PDF(Pubmed)
Abstract：
Heat shock protein 90 (Hsp90) is a conserved molecular chaperone responsible for the folding and maturation of nascent proteins. Hsp90 is regarded as a master regulator of protein homeostasis in the cell, and its inhibition affects the functions of a large array of client proteins. The classical Hsp90 inhibitor tanespimycin has shown potent antileishmanial activity. Despite the increasing importance of Hsp90 inhibition in the development of antileishmanial agents, the global effects of these inhibitors on the parasite proteome remain unknown. By combining tanespimycin treatment with bioorthogonal noncanonical amino acid tagging (BONCAT) metabolic labeling and isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic mass spectrometry, for the first time, we robustly profiled the relative changes in the synthesis of hundreds of parasite proteins as functions of dose and duration of the inhibitor treatment. We showed that Hsp90 inhibition dynamically regulates nascent protein synthesis in Leishmania mexicana, with many chaperones and virulence factors showing inhibitor concentration- and treatment duration-dependent changes in relative expression. Many ribosomal proteins showed a downregulation upon severe Hsp90 inhibition, providing the first protein-level evidence that Hsp90 inhibition affects the protein synthesis capacity of the ribosome in this organism. We also provide an unbiased target validation of tanespimycin in L. mexicana using live parasite photoaffinity labeling with a novel chemical probe and quantitative proteomic mass spectrometry. We showed that the classical Hsp90 inhibitor not only engages with its presumed target, Hsp83-1, in L. mexicana promastigotes but also affects multiple proteins involved in protein synthesis and quality control in the parasite. This study defines the Leishmania parasites\' response to Hsp90 inhibition at the level of nascent global protein synthesis and provides a rich resource for future studies on Leishmania spp. biology and antileishmanial drug development.IMPORTANCE Leishmania spp. are the causative agents of leishmaniasis, a poverty-related disease, which is endemic in >90 countries worldwide, affecting approximately 12 million people, with an estimated 700,000 to 1 million new cases and around 70,000 deaths annually. Inhibitors of the chaperone protein Hsp90 have shown promising antileishmanial activity. However, further development of the Hsp90 inhibitors as antileishmanials is hampered by a lack of direct information of their downstream effects on the parasite proteome. Using a combination of mass spectrometry-based quantitative proteomics and chemical and metabolic labeling, we provide the first protein-level evidence that Hsp90 inhibition affects global protein synthesis in Leishmania We also provide the precise relative quantitative changes in the expressions of hundreds of affected proteins as functions of both the concentration and duration of the inhibitor treatment. We find that Leishmania regulates its ribosomal proteins under Hsp90 inhibition while a set of virulence factors and chaperones are preferentially synthesized.
摘要：
热休克蛋白90（Hsp90）是一种保守的分子伴侣，负责新生蛋白质的折叠和成熟。Hsp90被认为是细胞内蛋白质稳态的主要调节因子，它的抑制作用会影响大量客户蛋白的功能。经典的Hsp90抑制剂tanespimycin已显示出有效的抗利什曼酶活性。尽管Hsp90抑制在抗利什曼原药的开发中越来越重要，这些抑制剂对寄生虫蛋白质组的整体影响仍然未知。通过将tanespimycin处理与生物正交非规范氨基酸标记（BONCAT）代谢标记和等量异位标签相结合，用于基于相对和绝对定量（iTRAQ）的定量蛋白质组学质谱，第一次,我们将数百种寄生虫蛋白合成的相对变化作为抑制剂治疗剂量和持续时间的函数。我们表明，Hsp90抑制动态调节墨西哥利什曼原虫的新生蛋白合成，许多伴侣和毒力因子显示抑制剂浓度和治疗持续时间依赖性的相对表达变化。许多核糖体蛋白在严重的Hsp90抑制后显示出下调，提供了第一个蛋白质水平的证据，证明Hsp90抑制会影响该生物体核糖体的蛋白质合成能力。我们还使用新型化学探针和定量蛋白质组学质谱，使用活寄生虫光亲和力标记，对墨西哥乳杆菌中的坦西比霉素进行了无偏目标验证。我们表明，经典的Hsp90抑制剂不仅与其假定的靶标相互作用，Hsp83-1，在墨西哥乳杆菌中，但也影响寄生虫中参与蛋白质合成和质量控制的多种蛋白质。这项研究定义了利什曼原虫寄生虫在新生的全球蛋白质合成水平上对Hsp90抑制的反应，并为利什曼原虫的未来研究提供了丰富的资源。生物学和抗利什曼酶药物的开发。重要性利什曼原虫属。是利什曼病的病原体，与贫困有关的疾病，在全球90多个国家流行，影响了大约1200万人，估计每年有70万至100万例新病例，约有7万人死亡。陪伴蛋白Hsp90的抑制剂已显示出有希望的抗利什曼酶活性。然而,由于缺乏有关其对寄生虫蛋白质组的下游作用的直接信息，因此阻碍了Hsp90抑制剂作为抗寄生虫的进一步开发。使用基于质谱的定量蛋白质组学以及化学和代谢标记的组合，我们提供了第一个蛋白质水平的证据，表明Hsp90抑制会影响利什曼原虫的整体蛋白质合成。我们还提供了数百种受影响蛋白质表达的精确相对定量变化，这些变化是抑制剂治疗浓度和持续时间的函数。我们发现利什曼原虫在Hsp90抑制下调节其核糖体蛋白，同时优先合成一组毒力因子和伴侣。
