Analysis of the mutant β-actin functions in the mutant actin transduced mouse

突变型肌动蛋白转导小鼠中突变型β-肌动蛋白的功能分析

基本信息

批准号：
13670817
负责人：
NUNOI Hiroyuki
金额：
$ 1.86万
依托单位：
宮崎医科大学
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670817/
关键词：
mutantβ-actin knock-in mouse neutrophil cytoskeletal disease Nox5 p41rox p5lnox

项目摘要

We reported a 12 year-old female patient with mutant β-actin. Her laboratory analysis showed thrombocytopenia, leukopenia and neutrophil dysfunctions. This β-actin mutation site was at a binding site for a profilin. Based on these studies, we showed the mutant actin work as a dominant negative inheritance and involved in the neutrophile dysfunctions, such as decreased chemotaxis and superoxide production (PNAS 1999). So far, cases of neutrophil actin dysfunction (NAD) and NAD with abnormal 47- and 89-kDa proteins (NAD 47/89) have been reported. Recently, a case with Rac 2 mutation that had neutrophil chemotactic dysfunction, were analyzed at the molecular level. We review these 4 cases as neutrophil cytoskeletal disease, because neutrophil dysfunctions of the patients are remarkable without an apparent systemic symptoms (Neutrophile cytoskeletal disease. Int J Hematol 74 : 119-24,2001)In this project, we tried to get this mutant β-actin expressed K562 cell line and analyze this mutantβ … More -actin function. But this mutant actin protein was expressed so small amount that we could not detect any inhibitory activity. Although we also tried to clone ES cell line with the mutantβ-actin Knock-in mouse, we could not get it during this project. Collaborating with Dr. Yamasaki who is the coauthor of our paper, we are still trying to get the mutantβ-actin knock-in mouse.Using in these technique developed forβ-actin project, we found the following facts concerning about neutrophile superoxide production. 1) In a collaboration with Dr. Sumimoto, we cloned human cDNAs for novel proteins homologous to gp91-phox, p47phox and p67phox, designated Nox 5, p41nox and p51nox, those are the catalytic and regulatory proteins of NADPH oxidase, respectively. (EMBO J. 21 : 6312-20,2002, JBC 2003 in press). 2) Collaborating with Dr. Sugimoto, mice transplanted bone marrow cells that was transduced a bicistronic retrovirus vector, Ha-MDR-IRES-gp91, showed 20-30% co-expression of P-glycoprotein and human gp91 in peripheral blood mononuclear cells for over 1 year. These results suggest that co-expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells (J Gene Med vol.4, 2003). Less

我们报告了一位 12 岁女性患者，她的实验室显示出血小板减少、白细胞减少和中性粒细胞功能障碍。根据这些研究，我们显示了该突变位点位于 profilin 的结合位点。肌动蛋白作为显性负遗传起作用，并参与中性粒细胞功能障碍，例如趋化性和超氧化物产生减少（PNAS 1999）。最近，我们对一例具有中性粒细胞趋化功能障碍的 Rac 2 突变病例进行了分子水平分析。病例为中性粒细胞骨架疾病，因为患者的中性粒细胞功能障碍显着，但没有明显的全身症状（中性粒细胞骨架疾病。Int J Hematol 74 : 119-24,2001）在这个项目中，我们试图获得表达突变型β-肌动蛋白的K562细胞系并分析这种突变型β…更多-肌动蛋白的功能，但是这种突变型肌动蛋白的表达量非常小，以至于我们无法检测到任何突变型肌动蛋白。虽然我们也尝试用突变型β-肌动蛋白敲入小鼠克隆ES细胞系，但在这个项目中我们无法与我们论文的合著者Yamasaki博士合作，我们仍在尝试获得突变型β-肌动蛋白敲入小鼠。利用为β-肌动蛋白项目开发的这些技术，我们发现了以下有关中性粒细胞超氧化物产生的事实： 1) 在与Sumimoto博士的合作中，我们克隆了人类cDNA。对于与 gp91-phox、p47phox 和 p67phox 同源的新蛋白质，指定为 Nox 5、p41nox 和 p51nox，这些是催化和 NADPH 氧化酶的调节蛋白（EMBO J. 21：6312-20，2002，JBC 2003 出版）2）与 Sugimoto 博士合作，将转导双顺反子逆转录病毒载体 Ha-的骨髓细胞移植到小鼠体内。 MDR-IRES-gp91，显示 P-糖蛋白和人 gp91 的共表达率为 20-30%这些结果表明，人类多药耐药基因 (MDR1) 与治疗基因的共表达为转基因细胞提供了选择性的生长优势（J Gene Med 第 4 卷，2003 年）。