BBB) scores was applied to evaluate the neurological functional recovery. Cresyl violet–stained was conducted evaluate the protective effect of EF-MSCs-Extracellular vesicles on lesion volume after SCI. ELISA, immunohistochemistry assay, TUNEL assay and western blotting were conducted to investigate the underlying molecular mechanisms. Our results demonstrated that the administration of EF-MSCs-Extracellular vesicles via tail vein injection improved neurological functional recovery and reduced the lesion volume after SCI. And systemic administration of EF-MSCs-Extracellular vesicles significantly inhibited NLRP3 inflammasome activation and reduced the expression of inflammatory cytokines. Additionally, the expression levels of proapoptotic protein Bax was decreased and antiapoptotic Bcl-2 was upregulated with the treatment of EF-MSCs-Extracellular vesicles after SCI. In summary, in current study, we demonstrated for the first time that the EF-MSCs-Extracellular vesicles can improve neurological functional recovery after SCI, and the underlying molecular mechanisms may partly through the inhibition of NLRP3 inflammasome activation.

    更新日期:2020-01-17
  • Daphnetin Ameliorates Experimental Autoimmune Encephalomyelitis Through Regulating Heme Oxygenase-1
    Neurochem. Res. (IF 2.782) Pub Date : 2020-01-16
    Dan Wang, Bo Zhu, Xiaoyi Liu, Qin Han, Weihong Ge, Wenping Zhang, Yin Lu, Qinan Wu, Liyun Shi

    Abstract To assess the potential role of daphnetin, a clinically used anti-inflammatory agent, on the development of the inflammatory and neurodegenerative disease, we investigated its immune regulatory function in a murine model of experimental autoimmune encephalomyelitis (EAE). Significantly, lower levels of pro-inflammatory cytokines including interleukin (IL)-17, interferon-γ, Il6, Il12a, and Il23a were observed in brains of daphnetin-treated EAE mice, compared with those in control littermates. We also confirmed that daphnetin suppressed the production of IL-1β, IL-6, and tumor necrosis factor-α in lipopolysaccharide-stimulated mouse BV2 microglial cells. Mechanistically, heme oxygenase-1 (HO-1), a canonical anti-oxidant and anti-inflammatory factor, was found to be substantially induced by daphnetin treatment in BV2 cells. Also, a significantly higher level of HO-1, accompanied by a decreased level of malondialdehyde, was observed in daphnetin-treated EAE mice. More importantly, the deletion of HO-1 in BV2 microglia largely abrogated daphnetin-mediated inhibition of the inflammatory response. Together, our data demonstrate that daphnetin has an anti-inflammatory and neuroprotective role during the pathogenesis of EAE, which is partially at least, dependent on its regulation of HO-1.

    更新日期:2020-01-17
  • Serotonin 5-HT1A and 5-HT1B receptors co-mediate the RU 24629-induced locomotor activity of male and female preweanling rats
    Pharmacol. Biochem. Behav. (IF 2.773) Pub Date : 2020-01-17
    Sanders A. McDougall; Jasmine A.M. Robinson; Esperanza Larios Ramirez; Henry A. Diaz

    The serotonin (5-HT) 1A/1B agonist RU 24969 robustly increases the locomotor activity of adult male rats and mice; however, studies using selective antagonists alternately report that 5-HT1A, 5-HT1B, or both receptor types mediate RU 24969's locomotor activating effects. The purpose of the present study was to extend these past findings by administering a selective 5-HT1 agonist and/or antagonists to male and female preweanling rats. This age group was tested because younger rats often exhibit psychopharmacological responses that are quantitatively or qualitatively different from adult rats. In a series of experiments, male and female preweanling rats were pretreated with vehicle, the 5-HT1A antagonist WAY 100635 (0.5, 1, 5, or 10 mg/kg), or the 5-HT1B antagonists NAS-181 (5 or 10 mg/kg) or SB 216641 (5 or 10 mg/kg) 30 min before assessment of locomotor activity. Rats were injected with saline or RU 24969 immediately prior to testing. Results showed that RU 24969 (0.625, 1.25, 2.5, or 5 mg/kg) significantly increased the locomotor activity of both male and female preweanling rats (no sex differences were apparent). Antagonism of either the 5-HT1A or the 5-HT1B receptor was sufficient to significantly reduce the locomotor activity of RU 24969-treated preweanling rats. Unexpectedly, NAS-181 did not act as a silent receptor antagonist, as both doses of NAS-181 significantly increased the locomotor activity of saline-treated preweanling rats. In sum, the present results show that both the 5-HT1A and 5-HT1B receptor systems mediate locomotion during the late preweanling period, and this mediation does not vary according to sex.

    更新日期:2020-01-17
  • Electrophysiological biomarkers of antidepressant response to ketamine in treatment-resistant depression: Gamma power and long-term potentiation
    Pharmacol. Biochem. Behav. (IF 2.773) Pub Date : 2020-01-17
    Jessica R. Gilbert; Carlos A. Zarate

    Over the last two decades, the discovery of ketamine's antidepressant properties has galvanized research into the neurobiology of treatment-resistant depression. Nevertheless, the mechanism of action underlying antidepressant response to ketamine remains unclear. This study reviews electrophysiological studies of ketamine's effects in individuals with depression as well as healthy controls, with a focus on two putative markers of synaptic potentiation: gamma oscillations and long-term potentiation. The review focuses on: 1) measures of gamma oscillations and power and their relationship to both acute, psychotomimetic drug effects as well as delayed antidepressant response in mood disorders; 2) changes in long-term potentiation as a promising measure of synaptic potentiation following ketamine administration; and 3) recent efforts to model antidepressant response to ketamine using novel computational modeling techniques, in particular the application of dynamic causal modeling to electrophysiological data. The latter promises to better characterize the mechanisms underlying ketamine's antidepressant effects.

