Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.
Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.
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DOI：
10.1371/journal.ppat.1011566
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发表时间：
2023-08
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影响因子：
6.7
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中科院分区：
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--
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作为一种专性胞内寄生虫，弓形虫必须将必需的营养物质从宿主细胞输入到寄生泡中。我们以前报道，寄生虫清除宿主内吞细胞器的胆固醇纳入膜和胆固醇酯储存在脂滴。在这项研究中，我们调查了弓形虫是否利用胆固醇作为前体的代谢产物，如类固醇的合成。在哺乳动物细胞中，类固醇生成发生在线粒体中，并涉及膜结合的I型细胞色素P450氧化酶，其通过与含有细胞色素b5结构域的血红素结合蛋白（例如膜相关孕酮受体（MAPR）家族的成员）相互作用而被激活。我们的LC-MS靶向脂质组学检测弓形虫中激素类固醇的选择性类别，其中抗炎性羟基双烯醇酮种类、脱氧皮质酮和脱氢表雄酮占优势。弓形虫基因组含有编码一种I型CYP 450酶（我们将其命名为TgCYP 450 mt）和一种MAPR（我们将其命名为TgMAPR）的同源物。我们发现，TgMAPR是一个血红素蛋白与血红素结合细胞色素b5结构域中的保守残基。TgCYP 450和TgMAPR都定位于cytokon，并在原位邻近连接试验中显示相互作用。弓形虫不能耐受cyp 450 mt的基因消除;因此，我们设计了一种条件性敲除菌株，并表明iΔ TgCYP 450 mt寄生虫在培养细胞中表现出生长障碍。可以产生mapr缺陷的寄生虫菌株;然而，ΔTgMAPR寄生虫遭受整体适应性差、质膜完整性丧失、线粒体嵴异常和细胞周期中S期异常长的问题。与野生型寄生虫相比，iΔ TgCYP 450 mt和ΔTgMAPR在小鼠中失去了毒力，代谢组学研究表明，这两种突变体的类固醇水平降低。这些观察结果表明，一个类固醇生成途径的运作，涉及一个进化保守的TgCYP 450 mt酶和它的结合伙伴TgMAPR的原生动物的精子。除了控制膜的流动性，胆固醇作为重要的生物分子，特别是类固醇激素合成的底物。类固醇生成涉及线粒体胆固醇代谢细胞色素P450被膜相关孕酮受体（MAPR）血红素蛋白激活。空泡内寄生虫弓形虫从宿主中清除胆固醇，我们的靶向脂质组学揭示了寄生虫中存在的双烯醇酮和选定的类固醇激素。我们研究了弓形虫中潜在的类固醇合成途径，通过表征弓形虫中的单个CYP 450（TgCYP 450 mt）和MAPR同源物（TgMAPR）。TgCYP 450 mt和TgMAPR均在胞浆中表达并相互作用。与Δ TgCYP 450 mt寄生虫不同，缺乏mapr的寄生虫在体外是存活的。条件性TgCYP 450 mt敲除寄生虫和ΔTgMAPR具有发育缺陷，毒性差，并且与WT寄生虫相比具有降低的类固醇水平。这些数据表明，弓形虫有能力合成一些类固醇的细胞色素P450的胆固醇代谢，并揭示类固醇合成是一个古老的途径，在真核细胞系中保守。
As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR. In addition to controlling membrane fluidity, cholesterol serves as a substrate for the synthesis of important biomolecules, in particular steroid hormones. Steroidogenesis involves mitochondrial cholesterol-metabolizing cytochrome P450s activated by membrane-associated progesterone receptor (MAPR) hemoproteins. The intravacuolar parasite Toxoplasma scavenges cholesterol from the host and our targeted lipidomics reveal the presence of pregnenolone and selected steroid hormones in the parasite. We investigated a potential steroidogenic pathway in Toxoplasma by characterizing the single CYP450 (TgCYP450mt) and the MAPR homolog (TgMAPR) in Toxoplama. Both TgCYP450mt and TgMAPR are expressed in the mitochondrion and interact with each other. Parasites lacking mapr are viable in vitro unlike ΔTgCYP450mt parasites. Conditional TgCYP450mt knock-out parasites and ΔTgMAPR suffer from developmental defects, are poorly virulent and have reduced levels of steroids compared to WT parasites. These data suggest that Toxoplasma has the ability to synthesize some steroids in the mitochondrion, using a cholesterol-metabolizing cytochrome P450 and reveal that steroidogenesis is an ancient pathway, conserved in eukaryotic lineages.
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影响因子：
5.1
作者：
Gutzeit O;Segal L;Korin B;Iluz R;Khatib N;Dabbah-Assadi F;Ginsberg Y;Fainaru O;Ross MG;Weiner Z;Beloosesky R
通讯作者：
Beloosesky R
影响因子：
--
作者：
Hand, RA;Jia, N;Craven, RJ
通讯作者：
Craven, RJ
影响因子：
4
作者：
Arrizabalaga, G;Boothroyd, JC
通讯作者：
Boothroyd, JC
影响因子：
6.4
作者：
Dou Z;McGovern OL;Di Cristina M;Carruthers VB
通讯作者：
Carruthers VB
影响因子：
64.5
作者：
Coppens, I;Dunn, JD;Joiner, KA
通讯作者：
Joiner, KA