Shin YoungHo, Lee Sungkyoung, Ku MyungHee, Kwak Min-Kyu, Kang Sa-Ouk
Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea.
Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea.
Int J Biochem Cell Biol. 2017 Nov;92:183-201. doi: 10.1016/j.biocel.2017.10.004. Epub 2017 Oct 12.
D-erythroascorbate peroxidase (EAPX1) deficiency causes glutathione deprivation, leading to the accumulation of methylglyoxal and reactive oxygen species (ROS), and especially, induction of cytochrome c peroxidase (Ccp1) in Candida albicans. Nevertheless, reciprocal effects between changes in Ccp1 activity and the antioxidative D-erythroascorbic acid- and glutathione-dependent redox status, which reflects methylglyoxal biosynthesis altering pathophysiology are unclear in eukaryotes. To elucidate the effect of CCP1 expression on EAPX1 and glutathione reductase (Glr1) activity-mediated D-erythroascorbic acid biosynthesis and redox homeostasis, the CCP1 gene was disrupted and overexpressed. First, we demonstrated both glutathione-independent and-dependent metabolite contents and their corresponding gene transcripts and enzyme activities (i.e., Ccp1, catalase-peroxidase [KatG], superoxide dismutase [Sod], Eapx1, and Glr1) in CCP1 mutants. Second, methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) and methylglyoxal-reducing oxidoreductase activity on glycolytic methylglyoxal and pyruvate production and NAD(P)H content were determined in these mutants. Contrary to our expectation, CCP1 disruption (42.19±3.22nmolOhmgwetcell) failed to affect cell respiration compared to the wild-type strain (41.62±7.11nmolOhmgwetcell) under cyanide treatment, and in contrast to hydrogen peroxide (HO) treatment (21.74±1.03nmol Ohmgwetcell). Additionally, Ccp1 predominantly detoxified HO rather than negligible scavenging activities towards methylglyoxal and other oxidants. CCP1 deficiency stimulated Sod and Adh1 activity but downregulated Glr1, Eapx1, catalase, and peroxidase activity while enhancing KatG, EAPX1, and GLR1 transcription by decreasing glutathione and D-erythroascorbic acid and increasing pyruvate. Noticeably, the ROS-accumulating CCP1-deficient mutant maintained steady-state levels of methylglyoxal, which was revealed to be regulated by methylglyoxal-oxidizing and -reducing activity with drastic changes in NAD(P)H. We confirmed and clarified our results by showing that CCP1/EAPX1 double disruptants underwent severe growth defects due to the D-erythroascorbic acid and glutathione depletion because of pyruvate overaccumulation. These observations were made in both budding and hyphal-growing CCP1 mutants. The revealed metabolic network involving Ccp1 and other redox regulators affected ROS and methylglyoxal through D-erythroascorbic acid and glutathione-dependent metabolites, thereby influencing dimorphism. This is the first report of the Ccp1-mediated D-erythroascorbic acid and glutathione biosynthesis accompanying methylglyoxal scavengers for full fungal virulence.
D - 赤藓糖型抗坏血酸过氧化物酶（EAPX1）缺乏会导致谷胱甘肽缺失，进而导致甲基乙二醛和活性氧（ROS）积累，尤其是在白色念珠菌中诱导细胞色素c过氧化物酶（Ccp1）的产生。然而，在真核生物中，Ccp1活性变化与反映甲基乙二醛生物合成改变病理生理学的抗氧化D - 赤藓糖型抗坏血酸和谷胱甘肽依赖性氧化还原状态之间的相互作用尚不清楚。为了阐明CCP1表达对EAPX1和谷胱甘肽还原酶（Glr1）活性介导的D - 赤藓糖型抗坏血酸生物合成和氧化还原稳态的影响，对CCP1基因进行了破坏和过表达。首先，我们展示了CCP1突变体中不依赖谷胱甘肽和依赖谷胱甘肽的代谢物含量及其相应的基因转录本和酶活性（即Ccp1、过氧化氢酶 - 过氧化物酶[KatG]、超氧化物歧化酶[Sod]、Eapx1和Glr1）。其次，测定了这些突变体中对糖酵解产生的甲基乙二醛和丙酮酸的甲基乙二醛氧化醇脱氢酶（Adh1）和甲基乙二醛还原氧化还原酶活性以及NAD(P)H含量。与我们的预期相反，在氰化物处理下，与野生型菌株（41.62±7.11nmol O/mg湿细胞）相比，CCP1破坏（42.19±3.22nmol O/mg湿细胞）未能影响细胞呼吸，与过氧化氢（H₂O₂）处理（21.74±1.03nmol O/mg湿细胞）相反。此外，Ccp1主要解毒H₂O₂，而对甲基乙二醛和其他氧化剂的清除活性可忽略不计。CCP1缺乏刺激了Sod和Adh1活性，但下调了Glr1、Eapx1、过氧化氢酶和过氧化物酶活性，同时通过降低谷胱甘肽和D - 赤藓糖型抗坏血酸并增加丙酮酸来增强KatG、EAPX1和GLR1转录。值得注意的是，积累ROS的CCP1缺陷突变体维持了甲基乙二醛的稳态水平，这表明其受甲基乙二醛氧化和还原活性调节，且NAD(P)H发生了剧烈变化。我们通过表明CCP1/EAPX1双破坏体由于丙酮酸过度积累导致D - 赤藓糖型抗坏血酸和谷胱甘肽耗竭而出现严重生长缺陷，证实并阐明了我们的结果。这些观察结果在出芽和菌丝生长的CCP1突变体中均有发现。所揭示的涉及Ccp1和其他氧化还原调节剂的代谢网络通过D - 赤藓糖型抗坏血酸和谷胱甘肽依赖性代谢物影响ROS和甲基乙二醛，从而影响二态性。这是关于Ccp1介导的D - 赤藓糖型抗坏血酸和谷胱甘肽生物合成以及甲基乙二醛清除剂对真菌完全毒力影响的首次报道。