    更新日期:2020-01-17
  • mGlu2/3 receptor antagonism: A mechanism to induce rapid antidepressant effects without ketamine-associated side-effects
    Pharmacol. Biochem. Behav. (IF 2.773) Pub Date : 2020-01-16
    Jeffrey M. Witkin

    The consensus that ketamine can produce rapid-onset antidepressant effects in patients combined with the recent approval of S(+)-ketamine (Esketamine) as an antidepressant, has fueled the search for other compounds that might recapitulate the remarkable therapeutic benefits of ketamine. At the same time, discovery efforts have been additionally directed toward minimization of the tolerability, side-effect, and safety issues associated with ketamine. The history of thought on the viability of metabotropic 2/3 (mGlu2/3) as a potential mechanism for inducing rapid-acting antidepressant effects is reviewed here. The biological basis for predicting antidepressant efficacy of mGlu2/3 receptor antagonists in depressed patients is also presented. This prediction is based upon convergent biochemical, neurochemical, electrophysiological, and behavioral data that indicate a striking homology in the substrates that underlie the effects of mGlu2/3 receptor antagonists and the known antidepressant ketamine. The data reviewed to date also demonstrate that the preclinical side-effect/tolerability and toxicology profile of mGlu2/3 receptor antagonists are not concerning. Finally, preclinical data on a relatively new mGlu2/3 receptor antagonist, LY3020371, and its orally-bioavailable prodrug, LY3027788, are reviewed. The data on this mechanism provides impressive optimism for successful translation of the mGlu2/3 receptor antagonist hypothesis into therapeutics for those suffering from depression.

    更新日期:2020-01-17
  • Biocontrol of tobacco black shank disease (Phytophthora nicotianae) by Bacillus velezensis Ba168
    Pestic. Biochem. Phys. (IF 2.87) Pub Date : 2020-01-16
    Dongsheng Guo; Chenhong Yuan; Yunyan Luo; YaHan Chen; Meihuan Lu; Guochan Chen; Guangwei Ren; Chuanbin Cui; Jiatao Zhang; Derong An
    更新日期:2020-01-17
  • Fecal metatranscriptomics and glycomics suggests that bovine milk oligosaccharides are fully utilized by healthy adults
    J. Nutr. Biochem. (IF 4.49) Pub Date : 2020-01-17
    Samuel T Westreich; Jaime Salcedo; Blythe Durbin-Johnson; Jennifer T Smilowitz; Ian Korf; David A. Mills; Daniela Barile; Danielle G Lemay

    Human milk oligosaccharides play a vital role in the development of the gut microbiome in the human infant. Although oligosaccharides derived from bovine milk (BMO) differ in content and profile with those derived from human milk (HMO), several oligosaccharide structures are shared between the species. BMO are a commercial alternative to HMO, but their fate in the digestive tract of healthy adult consumers is unknown. Healthy human subjects consumed two BMO doses over 11 day periods each and provided fecal samples. Metatranscriptomics of fecal samples was conducted to determine microbial and host gene expression in response to the supplement. Fecal samples were also analyzed by mass spectrometry to determine levels of undigested BMO. No changes were observed in microbial gene expression across all participants. Repeated sampling enabled subject-specific analyses: four of six participants had minor, yet statistically significant, changes in microbial gene expression. No significant change was observed in the gene expression of host cells exfoliated in stool. Levels of BMO excreted in feces after supplementation were not significantly different from baseline and were not correlated with dosage or expressed microbial enzyme levels. Collectively, these data suggest that BMO is fully fermented in the human gastrointestinal tract upstream of the distal colon. Additionally, the unaltered host transcriptome provides further evidence for the safety of BMO as a dietary supplement or food ingredient. Further research is needed to investigate potential health benefits of this completely fermentable prebiotic that naturally occurs in cow's milk.

    更新日期:2020-01-17
  • Receptor-mediated cell entry of Paramyxoviruses: mechanisms, and consequences for tropism and pathogenesis
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Chanakha K. Navaratnarajah, Alex R Generous, Iris Yousaf, Roberto Cattaneo

    Research in the last decade has uncovered many new paramyxoviruses, airborne agents that cause epidemic diseases in animals including humans. Most paramyxoviruses enter epithelial cells of the airway using sialic acid as receptor, and cause only mild disease. However, others cross the epithelial barrier and cause more severe disease. For some of these viruses, the host receptors have been identified, and the mechanisms of cell entry elucidated. The tetrameric attachment proteins of paramyxoviruses have vastly different binding affinities for their cognate receptors, which they contact through different binding surfaces. Nevertheless, all input signals are converted to the same output: conformational changes that trigger refolding of trimeric fusion proteins, and membrane fusion. Experiments with selectively receptor-blinded viruses inoculated into their natural hosts have provided insights into tropism, identifying the cells and tissues that support growth, and revealing the mechanisms of pathogenesis. These analyses also shed light on diabolically elegant mechanisms used by morbilliviruses, including measles virus, to promote massive amplification within the host, followed by efficient aerosolization and rapid spread through host populations. In another paradigm of receptor-facilitated severe disease, henipaviruses, including Nipah and Hendra viruses, use different members of one protein family to cause zoonoses. Specific properties of different paramyxoviruses, like neurotoxicity and immunosuppression, are now understood in the light of receptor specificity. We propose that research on the specific receptors for several newly identified members of the Paramyxoviridae family that may not bind sialic acid is needed to anticipate their zoonotic potential, and generate effective vaccines and antiviral compounds.

    更新日期:2020-01-17
  • DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Arthur Marivin, Marcin Maziarz, Jingyi Zhao, Vincent DiGiacomo, Isabel Olmos Calvo, Emily A Mann, Jason Ear, Juan B Blanco-Canosa, Elliott M. Ross, Pradipta Ghosh, Mikel Garcia-Marcos

    Besides being regulated by G protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE are the best characterized members of a group of cytoplasmic regulators that contain a Gα-binding-and-activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11 or G12/13), and promoting nucleotide exchange in vitro. However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remains to be investigated. Here, we show that DAPLE’s GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq. Unlike the nucleotide exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq. These findings indicate that GBA motifs have versatility in their G protein-modulating effect, i.e. they can bind to Gα subunits of different classes, and either stimulate or inhibit nucleotide exchange depending on the G protein subtype.

