Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.     

High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

Insulin-producing cells from human adipose tissue-derived mesenchymal stem cells detected by atomic force microscope

We successfully differentiated human adipose tissue-derived mesenchymal stem cells (haMSCs) into insulin-producing cells (IPCs) in vitro and did not use any insulin which might be absorbed by cells during in vitro culture. Expression of insulin gene was massively increased by 28,000-fold at day 12 compared with haMSCs (P < 0.05). IPCs could secrete insulin after glucose was stimulated. The higher the concentration of glucose, the more production of insulin was noted. We reported AFM images of IPCs for the first time. AFM images showed that the sizes of cells were similar to each other, and all IPC surface had a porous structure in the cytoplasm area. In sugar-free group, the size of holes was similar (diameter, 1,086.98 ± 156.70 nm; depth, 185.22 ± 52.14 nm). In higher sugar-stimulated group, there were more holes with bigger diameter and smaller depth. (diameter, 3,183.65 ± 2,229.18 nm; depth 109.42 ± 56.26 nm, P < 0.05). We found that the hole diameter and depth could change with the concentration of glucose in media. Concurrently, laser scanning confocal microscopy images indicated that cortical actin network beneath plasma membrane in IPCs was dense and continuous. After glucose stimulation, we found the actin web depolymerized and became discontinuous in IPCs. We speculated that diameter augmentation of holes located in the cytoplasm area in IPCs was one manifestation of excytosis increase.     

Metformin promotes survivin degradation through AMPK/PKA/GSK-3β-axis in non–small cell lung cancer

Metformin, a first-line antidiabetic drug, has been reported with anticancer activities in many types of cancer. However, its molecular mechanisms remain largely unknown. As a member of inhibitor of apoptosis proteins, survivin plays an important role in the regulation of cell death. In the present study, we investigated the role of survivin in metformin-induced anticancer activity in non–small cell lung cancer in vitro. Metformin mainly induced apoptotic cell death in A549 and H460 cell lines. It remarkably suppressed the expression of survivin, decreased the stability of this protein, then promoted its proteasomal degradation. Moreover, metformin greatly suppressed protein kinase A (PKA) activity and induced its downstream glycogen synthase kinase 3β (GSK-3β) activation. PKA activators, both 8-Br-cAMP and forskolin, significantly increased the expression of survivin. Consistently both GSK-3β inhibitor LiCl and siRNA restored the expression of survivin in lung cancer cells. Furthermore, metformin induced adenosine 5′-monophosphate-activated protein kinase (AMPK) activation. Suppression of the activity of AMPK with Compound C reversed the degradation of survivin induced by metformin, and meanwhile, restored the activity of PKA and GSK-3β. These results suggest that metformin kills lung cancer cells through AMPK/PKA/GSK-3β-axis–mediated survivin degradation, providing novel insights into the anticancer effects of metformin.     

高产申嗪霉素和吩嗪-1-酰胺的水稻根际铜绿假单胞菌PA1201分离、鉴定与应用潜力

【目的】从水稻根际筛选能高效抑制水稻病原菌的细菌,分析和鉴定其形态和生化特征,为开发新型绿色农药奠定基础。【方法】从水稻根际分离能以1-氨基环丙烷-1-羧酸(ACC)为唯一碳源的菌株,根据菌株形态和生化特性、16S r DNA序列比对和磷脂脂肪酸图谱,对该菌株进行鉴定。通过氯仿萃取抽提、高效液相色谱分析,确定菌株PA1201在PPM培养基和黄豆粉培养基中申嗪霉素和吩嗪-1-酰胺的产量。【结果】菌株PA1201能有效抑制水稻纹枯病菌和水稻白叶枯病菌的生长,属于铜绿假单胞菌(Pseudomonas aeruginosa sp.PA1201);PA1201产生两种抑菌代谢产物申嗪霉素和吩嗪-1-酰胺,在PPM和黄豆粉培养基中申嗪霉素的产量可达81.7 mg/L和926.9 mg/L,吩嗪-1-酰胺的产量亦可达18.1 mg/L和489.5 mg/L;PA1201产生大量胞外蛋白酶,对人肺腺癌细胞系A549和黑腹果蝇具有一定毒性。【结论】PA1201的申嗪霉素产量比现有生产菌株的出发菌株M18高3-4倍,还能产生另一种抑菌活性更高的衍生物吩嗪-1-酰胺,具有进一步开发的潜力。     

