Structure and function of peroxisomal ABC transporters and regulation of cellular lipid metabolism

过氧化物酶体ABC转运蛋白的结构和功能以及细胞脂质代谢的调节

• 批准号: 09672255
• 负责人: IMANAKA Tsuneo
• 金额: $ 1.92万
• 依托单位: Toyama Medical and Pharmaceutical University (1998)Teikyo University (1997)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672255/
• 关键词: peroxisome; ABC transporter; PMP70; ALDP; ATP; lipid rransport; acyl-CoA; fatty asic beta-oxidation; ABC・トランスポーター

ATP-binding cassette (ABC) transporters are a superfamily of membrane-bound proteins whose structure and function have been highly conserved from eubacteria to mammals, and catalyze the ATP-dependent transmembrane translocation of a wide variety of substrates, including anti -tumor drugs, amino acids, peptides, proteins, sugars, oligosaccharides, and several kind of lipids. To date, four peroxisomal ABC transporters have been identified in mammalian peroxisomes ; 70-kDa peroxisomal membrane protein (PMP70), adrenoleukodystrophy protein (ALDP), ALOP related protein (ALDRP) and PM P70 related protein (P70R). PMP70 is one of the major components of peroxisomal membranes and is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes.To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into CHO cells and established cell lin … More es stably expressing PM P70. The content of PM P70 in the transfectants increased about 5 fold when compared with the control cells. Subcellular fractionation study showed that over expressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by Immuno fluorescence and immuno electron microscopy. The number of immuno - gold particles on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3 fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes preparedfrom both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Further, we established three CHO cell lines stably expressing mutant PM P70 (missense mutants in EAA- like or Walker A motifs, and a deletion mutant in C-terminal segment including a part of Walker B motif). ln these cells, 13-oxidation of palmitic acid decreased markedly. These results suggest that PM P70 is involved in metabolic transport of long-chain acyl-CoA across peroxisomal membranes and increase of PM P70 is not associated with proliferation of peroxisomes. Less

ATP结合盒（ABC）转运蛋白是一类结构和功能高度保守的膜结合蛋白超家族，催化多种底物跨膜转运，包括抗肿瘤药物、氨基酸、肽、蛋白质、糖、寡糖和多种脂类。迄今为止，已在哺乳动物过氧化物酶体中鉴定出四种过氧化物酶体ABC转运蛋白：70-kDa过氧化物酶体膜蛋白（PMP 70）、肾上腺脑白质营养不良蛋白（ALDP）、ALOP相关蛋白（ALDRP）和PM P70相关蛋白（P70 R）。为了研究PMP 70在过氧化物酶体生物合成和功能中的作用，我们将大鼠PMP 70的cDNA转染CHO细胞，建立了细胞系，并将其转染到CHO细胞中，建立了细胞系。 ...更多信息 稳定表达PM P70。与对照细胞相比，转染细胞中PM P70的含量增加了约5倍。亚细胞分级分析表明，过表达的PMP 70富含过氧化物酶体。免疫荧光和免疫电子显微镜证实了过氧化物酶体的定位。过氧化物酶体上的免疫金颗粒数量在转染细胞中显著增加，但过氧化物酶体的大小和数量在转染细胞和对照细胞中基本相同。在转染子中棕榈酸的β-氧化增加约2-3倍，而二十四烷酸的氧化降低约30- 40%。当完整的过氧化物酶体从这两个细胞系与棕榈酰辅酶A孵育，氧化与ATP刺激，但刺激的程度是在转染细胞高于对照细胞。此外，我们建立了三个稳定表达突变PM P70的CHO细胞系（EAA样或步行者A基序中的错义突变体，以及包括部分步行者B基序的C-末端区段中的缺失突变体）。在这些细胞中，棕榈酸的13-氧化作用显著降低。这些结果表明，PM P70参与长链酰基CoA跨过氧化物酶体膜的代谢转运，并且PM P70的增加与过氧化物酶体的增殖无关。少

项目成果

Shimozawa, N.: "Genetic basis of peroxisome-assembly mutants of humans, chinese hamster ovary cells and yeast : identification of a new comple-mentation group of peroxisome-biogenesis disorders, apparently lacking peroxisomal membrane ghosts." Am.J.Hum.Ge

Shimozawa, N.：“人类、中国仓鼠卵巢细胞和酵母的过氧化物酶体组装突变体的遗传基础：鉴定出一个新的过氧化物酶体生物发生障碍互补组，显然缺乏过氧化物酶体膜鬼影。”

Imanaka,T.: "The 70-kDa peroxisomal membrane protein(PMP70),an ATP-binding cassette transporter" Cell Biochem.Biophys.(in press). (1999)

Imanaka,T.：“70-kDa 过氧化物酶体膜蛋白 (PMP70)，一种 ATP 结合盒转运蛋白”Cell Biochem.Biophys.（出版中）。

Imanaka, T.: "ABC protein and peroxisomal diseases. (Japanese)" Saishin Igaku. 54. 434-440 (1999)

Imanaka, T.：“ABC 蛋白和过氧化物酶体疾病。（日语）”Saishin Igaku。

Takahashi,Y.: "Spatial pattern of smooth muscle differentiation is specified by the epithelium in the stomach of mouse embryo." Developmental Dynamics. (in press). (1998)

Takahashi,Y.：“平滑肌分化的空间模式是由小鼠胚胎胃中的上皮细胞指定的。”

Imamura, A.: "Temperature-sensitive phenotypes of peroxisome assembly processes represent the milder forms of human peroxisome biogenesis disorders." Am.J.Hum.Genet.62. 1539-1543 (1998)

Imamura, A.：“过氧化物酶体组装过程的温度敏感表型代表了人类过氧化物酶体生物发生障碍的较温和形式。”