    更新日期:2020-01-17
  • HYBID and hyaluronan synthase coordinately regulate hyaluronan metabolism in histamine-stimulated skin fibroblasts
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Hiroyuki Yoshida, Mika Aoki, Aya Komiya, Yoko Endo, Keigo Kawabata, Tomomi Nakamura, Shingo Sakai, Tetsuya Sayo, Yasunori Okada, Yoshito Takahashi

    The immune-regulatory compound histamine is involved in the metabolism of the essential skin component hyaluronan (HA). We previously reported that histamine up-regulates the expression of hyaluronan-binding protein involved in hyaluronan depolymerization (HYBID, aka CEMIP or KIAA1199), which plays a key role in HA degradation. However, no information is available about histamine’s effects on HA synthase (HAS) expression, the molecular sizes of the HAs produced, and histamine receptors and their signaling pathways in skin fibroblasts. Moreover, histamine’s effects on photoaged skin remain elusive. Here, we show that histamine increases HA degradation by up-regulating HYBID and down-regulating HAS2 in human skin fibroblasts in a dose- and time-dependent manner and thereby decreases the total amounts and sizes of newly produced HA. Histamine H1 blocker abrogated the histamine effects on HYBID up-regulation, HAS2 suppression, and HA degradation. Histamine H1 agonist exhibited effects on HA levels, composition, and breakdown similar to those of histamine. Of note, blockade of protein kinase Cδ (PKCδ) or PI3K–AKT signaling abolished histamine-mediated HYBID stimulation and HAS2 suppression, respectively. Immunohistochemical experiments revealed a significant ~2-fold increase in tryptase-positive mast cells in photoaged skin, where HYBID and HAS2 expression levels were increased and decreased, respectively, compared with photoprotected skin. These results indicate that histamine controls HA metabolism by up-regulating HYBID and down-regulating HAS2 via distinct signaling pathways downstream of histamine receptor H1. They further suggest that histamine may contribute to photoaged skin damage by skewing HA metabolism toward degradation.

    更新日期:2020-01-17
  • Loss of voltage-gated proton channel Hv1 decreases insulin secretion and leads to hyperglycemia and glucose intolerance in mice
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Huimin Pang, Xudong Wang, Shiqun Zhao, Wang Xi, Jili Lv, Jiwei Qin, Qing Zhao, Yongzhe Che, Liangyi Chen, Shu Jie Li

    Insulin secretion by pancreatic islet β-cells is regulated by glucose levels and is accompanied by proton generation. The voltage-gated proton channel Hv1 is present in pancreatic β-cells and extremely selective for protons. However, whether Hv1 is involved in insulin secretion is unclear. Here, we demonstrate that Hv1 promotes insulin secretion of pancreatic β-cells and glucose homeostasis. Hv1-deficient mice displayed hyperglycemia and glucose intolerance due to reduced insulin secretion, but retained normal peripheral insulin sensitivity. Moreover, Hv1 loss contributed much more to severe glucose intolerance as the mice got older. The islets of Hv1-deficient and heterozygous mice were markedly deficient in glucose- and K+-induced insulin secretion. In perifusion assays, Hv1 deletion dramatically reduced both the first and second phase of glucose-stimulated insulin secretion (GSIS). Islet insulin and proinsulin contents were reduced, and histological analysis of pancreas slices revealed an accompanying modest reduction of β-cell mass in the Hv1-knockout mice. EM observations also indicated a reduction in insulin granule size, but not granule number or granule docking, in the Hv1-deficient mice. Mechanistically, Hv1 loss limited the capacity of glucose-induced membrane depolarization, accompanying the reduced ability of glucose to raise Ca2+ levels in islets, evidenced by a decreased duration of individual calcium oscillations. Moreover, Hv1 expression was significantly reduced in pancreatic β-cells from streptozotocin-induced diabetic mice, indicating that Hv1 deficiency is associated with β-cell dysfunction and diabetes. We conclude that Hv1 regulates insulin secretion and glucose homeostasis through a mechanism that depends on intracellular Ca2+ levels and membrane depolarization.

    更新日期:2020-01-17
  • Functional Binding of E-selectin to its Ligands is Enhanced by Structural Features Beyond its Lectin Domain
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Fajr A Aleisa, Kosuke Sakashita, Jae Man Lee, Dina B. AbuSamra, Bader Alwan, Shuho Nozue, Muhammad Tehseen, Samir M Hamdan, Satoshi Habuchi, Takahiro Kusakabe, Jasmeen S Merzaban

    Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- or L- selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

    更新日期:2020-01-17
  • A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Samantha K. Sarkar, Alexander C.Y. Foo, Angela Matyas, Ikhuosho Asikhia, Tanja Kosenko, Natalie K Goto, Ariela Vergara-Jaque, Thomas A. Lagace

    Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30%–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9’s prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N-terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish the PCSK9’s ability to bind LDL reported here support the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

    更新日期:2020-01-17
  • Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Friederike Hans, Hanna Glasebach, Philipp J. Kahle

    Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.

    更新日期:2020-01-17
  • Triple-cell lineage tracing by a dual reporter on a single allele
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Kuo Liu, Muxue Tang, Hengwei Jin, Qiaozhen Liu, Lingjuan He, Huan Zhu, Xiuxiu Liu, Ximeng Han, Yan Li, Libo Zhang, Juan Tang, Wenjuan Pu, Zan Lv, Haixiao Wang, Hongbin Ji, Bin Zhou

    Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter (R26-TLR), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre+Dre−, Cre−Dre+, and Cre+Dre+ cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity.

    更新日期:2020-01-17
  • Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Aarti Krishnan, Joachim Kloehn, Matteo Lunghi, Dominique Soldati-Favre

    The Apicomplexa phylum comprises diverse parasitic organisms that have evolved from a free-living ancestor. These obligate intracellular parasites exhibit versatile metabolic capabilities reflecting their capacity to survive and grow in different hosts and varying niches. Determined by nutrient availability, they either use their biosynthesis machineries or largely depend on their host for metabolite acquisition. Because vitamins cannot be synthesized by the mammalian host, the enzymes required for their synthesis in apicomplexan parasites represent a large repertoire of potential therapeutic targets. Here, we review recent advances in metabolic reconstruction and functional studies coupled to metabolomics that unravel the interplay between biosynthesis and salvage of vitamins and cofactors in apicomplexans. A particular emphasis is placed on Toxoplasma gondii, during both its acute and latent stages of infection.

    更新日期:2020-01-17
  • How to catch a HIF—the work of Gregg Semenza's lab on hypoxia-inducible factor 1
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Martin J. Spiering

    Molecular oxygen is essential for the viability and function of every cell of the animal body. Because skin, tissues, and organs impede oxygen diffusion directly from the air, molecular oxygen concentrations inside the body often are less than 5% (1), much lower than the 21% in Earth's atmosphere.As multicellular organisms evolved, this steep oxygen gradient necessitated the development of structures such as the circulatory system and of biochemical mechanisms that monitor and control oxygen levels in the body.A major player in sensing potentially harmful drops in cellular oxygen concentrations (hypoxia) is the transcription factor hypoxia-inducible factor 1 (HIF-1).2 HIF-1, and its close relative HIF-2, regulate many genes, including the erythropoietin (EPO) gene, which encodes a hormone that stimulates production of red blood cells (2, 3).One important milestone in uncovering HIF-1's pivotal role in oxygen sensing was its purification and biochemical characterization in the mid-1990s by the lab of Gregg Semenza (Fig. 1), a geneticist at Johns Hopkins University School of Medicine. This work was reported in two JBC papers recognized as Classics here (4, 5).jbc;295/3/715/F1F1F1Figure 1.Gregg Semenza and colleagues isolated and biochemically characterized the HIF-1 protein. Photo courtesy of Johns Hopkins Medicine.“JBC was the first choice,” says Semenza, referring to the publication of the first Classics article. “To me, that was a classic JBC paper.”A few years before this work, Semenza's team had found that hypoxia induces the binding of a nuclear protein to a 50-nucleotide-long enhancer region located in the 3′-flanking region of the EPO gene (2, 3). Although...

