Zinc metalloprotease families in Trypansoma brucei

描述(由申请人提供)：蛋白酶是使许多原生动物和蠕虫在哺乳动物和昆虫宿主体内寄生和生存的关键因素。将利用传统的基因缺失、RNA干扰(RNAi)和过表达等技术，对非洲布氏锥虫锌金属蛋白酶相关组的生物学特性和差异表达进行研究。三组金属蛋白酶TbMSP-A、TbMSP-B和TbMSP-C与利什曼原虫的主要表面蛋白水解酶(MSP，以前称为GP63)具有约33%的氨基酸同源性，并具有共同的锌结合基序。这三个基因家族的mRNAs都存在于血流锥体中，而TbMSP-B的mRNAs仅在原循环锥体中表达。它们的C端氨基酸序列表明，TbMSP-A和-B可能通过糖基磷脂酰肌醇(GPI)锚点与细胞膜结合，而TbMSP-C可能位于细胞质中或分泌。我们推测，根据TbMSP-A和TbMSP-B的序列预测的TbMSP-A和TbMSP-B在促进锥虫在宿主环境中的生存方面发挥了重要作用。 我们的初步数据使我们假设TbMSP-B是血流细胞中一种受翻译控制的表面蛋白，它参与了从血流到原循环细胞分化过程中变异的表面糖蛋白(VSG)的脱落。进一步的证据使我们假设TbMSP-A具有一种不同的蛋白分解功能，这在原周期细胞中是不需要的，尽管这一功能的确切性质尚未确定。在这一应用中，我们建议使用免疫定位、亚细胞分离、代谢标记、蛋白酶鉴定和基因突变等技术来确定TbMSP-A和TbMSP-B家族的亚细胞位置，确定它们的生物学功能，并阐明它们差异表达的分子机制。

DESCRIPTION (provided by the applicant): Proteases are critical factors enabling many protozoa and helminths to parasitize and survive in both mammalian and insect hosts. The biological properties and differential expression of related groups of zinc metalloproteases in the African trypanosome Trypanosoma brucei will be studied using the techniques of traditional gene deletion, RNA interference (RNAi), and overexpression for selected experiments. Three groups of metalloproteases, called TbMSP-A, TbMSP-B and TbMSP-C, each has about 33% amino acid identity with the major surface protease (MSP, formerly called GP63) of Leishmania, and share a common zinc binding motif. The mRNAs of all three gene families occur in bloodstream trypanosomes, whereas only the mRNA of TbMSP-B is expressed in procyclic trypanosomes. Their C-terminal amino acid sequences suggest that TbMSP-A and -B are likely associated with a cellular membrane via a glycosylphosphatidylinositol (GPI) anchor, whereas TbMSP-C may be either located in the cytoplasm or secreted. We hypothesize that those TbMSPs predicted on the basis of their sequences to traffic to the trypanosome surface, i.e., TbMSP-A and TbMSP-B, serve important functions in facilitating survival of trypanosomes in the host environment. Our preliminary data lead us to hypothesize that TbMSP-B is a surface protease under translational control in bloodstream cells, which participates in shedding of the variant surface glycoprotein (VSG) during differentiation from bloodstream to procyclic cells. Further evidence leads us to hypothesize that TbMSP-A serves a distinct proteolytic function that is not required in procyclic cells, although the exact nature of this function has yet to be identified. In this application we propose to use the techniques of immunolocalization, sub-cellular fractionation, metabolic labeling, protease characterization, and gene mutagenesis to identify the sub-cellular locations of the TbMSP-A and TbMSP-B families, to determine their biological functions, and to elucidate the molecular mechanisms regulating their differential expression.