Development of Chemical Proteomics for the Folateome and Analysis of the Kinetoplastid Folateome

The folate pathway has been extensively
studied in a number of organisms, with its essentiality exploited
by a number of drugs. However, there has been little success in developing
drugs that target folate metabolism in the kinetoplastids. Despite
compounds being identified which show significant inhibition of the
parasite enzymes, this activity does not translate well into cellular
and animal models of disease. Understanding to which enzymes antifolates
bind under physiological conditions and how this corresponds to the
phenotypic response could provide insight on how to target the folate
pathway in these organisms. To facilitate this, we have adopted a
chemical proteomics approach to study binding of compounds to enzymes
of folate metabolism. Clinical and literature antifolate compounds
were immobilized onto resins to allow for “pull down”
of the proteins in the “folateome”. Using competition
studies, proteins, which bind the beads specifically and nonspecifically,
were identified in parasite lysate (Trypanosoma brucei and Leishmania major) for each antifolate compound.
Proteins were identified through tryptic digest, tandem mass tag (TMT)
labeling of peptides followed by LC-MS/MS. This approach was further
exploited by creating a combined folate resin (folate beads). The
resin could pull down up to 9 proteins from the folateome. This information
could be exploited in gaining a better understanding of folate metabolism
in kinetoplastids and other organisms.

叶酸通路（folate pathway）已在多种生物体中得到广泛研究，其核心生理功能已被诸多药物开发所利用。然而，靶向动质体（kinetoplastids）叶酸代谢的药物开发却鲜有斩获。尽管已发现可显著抑制寄生虫酶活性的抗叶酸化合物，但这类活性无法很好地转化为疾病细胞模型与动物模型中的药效。明确生理条件下抗叶酸化合物结合的具体酶类，以及该结合与表型响应的关联机制，可为靶向这类生物体的叶酸通路提供研究思路。为推进相关研究，我们采用化学蛋白质组学方法，探究化合物与叶酸代谢酶的结合情况。将临床及文献报道的抗叶酸化合物固定于树脂载体，以实现对“叶酸组（folateome）”中蛋白质的“pull down（下拉富集）”。通过竞争实验，我们在布氏锥虫（Trypanosoma brucei）和利什曼原虫（Leishmania major）的寄生虫裂解液中，分别鉴定出每种抗叶酸化合物特异性及非特异性结合磁珠的蛋白质。蛋白质鉴定采用胰蛋白酶酶解、肽段串联质量标签（Tandem Mass Tag, TMT）标记，随后进行液相色谱-串联质谱（LC-MS/MS）分析。我们进一步开发了复合叶酸树脂（folate beads），该树脂可从叶酸组中富集多达9种蛋白质。该研究方法可为深入理解动质体及其他生物体的叶酸代谢机制提供助力。