Fine-tuning of the Hsc70-based Human Protein Disaggregase Machinery by the Distinctive C-terminal Extension of Apg2

Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). Together with DnaJB1, Apg2 serves to nucleate Hsc70 molecules into sites where productive entropic pulling forces can be developed. During aggregate reactivation, Apg2 performs as a specialized nucleotide exchange factor, but the origin of its specialization is poorly defined. Here we report on the role of the distinctive C-terminal extension present in Apg2 and other metazoan homologs. We found that the first part of this Apg2 subdomain, with propensity to adopt α-helical structure, interacts with the nucleotide binding domain of Hsc70 in a nucleotide-dependent manner, contributing significantly to the stability of the Hsc70:Apg2 complex. Moreover, the second intrinsically disordered segment of Apg2 C-terminal extension plays an important role as a downregulator of nucleotide exchange. An NMR analysis showed that the interaction with Hsc70 nucleotide binding domain modifies the chemical environment of residues located in important functional sites such as the interface between lobe I and II and the nucleotide binding site. Our data indicate that Apg2 C-terminal extension is a fine-tuner of human Hsc70 activity that optimizes the substrate remodeling ability of the chaperone system.     

Drosophila female germline stem cells undergo mitosis without nuclear breakdown

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Microwave-And Ultrasound-Assisted Synthesis of Some Acyclonucleobases Based on a Uracil Moiety Using Dmap as Base

In this study, 1-(bromoalkanoyl)-1H-pyrimidine-2,4-diones, (2,4-dioxo-pyrimidin-1-yl)-oxo-alkanoic acids, and bis(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-alkanones were successfully prepared via electrophilic substitution of uracil and its derivatives. High yields and pure products were obtained when microwave and ultrasound methodologies were used for undertaking the reactions. Importantly, the use of 4-dimethylaminopyridine in the present investigation gave rise to higher conversions of the starting material and afforded facile access to regioselective N-1 products.     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.     

H.pylori可塑区tfs3a分泌系统virB2基因功能

目的对virB2基因编码蛋白进行分析,为virB2基因及其编码蛋白功能提供实验依据。方法利用多种生物学软件以及网站对VirB2蛋白的结构和功能进行分析预测,VirB2蛋白序列通过基因推导获得并由生物公司合成,然后通过免疫动物实验制备鼠抗VirB2蛋白多克隆抗体,同时设计进行VirB2蛋白细胞毒试验(MTT法)。结果virB2基因编码蛋白属于疏水性蛋白,为鞭毛样结构,有较强的细胞毒作用。结论对VirB2蛋白的结构和功能进行了分析预测,证明VirB2蛋白在H.pylori相关的致病性特别是引起胃黏膜炎症方面起到一定的作用,能够为研究H.pylori致病机制提供帮助。     

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in Human hepatoma cells

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H₂O₂-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H₂O₂-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H₂O₂-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H₂O₂-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H₂O₂-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.     

Insulin-releasing and cytotoxic properties of the frog skin peptide,tigerinin-1R: a structure–activity study

The frog skin host-defense peptide tigerinin-1R (RVCSAIPLPICH.NH₂) is insulinotropic both in vitro and in vivo. This study investigates the effects on insulin release and cytotoxicity of changes in cationicity and hydrophobicity produced by selected substitutions of amino acids by l-arginine, l-lysine and l-tryptophan. The [A5W], [L8W] and [I10W] analogs produced a significant (P < 0.01) increase in the rate of insulin release from BRIN-BD11 rat clonal β cells at concentration of 0.01 nM compared with 0.1 nM for tigerinin-1R. The increase in the rate of insulin release produced by a 3 μM concentration of the [S4R], [H12K], and [I10W] analogs from both BRIN-BD11 cells and mouse islets was significantly greater (P < 0.05) than that produced by tigerinin-1R. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM indicating that plasma membrane integrity had been preserved. [A5W] tigerinin-1R was the only analog tested that showed cytotoxic activity against human erythrocytes (LC₅₀ = 265 ± 16 μM) and inhibited growth of Escherichia coli (MIC = 500 μM) and Staphylococcus aureus (MIC = 250 μM). The circular dichroism spectra of tigerinin-1R and [A5W] tigerinin-1R indicate that the peptides adopt a mixture of β-sheet, random coil and reverse β-turn conformations in 50% trifluoroethanol/water and methanol/water. Administration of [S4R] tigerinin-1R (75 nmol/kg body weight) to high-fat fed mice with insulin resistance significantly (P < 0.05) enhanced insulin release and improved glucose tolerance over a 60 min period following an intraperitoneal glucose load. The study supports the claim that tigerinin-1R shows potential for development into novel therapeutic agents for treatment of type 2 diabetes mellitus.     

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover

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Intrinsic Bent DNA Sites in the Developmentally Amplified C3-22 Gene Promoter of Rhynchosciara americana (Diptera: Sciaridae)

The Rhynchosciara americana C3-22 gene is located in an amplified domain and is developmentally expressed. The aim of the present work was to identify intrinsically bent DNA sites in a segment containing the gene promoter and downstream sequence. The results indicated that this gene is flanked by intrinsically bent DNA sites. Three bent DNA sites (b^?3, b^?2, and b^?1) were localized in the promoter, and one was localized downstream of the gene (b⁺¹). These sites had helical parameters that confirmed the curved structure, as well as segments with left-handed superhelical writhe. In silico analysis of the promoters of four other insect genes, which encode secreted polypeptides, showed that they all had curved structures and similar helical parameters. Correlation with other results indicates that the detected intrinsically bent DNA sites that flank the C3-22 gene might be a consensus feature of the gene structure in the amplified domains.