    更新日期:2020-01-17
  • The Arabidopsis AtGCD3 protein is a glucosylceramidase that preferentially hydrolyzes long-acyl-chain glucosylceramides
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Guang-Yi Dai, Jian Yin, Kai-En Li, Ding-Kang Chen, Zhe Liu, Fang-Cheng Bi, Chan Rong, Nan Yao

    Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

    更新日期:2020-01-17
  • Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Kieu T. M. Pham, Ziyin Li

    The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei. Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

    更新日期:2020-01-17
  • The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Christopher J. Hosford, Anthony Q. Bui, Joshua S. Chappie

    McrBC is a two-component, modification-dependent restriction system that cleaves foreign DNA-containing methylated cytosines. Previous crystallographic studies have shown that Escherichia coli McrB uses a base-flipping mechanism to recognize these modified substrates with high affinity. The side chains stabilizing both the flipped base and the distorted duplex are poorly conserved among McrB homologs, suggesting that other mechanisms may exist for binding modified DNA. Here we present the structures of the Thermococcus gammatolerans McrB DNA-binding domain (TgΔ185) both alone and in complex with a methylated DNA substrate at 1.68 and 2.27 Å resolution, respectively. The structures reveal that TgΔ185 consists of a YT521-B homology (YTH) domain, which is commonly found in eukaryotic proteins that bind methylated RNA and is structurally unrelated to the E. coli McrB DNA-binding domain. Structural superposition and co-crystallization further show that TgΔ185 shares a conserved aromatic cage with other YTH domains, which forms the binding pocket for a flipped-out base. Mutational analysis of this aromatic cage supports its role in conferring specificity for the methylated adenines, whereas an extended basic surface present in TgΔ185 facilitates its preferential binding to duplex DNA rather than RNA. Together, these findings establish a new binding mode and specificity among McrB homologs and expand the biological roles of YTH domains.

    更新日期:2020-01-17
  • Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Caroline C. Farrington, Eric Yuan, Sahar Mazhar, Sudeh Izadmehr, Lauren Hurst, Brittany L. Allen-Petersen, Mahnaz Janghorban, Eric Chung, Grace Wolczanski, Matthew Galsky, Rosalie Sears, Jaya Sangodkar, Goutham Narla

    The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix–loop–helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non–small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.

    更新日期:2020-01-17
  • Crystal structure of phytochromobilin synthase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phytochrome
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Masakazu Sugishima, Kei Wada, Keiichi Fukuyama, Ken Yamamoto

    Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.

    更新日期:2020-01-17
  • Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Shraboni Ghosh, Nitin Uttam Kamble, Pooja Verma, Prafull Salvi, Bhanu Prakash Petla, Shweta Roy, Venkateswara Rao, Abhijit Hazra, Vishal Varshney, Harmeet Kaur, Manoj Majee

    Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

    更新日期:2020-01-17
  • The machinery for endocytosis of epidermal growth factor receptor coordinates the transport of incoming hepatitis B virus to the endosomal network
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Masashi Iwamoto, Wakana Saso, Kazane Nishioka, Hirofumi Ohashi, Ryuichi Sugiyama, Akihide Ryo, Mio Ohki, Ji-Hye Yun, Sam-Yong Park, Takayuki Ohshima, Ryosuke Suzuki, Hideki Aizaki, Masamichi Muramatsu, Tetsuro Matano, Shingo Iwami, Camille Sureau, Takaji Wakita, Koichi Watashi

    Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

    更新日期:2020-01-17
  • Structural insights into the mechanism of c-di-GMP–bound YcgR regulating flagellar motility in Escherichia coli
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Yan-Jie Hou, Wen-Si Yang, Yuan Hong, Ying Zhang, Da-Cheng Wang, De-Feng Li

    The motile-sessile transition is critical for bacterial survival and growth. Cyclic-di-GMP (c-di-GMP) plays a central role in controlling this transition and regulating biofilm formation via various effectors. As an effector of c-di-GMP in Escherichia coli and related species, the PilZ domain–containing protein YcgR responds to elevated c-di-GMP concentrations and acts on the flagellar motor to suppress bacterial motility in a brakelike fashion, which promotes bacterial surface attachment. To date, several target proteins within the motor, MotA, FliG, and FliM, along with different regulatory mechanisms have been reported. However, how YcgR acts on these components remains unclear. Here, we report that activated YcgR stably binds to MotA at the MotA-FliG interface and thereby regulates bacterial swimming. Biochemical and structural analyses revealed that c-di-GMP rearranges the PilZ domain configuration, resulting in the formation of a MotA-binding patch consisting of an RXXXR motif and the C-tail helix α3. Moreover, we noted that a conserved region in the YcgR-N domain, which is independent of MotA interaction, is necessary for motility regulation. On the basis of these findings, we infer that the YcgR-N domain is required for activity on other motor proteins. We propose that activated YcgR appends to MotA via its PilZ domain and thereby interrupts the MotA-FliG interaction and simultaneously interacts with other motor proteins via its YcgR-N domain to inhibit flagellar motility. Our findings suggest that the mode of interaction between YcgR and motor proteins may be shared by other PilZ family proteins.

