14-3-3参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径研究

本室以前已经报道了G蛋白偶联受体APJ的内源性配体多肽,apelin-13,通过激活ERK1/2促进大鼠血管平滑肌细胞增殖.本文研究14-3-3信号蛋白是否参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径,探讨apelin/APJ系统的细胞信号转导机制.组织贴块法培养大鼠胸主动脉VSMCs;Western blotting方法检测14-3-3、pRaf-1、Raf-1、pERK1/2、ERK1/2、cyclinD1、cyclinE的表达;MTT方法观察14-3-3抑制剂Difopein对VSMCs的增殖作用;免疫共沉淀方法检测14-3-3和Raf-1蛋白复合物的形成.Western blotting方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,以2μmol/L最为明显;2μmol/L apelin-13时间依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,在4 h增加最为显著;14-3-3蛋白抑制剂Difopein明显抑制apelin-13诱导的Raf-1磷酸化、ERK1/2磷酸化、cyclinD1及cyclinE表达;免疫共沉淀方法发现apelin-13诱导14-3-3与Raf-1结合增加,而Difopein明显抑制两者结合;MTT法显示Difopein明显抑制apelin-13诱导的血管平滑肌细胞增殖.上述结果表明,Apelin-13通过14-3-3/Raf-1复合物-ERK1/2信号转导通路促进大鼠血管平滑肌细胞增殖.     

Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.     

Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages

The colony-forming ability of Escherichia coli genetically engineered to produce eicosapentaenoic acid (EPA) grown in 3mM hydrogen peroxide (H(2)O(2)) was similar to that of untreated cells. It was rapidly lost in the absence of EPA. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The fatty acid composition of the transformants was unaffected by H(2)O(2) treatment, but the amount of fatty acids decreased in cultures of cells lacking EPA and increased in cultures of cells producing EPA, suggesting that cellular EPA is stable in the presence of H(2)O(2) in vivo and may protect cells directly against oxidative damage. We discuss the possible role of EPA in partially blocking the penetration of H(2)O(2) into cells through membranes containing EPA.     

Exercise enhancement by RGS14 disruption is mediated by brown adipose tissue

Enhanced exercise capacity is not only a feature of healthful aging, but also a therapy for aging patients and patients with cardiovascular disease. Disruption of the Regulator of G Protein Signaling 14 (RGS14) in mice extends healthful lifespan, mediated by increased brown adipose tissue (BAT). Accordingly, we determined whether RGS14 knockout (KO) mice exhibit enhanced exercise capacity and the role of BAT in mediating exercise capacity. Exercise was performed on a treadmill and exercise capacity was assessed by maximal running distance and work to exhaustion. Exercise capacity was measured in RGS14 KO mice and their wild types (WT), and also in WT mice with BAT transplantation from RGS14 KO mice or from other WT mice. RGS14 KO mice demonstrated 160 ± 9% increased maximal running distance and 154 ± 6% increased work to exhaustion, compared to WT mice. RGS14 KO BAT transplantation to WT mice, resulted in a reversal of phenotype, with the WT mice receiving the BAT transplant from RGS14 KO mice demonstrating 151 ± 5% increased maximal running distance and 158 ± 7% increased work to exhaustion, at three days after BAT transplantation, compared to RGS14 KO donors. BAT transplantation from WT to WT mice also resulted in increased exercise performance, but not at 3 days, but only at 8 weeks after transplantation. The BAT induced enhanced exercise capacity was mediated by (1) mitochondrial biogenesis and SIRT3; (2) antioxidant defense and the MEK/ERK pathway, and increased hindlimb perfusion. Thus, BAT mediates enhanced exercise capacity, a mechanism more powerful with RGS14 disruption.     

A conserved role for the zinc finger polyadenosine RNA binding protein,ZC3H14, in control of poly(A) tail length

The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.