溶酶体跨膜蛋白175对HeLa细胞线粒体功能的调控作用OA

AIM:To investigate the impact of lysosomal transmembrane protein 175(TMEM175)knockout on mitochondrial function in HeLa cells.METHODS:A HeLa cell line was cultured in passage and assigned to two groups:wild type(WT)and TMEM175 knockout(KO).Western blot was conducted to assess the protein expression of TMEM175 and lysosome-associated molecules,including lysosomal-associated membrane protein 1(LAMP1),LAMP2,and transcription factor EB(TFEB).Changes in the lysosomal acidity were measured by LysoSensor staining.The mito-chondrial membrane potential and reactive oxygen species(ROS)levels were determined with JC-1 and Mito-SOX probes,respectively.The mitochondrial oxygen consumption rate and ATP content were evaluated using the O2K high-resolution mitochondrial respiration assay system and ATP assay.Moreover,Western blot was performed to analyze the expression of key proteins involved in mitochondrial dynamics and energy metabolism,such as mitofusin2(Mfn2),optic atrophy 1(OPA1),dynamin-related protein 1(Drp1),AMP-activated protein kinase(AMPK),and the components of the electron transport chain.RESULTS:When compared to the WT group,TMEM175 protein expression was significantly reduced in KO cells(P<0.01),which exhibited increased lysosomal acidity(P<0.05),decreased LAMP1 protein expression(P<0.05),and elevated levels of LAMP2 and TFEB phosphorylation(P<0.05).Moreover,TMEM175 KO reduced the mito-chondrial membrane potential(P<0.01)and increased the mitochondrial ROS levels(P<0.01).Mitochondrial basal res-piration and leak respiration capacities were impaired(P<0.01),whereas ATP-linked respiration and ATP content were increased(P<0.05 or P<0.01).No significant differences were detected in the protein expressions of Mfn2 and OPA1 be-tween WT and KO cells(P>0.05).However,in the KO group,the phosphorylation of Drp1 showed an increase(P<0.05),and both the expression and activity of AMPK were significantly elevated(P<0.05),whereas the protein expres-sion of mitochondrial complexes I and IV was reduced(P<0.05 or P<0.01).CONCLUSION:The TMEM175 KO in HeLa cells induces lysosomal acidification,alters the expression of lysosome-associated proteins,and impairs mitochondrial function while activating the Drp1 and AMPK signaling.