ENDOPLASMIC RETICULUM OF TRYPANOSOMATIDS

描述(由申请人提供)：锥虫表面蛋白(例如，布鲁氏锥虫的变种表面糖蛋白，VSG)对于确定感染是关键的。进入内质网(ER)是这些多肽进入胞外途径的关键步骤。因此，ER蛋白易位是寄生虫体内一条非常重要的途径。我们的长期目标是确定锥虫内质网进口机制的特征，因为内质网蛋白进口的大多数基本性质尚未确定。真核生物内质网中蛋白质的转位依赖于N端信号序列、信号识别颗粒(SRP)、SRP受体(5R)和转位(由5ec61p复合体形成)。蛋白质输入发生在共翻译或翻译后，人们普遍认为信号肽可以在所有生物家族中互换。我们在信号序列识别中发现了物种特异性：来自布氏毛滴虫和利什曼原虫的信号肽不能有效地(如果有的话)与脊椎动物微粒体输入机器一起工作。我们目前的目标是开发一种能够从锥虫体内导入蛋白质的体外系统，因为狗的微粒体系统对此毫无价值。为此，将对布鲁氏锥虫的膜进行分级，并针对VSG的进口进行优化。布鲁氏锥虫微体系统的建立将有助于表征(I)锥虫信号肽的性质，(Ii)描述布鲁氏锥虫易位系统的生化性质，以及(Iii)鉴定宿主和布氏锥虫易位机制之间的分子差异。了解锥虫和哺乳动物宿主之间信号肽选择的物种特异性的分子基础是重要的，因为这些信息可能为抗锥虫药物的基本发展提供线索。

DESCRIPTION (provided by the applicant): Trypanosomatid surface proteins (e.g., variant surface glycoprotein, VSG of Trypanosoma brucei) are critical for establishment of infections. Import into the endoplasmic reticulum (ER) is the committed step or entry into the exocytic pathway for these polypeptides. Hence, ER protein translocation is a very important pathway in the parasite. Our long-term objective is to characterize the trypanosomatid ER import machinery, as most fundamental properties of the ER protein import have not been established. Translocation of proteins into the ER of eukaryotes is dependent on an N-terminal signal sequence, signal recognition particle (SRP), SRP receptor (5R), and a translocon (formed by the 5ec61 p complex). Protein import takes place either co-translationally or post-translationally, and it is widely held that signal peptides are interchangeable across all biological families We have found species-specificity in signal sequence recognition: signal peptides from T. brucei and Leishmania do not operate efficiently (if at all) with vertebrate microsomal import machineries. Our immediate goal is to develop an in vitro system that is capable of importing proteins from trypanosomes, since the canine microsomal system is worthless for this purpose. To this end, membranes from T. brucei will be fractionated, and optimized for import of VSG. Availability of a T. brucei microsomal system will facilitate characterization of (i) properties of trypanosome signal peptides, (ii) delineation of the biochemical properties of the T. brucei translocation system, and (iii) identification of molecular differences between host and T. brucei translocation machineries. Understanding the molecular basis of the species-specificity of signal peptide selection between a trypanosome and a mammalian host is important, because the information may provide leads for rationale development of anti-trypanosome drugs.