    更新日期:2020-01-17
  • So many roads traveled: A career in science and administration
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    James R. Halpert

    I have traveled many roads during my career. After spending my first 19 years in Los Angeles, I became somewhat of an academic nomad, studying and/or working in six universities in the United States and three in Sweden. In chronological order, I have a B.A. in Scandinavian languages and literature from UCLA, a Ph.D. in biochemistry from Uppsala University, and an M.S. in toxicology from the Karolinska Institute. I have been in schools of natural science, pharmacy, and medicine and have worked in multiple basic science departments and one clinical department. I have served as a research-track and tenured faculty member, department chair, associate dean, and dean. My research has spanned toxinology, biochemistry, toxicology, and pharmacology. Through all the moves, I have gained much and lost some. For the past 40 years, my interest has been cytochrome P450 structure-function and structure-activity relationships. My lab has focused on CYP2B enzymes using X-ray crystallography, site-directed mutagenesis, deuterium-exchange MS, isothermal titration calorimetry, and computational methods in conjunction with a variety of functional assays. This comprehensive approach has enabled detailed understanding of the structural basis of the remarkable substrate promiscuity of CYP2B enzymes. We also have investigated the mechanisms of CYP3A4 allostery using biophysical and advanced spectroscopic techniques, and discovered a pivotal role of P450-P450 interactions and of multiple-ligand binding. A major goal of this article is to provide lessons that may be useful to scientists in the early and middle stages of their careers and those more senior scientists contemplating an administrative move.

    更新日期:2020-01-17
  • Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Zhong Li, Yuanyuan Jiang, F. Peter Guengerich, Li Ma, Shengying Li, Wei Zhang

    Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.

    更新日期:2020-01-17
  • Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Dzmitry Sinitski, Katrin Gruner, Markus Brandhofer, Christos Kontos, Pascal Winkler, Anja Reinstädler, Priscila Bourilhon, Zhangping Xiao, Robbert Cool, Aphrodite Kapurniotu, Frank J. Dekker, Ralph Panstruga, Jürgen Bernhagen

    Human macrophage migration-inhibitory factor (MIF) is an evolutionarily-conserved protein that has both extracellular immune-modulating and intracellular cell-regulatory functions. MIF plays a role in various diseases, including inflammatory diseases, atherosclerosis, autoimmunity, and cancer. It serves as an inflammatory cytokine and chemokine, but also exhibits enzymatic activity. Secreted MIF binds to cell-surface immune receptors such as CD74 and CXCR4. Plants possess MIF orthologs but lack the associated receptors, suggesting functional diversification across kingdoms. Here, we characterized three MIF orthologs (termed MIF/d-dopachrome tautomerase–like proteins or MDLs) of the model plant Arabidopsis thaliana. Recombinant Arabidopsis MDLs (AtMDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using either p-hydroxyphenylpyruvate or dopachrome methyl ester as substrate. Site-specific mutagenesis suggests that this is due to a distinct amino acid difference at the catalytic cavity-defining residue Asn-98. Surprisingly, AtMDLs bind to the human MIF receptors CD74 and CXCR4. Moreover, they activate CXCR4-dependent signaling in a receptor-specific yeast reporter system and in CXCR4-expressing human HEK293 transfectants. Notably, plant MDLs exert dose-dependent chemotactic activity toward human monocytes and T cells. A small molecule MIF inhibitor and an allosteric CXCR4 inhibitor counteract this function, revealing its specificity. Our results indicate cross-kingdom conservation of the receptor signaling and leukocyte recruitment capacities of human MIF by its plant orthologs. This may point toward a previously unrecognized interplay between plant proteins and the human innate immune system.

    更新日期:2020-01-17
  • PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Nicholas E. Powers, Benjamin Swartzwelter, Carlo Marchetti, Dennis M. de Graaf, Alexandra Lerchner, Martin Schlapschy, Rajiv Datar, Uli Binder, Carl K. Edwards, Arne Skerra, Charles A. Dinarello

    Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS–IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS–IL-1Ra molecules, PAS600–IL-1Ra and PAS800–IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600–IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600–IL-1Ra and PAS800–IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800–IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800–IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600–IL-1Ra, and PAS800-IL-Ra reduced IL-1α–induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans–induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS–IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS–IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

    更新日期:2020-01-17
  • In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Yunrong Gao, Dongdong Cao, Hyunjun Max Ahn, Anshuman Swain, Shaylan Hill, Claire Ogilvie, Matthew Kurien, Taha Rahmatullah, Bo Liang

    The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

    更新日期:2020-01-17
  • Correction: Ctp1 protein–DNA filaments promote DNA bridging and DNA double-strand break repair.
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Sara N. Andres, Zimeng M. Li, Dorothy A. Erie, R. Scott Williams

    VOLUME 294 (2019) PAGES 3312–3320Due to publisher error, F. Peter Guengerich is listed as the Editor. The correct Editor for this article is Patrick Sung.

    更新日期:2020-01-17
  • Correction: A nitric oxide synthase–like protein from Synechococcus produces NO/NO3− from l-arginine and NADPH in a tetrahydrobiopterin- and Ca2+-dependent manner.
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Angela L. Picciano, Brian R. Crane

    VOLUME 294 (2019) PAGES 10708–10719There was an error in the title. The abbreviation “NAPDH” should be “NADPH.”

    更新日期:2020-01-17
  • Correction: An alternative retinoic acid-responsive Stra6 promoter regulated in response to retinol deficiency.
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Kristian B. Laursen, Vasundhra Kashyap, Joseph Scandura, Lorraine J. Gudas

    VOLUME 290 (2015) PAGES 4356–4366The wrong images were used for the long form of Stra6 and RARγ in Fig. 4D. These errors have now been corrected and do not affect the results or conclusions of this work.jbc;295/3/898/F4F1F4Figure 4.

    更新日期:2020-01-17
  • Temperature-Induced Formation of Uniform Polymer Nanocubes Directly in Water
    Biomacromolecules (IF 5.667) Pub Date : 2020-01-16
    Carlos Fitzgerald Grandes Reyes; Sung-Po R. Chen; Valentin A. Bobrin; Zhongfan Jia; Michael J. Monteiro
    更新日期:2020-01-17
  • Surface Charge Switchable Polymer/DNA Nanoparticles Responsive to Tumor Extracellular pH for Tumor-Triggered Enhanced Gene Delivery
    Biomacromolecules (IF 5.667) Pub Date : 2020-01-16
    Ying Jie Ooi; Yuting Wen; Jingling Zhu; Xia Song; Jun Li

