Using Lipoamidase as a Novel Probe To Interrogate the Importance of Lipoylation in Plasmodium falciparum.
Using Lipoamidase as a Novel Probe To Interrogate the Importance of Lipoylation in Plasmodium falciparum.
复制标题
DOI：
10.1128/mbio.01872-18
复制
发表时间：
2018-11-20
期刊：
影响因子：
6.4
通讯作者：
Prigge ST
中科院分区：
文献类型：
--
作者：
Jhun H;Walters MS;Prigge ST
Lipoate是少数对中枢代谢很重要的酶的必需辅因子。疟疾寄生虫需要从人类宿主中清除的硫辛酸来生长和存活;然而，尚不清楚为什么这种辅因子如此重要。为了解决这个问题，我们设计了一个基于细菌酶脂酰胺酶（Lpa）的硫辛酸活性探针。这种探针在不同亚细胞位置的表达，使我们能够定义为隔室住房必需的脂肪酸代谢的peption。为了进一步了解硫辛酸在细胞中的具体用途，我们设计了一系列催化衰减探针，并将探针与化学旁路系统结合使用。这些研究表明，两个脂酰化的蛋白质需要寄生虫的生存。我们能够在不同的亚细胞区室中表达具有不同催化能力的Lpa，并由不同的启动子驱动，证明了这种工具的多功能性，并表明它可以用作其他生物体中硫辛酸代谢的探针。Lipoate是一种氧化还原活性辅因子，与氧化代谢的关键酶共价结合。恶性疟原虫是营养缺陷型的硫辛酸在红细胞内的阶段，但它是不知道是否需要连接到蛋白质的硫辛酸或是否需要在一个特定的亚细胞区室的寄生虫。为了解决这些问题，我们使用了一种称为脂酰胺酶（Lpa）的酶作为硫辛酸代谢的探针。Lpa首先在粪肠球菌中被描述，它特异性地切割蛋白质结合的硫辛酸，使需要这种辅因子的酶失活。具有酶活性的Lpa可以在恶性疟原虫的胞质溶胶中表达，而对蛋白质脂酰化或寄生虫生长没有任何影响。同样，Lpa可以在顶质体中表达，虽然蛋白质脂酰化减少，但寄生虫生长不受抑制。相比之下，虽然Lpa的失活突变体可以在peption中表达，但活性酶不能。我们设计了一个减毒突变体的LPA，并发现这种酶可以在寄生虫的寄生虫，但只有在与化学旁路系统。这些研究表明，乙酰辅酶A生产和H蛋白的隐蔽功能都是寄生虫生存所必需的。我们的研究验证了Lpa作为一种新的代谢探针，可用于其他系统，并提供了新的见解线粒体代谢的关键方面，负责在疟疾寄生虫的硫辛酸营养缺陷型。
Lipoate is an essential cofactor for a small number of enzymes that are important for central metabolism. Malaria parasites require lipoate scavenged from the human host for growth and survival; however, it is not known why this cofactor is so important. To address this question, we designed a probe of lipoate activity based on the bacterial enzyme lipoamidase (Lpa). Expression of this probe in different subcellular locations allowed us to define the mitochondrion as the compartment housing essential lipoate metabolism. To gain further insight into the specific uses of lipoate in the mitochondrion, we designed a series of catalytically attenuated probes and employed the probes in conjunction with a chemical bypass system. These studies suggest that two lipoylated proteins are required for parasite survival. We were able to express Lpa with different catalytic abilities in different subcellular compartments and driven by different promoters, demonstrating the versatility of this tool and suggesting that it can be used as a probe of lipoate metabolism in other organisms. Lipoate is a redox active cofactor that is covalently bound to key enzymes of oxidative metabolism. Plasmodium falciparum is auxotrophic for lipoate during the intraerythrocytic stages, but it is not known whether lipoate attachment to protein is required or whether attachment is required in a specific subcellular compartment of the parasite. To address these questions, we used an enzyme called lipoamidase (Lpa) as a probe of lipoate metabolism. Lpa was first described in Enterococcus faecalis, and it specifically cleaves protein-bound lipoate, inactivating enzymes requiring this cofactor. Enzymatically active Lpa could be expressed in the cytosol of P. falciparum without any effect on protein lipoylation or parasite growth. Similarly, Lpa could be expressed in the apicoplast, and although protein lipoylation was reduced, parasite growth was not inhibited. By contrast, while an inactive mutant of Lpa could be expressed in the mitochondrion, the active enzyme could not. We designed an attenuated mutant of Lpa and found that this enzyme could be expressed in the parasite mitochondrion, but only in conjunction with a chemical bypass system. These studies suggest that acetyl-CoA production and a cryptic function of the H protein are both required for parasite survival. Our study validates Lpa as a novel probe of metabolism that can be used in other systems and provides new insight into key aspects of mitochondrial metabolism that are responsible for lipoate auxotrophy in malaria parasites.