Klaus Christina, Kaemmerer Elke, Reinartz Andrea, Schneider Ursula, Plum Patrick, Jeon Min Kyung, Hose Josephine, Hartmann Franziska, Schnölzer Martina, Wagner Norbert, Kopitz Jürgen, Gassler Nikolaus
Institute of Pathology, RWTH Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.
Cell Tissue Res. 2014 Jul;357(1):267-78. doi: 10.1007/s00441-014-1826-8. Epub 2014 Apr 29.
Acyl-CoA synthetase 5 (ACSL5), a mitochondrially localized enzyme, catalyzes the synthesis of long-chain fatty acid thioesters and is physiologically involved in pro-apoptotic sensing of enterocytes. The aim of the present study is to identify an ACSL5-dependent regulation of mitochondrially expressed proteins and the characterization of related pathways in normal and diseased human intestinal mucosa. Proteomics of isolated mitochondria from ACSL5 transfectants and CaCo2 controls were performed. ACSL5-dependent protein synthesis was verified with quantitative reverse transcription plus the polymerase chain reaction, Western blotting, short-interfering-RNA-mediated gene silencing and additional cell culture experiments. Lipid changes were analyzed with tandem mass spectrometry. ACSL5-related pathways were characterized in normal mucosa and sporadic adenocarcinomas of the human intestine. In CaCo2 cells transfected with ACSL5, mortalin (HSPA9) was about two-fold increased in mitochondria, whereas cytoplasmic mortalin levels were unchanged. Disturbance of acyl-CoA/sphingolipid metabolism, induced by ACSL5 over-expression, was characterized as crucial. ACSL5-related over-expression of mitochondrial mortalin was found in HEK293 and Lovo (wild-type TP53 [tumor protein p53]) and CaCo2 (p53-negative; TP53 mutated) cells but not in Colo320DM cells (mutated TP53). In normal human intestinal mucosa, an increasing gradient of both ACSL5 and mortalin from bottom to top was observed, whereas p53 (wild-type TP53) decreased. In sporadic intestinal adenocarcinomas with strong p53 immunostaining (mutated TP53), ACSL5-related mortalin expression was heterogeneous. ACSL5-induced mitochondrial mortalin expression is assumed to be a stress response to ACSL5-related changes in lipid metabolism and is regulated by the TP53 status. Uncoupling of ACSL5 and mitochondrial mortalin by mutated TP53 could be important in colorectal carcinogenesis.
酰基辅酶A合成酶5（ACSL5）是一种定位于线粒体的酶，催化长链脂肪酸硫酯的合成，在生理上参与肠细胞的促凋亡感知。本研究的目的是确定ACSL5对线粒体表达蛋白的依赖性调节以及正常和患病人类肠黏膜中相关途径的特征。对来自ACSL5转染细胞和CaCo2对照的分离线粒体进行了蛋白质组学分析。通过定量逆转录加聚合酶链反应、蛋白质免疫印迹法、短干扰RNA介导的基因沉默以及其他细胞培养实验验证了ACSL5依赖性蛋白质合成。用串联质谱法分析脂质变化。在正常黏膜和人类肠道散发性腺癌中对ACSL5相关途径进行了特征描述。在转染了ACSL5的CaCo2细胞中，线粒体中的mortalin（热休克蛋白家族A成员9，HSPA9）增加了约两倍，而细胞质中的mortalin水平未变。ACSL5过表达诱导的酰基辅酶A/鞘脂代谢紊乱被认为至关重要。在人胚肾293细胞、Lovo细胞（野生型TP53 [肿瘤蛋白p53]）和CaCo2细胞（p53阴性；TP53突变）中发现了与ACSL5相关的线粒体mortalin过表达，但在Colo320DM细胞（TP53突变）中未发现。在正常人类肠黏膜中，观察到从底部到顶部ACSL5和mortalin均呈递增梯度，而p53（野生型TP53）则减少。在p53免疫染色强的散发性肠腺癌（TP53突变）中，与ACSL5相关的mortalin表达是异质性的。ACSL5诱导的线粒体mortalin表达被认为是对ACSL5相关脂质代谢变化的应激反应，并受TP53状态调节。TP53突变导致ACSL5与线粒体mortalin解偶联可能在结直肠癌发生中起重要作用。