    A tumor-targeted surface charge switchable polymeric gene delivery system with the function of switching surface charge upon reaching tumor site owing to the tumor extracellular pH (pHe) was developed. The delivery system was fabricated by two steps. First, the positively charged polyplexes nanoparticles were formed between β-cyclodextrin-oligoethylenimine star polymer (CD-OEI) and plasmid DNA (pDNA). Next, the CD-OEI/pDNA polyplex nanoparticles were coated with a pHe-responsive anionic polymer via electrostatic interaction to form ternary complexes. The pHe-responsive anionic polymer was a block copolymers of poly(ethylene glycol) (PEG) and poly(2-aminoethyl methacrylate) (pAEMA) modified with 2,3-dimethylmaleic anhydride (denoted as PPD). The coating polymer was mixed with a small amount of pHe-insensitive PEG-pAEMA modified with succinic anhydride (denoted as PPS), giving a balanced negatively charged and PEG-shielded surface with pHe-responsive property for achieving the expected tumor-triggered enhanced gene delivery. At physiological pH 7.4, owing to the charge shielding of anionic surface coating and the PEGylation, the negatively charged CD-OEI/pDNA/PPD+PPS polyplex complexes could avoid undesirable interaction with serum proteins and non-targeted components. However, the amide bond of PPD was sensitive to pH changes and could be easily hydrolyzed under acidic pHe (< 6.8) to expose the primary amine group due to nucleophile catalysis by the carboxylic acid. The PEG block in the copolymers was used to further enhance the surface shielding effect. Our data showed that excellent particle salt stability and serum tolerance was achieved through the PPD+PPS surface coating. The CD-OEI/pDNA/PPD+PPS complexes achieved lower cellular uptake and transfection efficiency at neutral pH 7.4, while exhibiting comparable cellular uptake and transfection efficiency at acidic pHe 6.5 as compared to the uncoated polyplexes, indicating that the surface charge switching worked well. Considering the potential of the CD-OEI star polymer for further functionalization through supramolecular assembly, the surface charge switchable CD-OEI/pDNA/PPD+PPS nanoparticle system may be promising for tumor-targeted gene therapy applications.

    更新日期:2020-01-17
  • On the Helicity of Peptoid Ions in the Gas Phase
    Biomacromolecules (IF 5.667) Pub Date : 2020-01-16
    Sébastien Hoyas; Emilie Halin; Vincent Lemaur; Julien De Winter; Pascal Gerbaux; Jérôme Cornil

    Peptoids are attractive substitutes for peptides in several research areas, especially when they adopt a helical structure. The chain-size evolution of the secondary structure of the widely studied Nspe peptoids is here analyzed by means of the ion mobility mass spectrometry technique increasingly used as a powerful analytical tool and is further supported by theoretical modelling. We conclude that the helical shape of the peptoids prevailing in solution is lost in gas phase by the need to screen the positive charge borne by the peptoid even though the collisional cross sections are close to the values expected for helical systems. We further illustrate that trend line analyses predicting molecular shapes from fits of the size evolution of cross-sections can be very misleading since they critically depend on the range of polymerization degrees under study.

    更新日期:2020-01-17
  • Antioxidant and UV-blocking leather-inspired nanocellulose-based films with high wet strength
    Biomacromolecules (IF 5.667) Pub Date : 2020-01-16
    Konstantin Kriechbaum; Lennart Bergström

    The mechanical performance in the wet state needs to be significantly improved and the intrinsic functionalities should be fully utilized to promote the replacement of fossil-based plastics with renewable bio-based materials. We demonstrate a leather-inspired approach to produce multifunctional materials with a high wet strength that is based on tannin-induced precipitation of gelatin grafted onto surface-modified cellulose nanofibrils (CNF). The leather-inspired CNF-based films had a wet tensile strength of 33 MPa, a Young’s modulus of 310 MPa, and a strain at failure of 22%, making the wet materials stronger than e.g. dry conventional low-density polyethylene and more ductile than paper-based food packaging materials. The tannin-containing films displayed excellent antioxidant and UV-blocking properties, rapidly scavenging more than 90% of added free radicals and absorbing 100% of light in the UV-B/UV-C range. This work illustrates the prospect of combining renewable materials in a leather-inspired approach to form wet strong and multifunctional films with potential application in food packaging.

    更新日期:2020-01-17
  • Conversion of an injectable MMP-degradable hydrogel into core-crosslinked micelles
    Biomacromolecules (IF 5.667) Pub Date : 2020-01-16
    Marzieh Najafi; Hamed Asadi; Joep van den Dikkenberg; Mies J van Steenbergen; Marcel H.A.M. Fens; Wim E. Hennink; Tina Vermonden

    In this study, a new type of injectable hydrogel called “HyMic” that can convert into core crosslinked (CCL) micelles upon exposure to matrix metalloproteinases (MMP’s), was designed and developed for drug delivery applications. HyMic is composed of CCL micelles connected via an enzyme cleavable linker. To this end, two complementary ABA block copolymers with polyethylene glycol (PEG) as B block were synthesized using atom transfer radical polymerization (ATRP). The A blocks were composed of a random copolymer of N-isopropylacrylamide (NIPAM) and either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys) or N-(2-hydroxypropyl) methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA). Mixing the aqueous solutions of the obtained polymers and rising the temperature above the cloud point of the PNIPAM block resulted in the self-assembly of these polymers into flower-like micelles composed of a hydrophilic PEG shell and hydrophobic core. The micellar core was crosslinked by native chemical ligation between the cysteine (in HPMA-Cys) and thioester (in HPMA-ETSA) functionalities. A slight excess of thioester to cysteine groups (molar ratio 3:2) was used to allow further chemical reactions exploiting the unreacted thioester groups. The obtained micelles displayed a Z-average diameter of 80±1 nm (PDI 0.1), and ζ-potential of -4.2±0.4 mV and were linked using two types of pentablock copolymers of P(NIPAM-co-HPMA-Cys)-PEG-Peptide-PEG-P(NIPAM-co-HPMA-Cys) (Pep-NC) to yield hydrogels. The pentablock copolymers were synthesized using a PEG-peptide-PEG ATRP macroinitiator and the peptide midblock (lysine-glycine-proline-glutamine-isoleucine-phenylalanine-glycine-glutamine-lysine (Lys-Gly-Pro-Gln-Gly-Ile-Phe-Gly-Gln-Lys)) consisted of either L or D amino acids (L-Pep-NC or D-Pep-NC), of which the L-amino acid sequence is a substrate for matrix metalloproteases 2 and 9 (MMPs 2 and 9). Upon mixing of the CCL micelles and the linker (L/D-Pep-NC), the cysteine functionalities of the L/D-Pep-NC reacted with remaining thioester moieties in the micellar core via native chemical ligation yielding a hydrogel within 160 minutes as demonstrated by rheological measurements. As anticipated, the gel crosslinked with L-Pep-NC was degraded in 7-45 days upon exposure to metalloproteases in a concentration dependent manner, while the gel crosslinked with the D-Pep-NC remained intact even after 2 months. Dynamic light scattering analysis of the release medium revealed the presence of nanoparticles with a Z-average diameter of ~120 nm (PDI <0.3) and ζ-potential of ~-3 mV indicating release of core crosslinked micelles upon HyMic exposure to metalloproteases. An in-vitro study demonstrated that the released CCL micelles were taken up by HeLa cells. Therefore, HyMic as an injectable and enzyme degradable hydrogel displaying controlled and on-demand release of CCL micelles has potential for intracellular drug delivery in tissues with upregulation of MMPs.

