目的:鑒定并闡述6個ACADVL新發(fā)突變的對細胞和分子功能的影響,為后期分子診斷和臨床病例管理提供科學(xué)依據(jù)。創(chuàng)新點:本文從分子功能和結(jié)構(gòu)的角度闡述6個新位點對蛋白和細胞功能的不同影響。方法:通過體外構(gòu)建突變載體建立各突變的過表達細胞系,檢測各細胞系脂肪酸代謝能力、線粒體呼吸鏈功能、線粒體產(chǎn)三磷酸腺苷（ATP）能力、活性氧（ROS）和凋亡水平。同時檢測了新發(fā)突變對蛋白二聚體穩(wěn)定性的影響并用分子動力學(xué)模擬了突變蛋白構(gòu)像的改變。結(jié)論:突變（c.541C>T、c.863T>G、c.895A>G、c.1238T>C、c.1276G>A和c.1505T>A）過表達細胞系均表現(xiàn)出脂肪酸代謝障礙,線粒體呼吸鏈功能障礙,ATP產(chǎn)生水平下降和線粒體ROS水平上升。在無糖條件下,各細胞系的凋亡水平上升。各突變影響了極長鏈�；o酶A脫氫酶（VLCAD）蛋白二聚體的穩(wěn)定性,分子動力學(xué)模擬預(yù)測每個突變均會導(dǎo)致VLCAD蛋白結(jié)構(gòu)的發(fā)生重要構(gòu)象變化。 

【文章頁數(shù)】：17 頁

【文章目錄】：
1 Introduction
2 Materials and methods
    2.1 Patients and novel variants
    2.2 Vector construction and cell culture
    2.3 Mitochondrial respiration assay
    2.4 FAO of cellular respiration and FAO flow cytometry
    2.5 ATP and mitochondrial superoxide measurements
    2.6 Western blotting
    2.7 Apoptosis assay
    2.8 Blue native PAGE
    2.9 Molecular dynamics simulation analyses
    2.1 0 Statistical analysis
3 Results
    3.1 Influence of variants on FAO capacity
    3.2 Influences of variants on mitochondrial res-piratory function,ATP production,and ROS
    3.3 Cells containing mutant VLCAD prone to apoptosis without glucose
    3.4 Influences of variants on the stability of VLCAD dimer and its MD simulation
4 Discussion
5 Conclusions
Contributors
Compliance with ethics guidelines
List of electronic supplementary materials



本文編號：3666043