非生物胁迫下耐辐射异常球菌drB0118基因功能分析

【目的】鉴定和确定被预测为编码干燥相关蛋白的耐辐射异常球菌(Deinococcus radiodurans) drB0118基因功能,探讨该基因对盐、渗透和氧化胁迫抗性的作用。【方法】构建drB0118基因缺失突变株(ΔB0118),通过氯化钠、D-山梨糖醇和过氧化氢等胁迫冲击实验及氧化胁迫条件下qRT-PCR分析,研究drB0118突变对非生物胁迫反应及氧化胁迫相关基因表达的影响。【结果】drB0118突变导致菌株对NaCl和D-sorbitol胁迫的抗性降低;对氧化胁迫(H2O2)敏感;qRT-PCR分析显示,drB0118突变引起氧化胁迫抗性基因pod和oxyR分别下调4倍和10倍。【结论】D. radiodurans中drB0118参与了盐、渗透和氧化等多种非生物胁迫反应。     

The Successful Diagnosis and Typing of Systemic Amyloidosis Using A Microwave-Assisted Filter-Aided Fast Sample Preparation Method and LC/MS/MS Analysis

Laser microdissection followed by mass spectrometry has been successfully used for amyloid typing. However, sample contamination can interfere with proteomic analysis, and overnight digestion limits the analytical throughput. Moreover, current quantitative analysis methods are based on the spectrum count, which ignores differences in protein length and may lead to misdiagnoses. Here, we developed a microwave-assisted filter-aided sample preparation (maFASP) method that can efficiently remove contaminants with a 10-kDa cutoff ultrafiltration unit and can accelerate the digestion process with the assistance of a microwave. Additionally, two parameters (P- and D-scores) based on the exponentially modified protein abundance index were developed to define the existence of amyloid deposits and those causative proteins with the greatest abundance. Using our protocol, twenty cases of systemic amyloidosis that were well-typed according to clinical diagnostic standards (training group) and another twenty-four cases without subtype diagnoses (validation group) were analyzed. Using this approach, sample preparation could be completed within four hours. We successfully subtyped 100% of the cases in the training group, and the diagnostic success rate in the validation group was 91.7%. This maFASP-aided proteomic protocol represents an efficient approach for amyloid diagnosis and subtyping, particularly for serum-contaminated samples.     

PtdIns(4,5)P2 and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance

Phagocytosis requires phosphoinositides (PIs) as both signaling molecules and localization cues. How PIs coordinate to control phagosomal sealing and the accompanying switch of organelle identity is unclear. In this study, we followed dynamic changes in PIs during apoptotic cell clearance in Caenorhabditis elegans. We found that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂) and phosphatidylinositol-3-phosphate (PtdIns3P), which accumulate transiently on unsealed and fully sealed phagosomes, respectively, are both involved in phagosome closure. We identified PtdIns3P phosphatase MTM-1 as an effector of PtdIns(4,5)P₂ to promote phagosomal sealing. MTM-1 coordinates with the class II PI3 kinase PIKI-1 to control PtdIns3P levels on unsealed phagosomes. The SNX9 family protein LST-4 is required for sealing, and its association with unsealed phagosomes is regulated by PtdIns(4,5)P₂, PIKI-1, and MTM-1. Loss of LST-4 or its retention on phagosomes disrupts sealing and suppresses PtdIns3P accumulation, indicating close coupling of the two events. Our findings support a coincidence detection mechanism by which phagosomal sealing is regulated and coupled with conversion from PtdIns(4,5)P₂ enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes.     

A p7 Ion Channel-derived Peptide Inhibits Hepatitis C Virus Infection in Vitro

Viral infection is an early stage of its life cycle and represents a promising target for antiviral drug development. Here we designed and characterized three peptide inhibitors of hepatitis C virus (HCV) infection based on the structural features of the membrane-associated p7 polypeptide of HCV. The three peptides exhibited low toxicity and high stability while potently inhibiting initial HCV infection and suppressed established HCV infection at non-cytotoxic concentrations in vitro. The most efficient peptide (designated H2-3), which is derived from the H2 helical region of HCV p7 ion channel, inhibited HCV infection by inactivating both intracellular and extracellular viral particles. The H2-3 peptide inactivated free HCV with an EC₅₀ (50% effective concentration) of 82.11 nm, which is >1000-fold lower than the CC₅₀ (50% cytotoxic concentration) of Huh7.5.1 cells. H2-3 peptide also bound to cell membrane and protected host cells from viral infection. The peptide H2-3 did not alter the normal electrophysiological profile of the p7 ion channel or block viral release from Huh7.5.1 cells. Our work highlights a new anti-viral peptide design strategy based on ion channel, giving the possibility that ion channels are potential resources to generate antiviral peptides.