    更新日期:2020-01-17
  • Zwitterionic polypeptides: Chemoenzymatic synthesis and loosening function for cellulose crystals
    Biomacromolecules (IF 5.667) Pub Date : 2020-01-16
    Kousuke Tsuchiya; Neval Yilmaz; Takaaki Miyamoto; Hiroyasu Masunaga; Keiji Numata

    A polypeptide with a GlyHisGly repeating sequence containing zwitterionic structures that effectively interact with cellulose was synthesized by chemoenzymatic polymerization followed by postfunctionalization of the side chains of the His residues to afford imidazolium butyrate. The resulting zwitterionic polypeptide effectively dissociated bundles of tunicate cellulose microcrystals, even when the conditions were mild and the concentration of the polypeptide was low. Polypeptide treatment also affected the morphology of the cell walls in cultured plant cells, and the cellulose microfibril networks and amorphous polysaccharide layer were dissociated according to atomic force microscopy (AFM). The zwitterionic polypeptide treatment did not change the crystal structure of the cellulose microcrystals. Analysis of the mechanical properties of the cellulose microcrystals by force curve measurements using AFM revealed that the elastic modulus of the cellulose microcrystals increased after treatment with the zwitterionic polypeptide, indicating that the amorphous part of the cellulose microcrystals was removed by interactions with the polypeptide. At a concentration of the polypeptide that enabled the dissociation of the cellulose network, the zwitterionic polypeptide showed negligible cytotoxicity to the plant cells. The mild and noncytotoxic technique for loosening cellulose microfibrils/microcrystals that was developed in this study has tremendous significance for the modification of cellulose in terms of polymer chemistry, material science and plant biotechnology.

    更新日期:2020-01-17
  • Designed Alteration of Binding Affinity in Structure-Switching Aptamers through the Use of Dangling Nucleotides
    Biochemistry (IF 2.952) Pub Date : 2020-01-16
    Sladjana Slavkovic; Sophie R. Eisen; Philip E. Johnson
    更新日期:2020-01-17
  • Structural Basis of the Substrate Selectivity of Viperin
    Biochemistry (IF 2.952) Pub Date : 2020-01-16
    Michael K. Fenwick; Dan Su; Min Dong; Hening Lin; Steven E. Ealick
    更新日期:2020-01-17
  • Cholesterol Modulates the Formation of the Aβ Ion Channel in Lipid Bilayers
    Biochemistry (IF 2.952) Pub Date : 2020-01-16
    Qi Gao; Guangfu Wu; King Wai Chiu Lai
    更新日期:2020-01-17
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  • Perfluorinated Probes for Noncovalent Protein Recognition and Isolation
    Bioconjugate Chem. (IF 4.349) Pub Date : 2020-01-16
    Ivan Bassanini; Corinna Galli; Erica E. Ferrandi; Fabiana Vallone; Annapaola Andolfo; Sergio Romeo
    更新日期:2020-01-17
  • Triazolecarbaldehyde Reagents for One‐Step N‐Terminal Protein Modification
    ChemBioChem (IF 2.593) Pub Date : 2020-01-16
    Akira Onoda; Nozomu Inoue; Eigo Sumiyoshi; Takashi Hayashi
    更新日期:2020-01-17
  • Site Directed Disulfide PEGylation of Interferon-beta-1b With Fork Peptide Linker
    Bioconjugate Chem. (IF 4.349) Pub Date : 2020-01-17
    Shayan Abbasi; Homa Farahani; Hossein Lanjanian; Mohammad Taheri; Loghman Firoozpour; Jamshid Davoodi; Sama Pirkalkhoran; Gholamhossein Riazi; Shahriar Pooyan

    The attachment of PEG to biopharmaceuticals has been applied for enhancement of bioavailability and improved stability. The PEG polymer is highly hydrated; thus effective attachment to inaccessible sites could be hindered. We have devised a scheme to address this issue by introducing a considerable distance between PEG and protein by addition of a linear peptide, appended to long chained reactive linkers. Secondly, the position of PEG conjugation directly affects biological activity. Accordingly, disulfide bond could be considered as an ideal choice for site directed PEGylation; but reactivity of both thiol moieties to bridging reagent is critical for maintenance of protein structure. In our design, a fork structure with two arms provides essential flexibility to account for dissociation of reduced cysteines. An efficient yield for disulfide PEGylation of IFN-β1b was attained and specificity, biophysical characterization, biological activity and pharmacokinetics were surveyed.

    更新日期:2020-01-17
  • Mechanism of diol dehydration by a promiscuous radical‐SAM enzyme homologue of the antiviral enzyme viperin (RSAD2)
    ChemBioChem (IF 2.593) Pub Date : 2020-01-17
    Kourosh Honarmand Ebrahimi; Jack Rowbotham; James McCullagh; William S James

    3´‐deoxy nucleotides are an important class of drugs because they interfere with metabolism of nucleotides and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds have largely been produced via multistep chemical synthesis and an enzyme with the ability to catalyse removal of 3´‐deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, high‐resolution mass spectrometry, and NMR spectroscopy we demonstrate that a thermostable fungal radical S‐adenosylmethionine (SAM) enzyme with similarity to the vertebrate antiviral enzyme viperin (RSAD2) can catalyze transformation of CTP, UTP, and 5‐bromo‐UTP to their 3ʹ‐deoxy‐3′,4ʹ‐didehydro analogues. We show that unlike the fungal enzyme human viperin can only catalyse transformation of CTP. Using electron paramagnetic resonance (EPR) spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical‐SAM enzymes. We predict that our work will help in utilizing a new class of radical‐SAM enzymes for biocatalytic synthesis of 3ʹ‐deoxy nucleotide/nucleoside analogues.

    更新日期:2020-01-17
  • Molecular imaging for cancer immunotherapy: Seeing is believing
    Bioconjugate Chem. (IF 4.349) Pub Date : 2020-01-17
    MADIHA SAEED; Zhiai Xu; Bruno G. De Geest; Huixiong Xu; Haijun Yu

    The importance of the immune system in cancer therapy has been reaffirmed by the success of immune checkpoint blockade. The complex tumor microenvironment and its interaction with the immune system, however, remain having mysteries. Molecular imaging may shed light on fundamental aspects of the immune response to elucidate the mechanism of cancer immunotherapy. In this review, we discuss various imaging approaches that offer in-depth insight into the tumor microenvironment, checkpoint blockade therapy, and T cell-mediated antitumor immune responses. Recent advances in the molecular imaging modalities, including magnetic resonance imaging (MRI), positron electron tomography (PET) and optical imaging (e.g., fluorescence and intravital imaging) for in situ tracking of the immune response are discussed. It is envisaged that the integration of imaging with immunotherapy may broaden our understanding to predict a particular antitumor immune response.

    更新日期:2020-01-17
  • Comparison of the biocatalytic activity of some halotolerant yeasts in freshwater and seawater
    ChemBioChem (IF 2.593) Pub Date : 2020-01-17
    Cecilia Andreu; Marcel·lí del Olmo

    The application of Green Chemistry concepts in catalysis has considerably increased in recent years, and the interest in using sustainable solvents in the chemical industry is growing. One of the recent proposals to fall in line with this is to employ seawater as a solvent in biocatalytic processes. This involves selecting halotolerant strains capable of carrying out chemical conversions in the presence of the salt concentrations found in this solution. Recent studies by our group have revealed the interest in using strains belonging to Debaryomyces and Schwannyomyces for catalytic processes run in this medium. In this work we select several strains based on their halotolerance to widen the scope of this strategy. We consider them for the monoreduction of 1‐phenylpropane‐1,2‐dione, a well‐characterized reaction that produces intermediates of pharmaceutical interest. The results obtained herein indicate that using seawater as a solvent for this reaction is possible, and it even offers advantages for stereoselectivity and biocatalyst reuse with some strains. The data found for S. cerevisiae FY86 and K. marxianus were particularly interesting.

    更新日期:2020-01-17
  • Highly Sensitive Genosensing Coupling Rolling Circle Amplification with Multiple DNAzyme Cores for DNA Walking
    Bioconjugate Chem. (IF 4.349) Pub Date : 2020-01-17
    Hua Chai; Mingyuan Wang; Chongyu Zhang; Yuguo Tang; Peng Miao

    Herein, a highly sensitive electrochemical genosensor is proposed by the construction of novel DNA walking machine. Generally, a number of tetrahedral DNA (TDNA) supported tracks and walkers are co-modified on the electrode surface. DNA walking is inhibited in the absence of target DNA. After the interaction between DNA walker strand and target DNA, a single-stranded primer sequence could be released, which initiates subsequent rolling circle amplification (RCA). The generated long single-stranded product contains multiple DNAzyme cores, which facilitate highly efficient cleavage of track strands and subsequent DNA walking. The electrode then loses the ability to localize silver nanoparticles (AgNPs) as the electrochemical species. Thus, by recording the reduced silver stripping current, a highly sensitive method for the detection of DNA is fabricated. Under optimal conditions, it achieves an admirable sensitiv-ity with the limit of detection as low as 0.1 fM. Satisfactory specificity is also guaranteed. In addition, the practicality is further con-firmed by applying human serum samples, which show great potential utility for clinical diagnosis.

    更新日期:2020-01-17
  • Biosynthesis and Structure Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin
    ChemBioChem (IF 2.593) Pub Date : 2020-01-16
    Simon Sieber; Christophe Daeppen; Christian Jenul; Vidya Mannancherril; Leo Eberl; Karl Gademann

    Only a few natural products possessing a diazeniumdiolate have been isolated and usually these compounds display a broad range of biological activities. Only recently the first diazeniumdiolate natural product biosynthetic gene cluster was identified in Burkholderia cenocepacia H111, which produces the fungicide (—)‐fragin and the signal molecule (rac)‐valdiazen. In this study, L‐valine was identified as the initial substrate of (—)‐fragin biosynthesis by feeding experiments using isotopically labeled amino acids. The formation of the diazeniumdiolate was chemically studied by several proposed intermediates. Our results indicate that the functional group is formed during an early stage of the biosynthesis. Furthermore, an oxime compound was identified as a degradation product of (—)‐fragin and was also observed in the crude extract of the wild type strain. Moreover, structure‐activity relationship analysis revealed that each moiety of (—)‐fragin is essential for its biological activity.

    更新日期:2020-01-17
  • Activated plasmonic nanoaggregates for dark-field situ imaging for HER2 protein imaging on cells surface
    Bioconjugate Chem. (IF 4.349) Pub Date : 2020-01-16
    Yingshu Guo; Fei Liu; Yinhua Hu; Xiaofei Zheng; Xiuping Cao; Yanxi Zhu; Xiaoru Zhang; Dongjiao Li; Zhenhua Zhang; Si-kai Chen

    Dark-field microscopy (DFM) based on localized surface plasmon resonance (LSPR) was used for observation of experimental phenomena, which is a hopeful non-damaged and non-photo bleaching biological imaging technique. In this strategy, plasma nanoaggregates with stronger scattering efficiency were formed in the presence of the target, causing a “turn-on” phenomenon, when asymmetry modified AuNPs were introduced as probes with zero LSPR background. First, Au1-N3 probe and Au2-C≡C probe was designed for the cycloaddition between azide- and alkyne- to form AuNP dimers under the catalytic action by Cu+ which obtained from the reduction of Cu2+ by sodium ascorbate. The two kinds of probes were used for the detection of Cu2+ in rat serum successfully. Then, for applying this concept to protein on cells, DNA and antibody were modified on the probes. DNA1/Au1-N3 probe and anti-HER2/Au2-C≡C probe were proposed for HER2 protein DFM on cells. By designing aptamer sequence in primer, the rolling circle amplification (RCA) was introduced in HER2 DFM on cell, and the image signal was much brighter than no-RCA. The unique design made it was more easily to discriminate the target signal from background noise in cell DFM. This method might be used in the fields of molecular diagnostics and cell imaging.

    更新日期:2020-01-17
  • Designed Protein Cages as Scaffolds for Building Multienzyme Materials
    ACS Synth. Biol. (IF 5.571) Pub Date : 2020-01-17
    Scott A. McConnell; Kevin A. Cannon; Christian Morgan; Rachel McAllister; Brendan R. Amer; Robert T. Clubb; Todd O. Yeates
    更新日期:2020-01-17
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