Zinc metalloprotease families in Trypansoma brucei

描述（由申请方提供）：蛋白酶是使许多原生动物和蠕虫能够在哺乳动物和昆虫宿主中寄生和存活的关键因素。非洲锥虫布氏锥虫锌金属蛋白酶的相关群体的生物学特性和差异表达将使用传统的基因缺失，RNA干扰（RNAi），和选择性实验过表达的技术进行研究。三组金属蛋白酶，称为TbMSP-A、TbMSP-B和TbMSP-C，每一组与利什曼原虫的主要表面蛋白酶（MSP，以前称为GP 63）具有约33%的氨基酸同一性，并且共享共同的锌结合基序。所有三个基因家族的mRNA都存在于血流锥虫中，而只有TbMSP-B的mRNA在前环锥虫中表达。它们的C-末端氨基酸序列表明TbMSP-A和-B可能通过糖基磷脂酰肌醇（GPI）锚与细胞膜结合，而TbMSP-C可能位于细胞质中或分泌。我们假设那些TbMSP基于它们的序列预测运输到锥虫表面，即，TbMSP-A和TbMSP-B在促进锥虫在宿主环境中的存活方面发挥重要作用。 我们的初步数据使我们假设TbMSP-B是在血流细胞中受翻译控制的表面蛋白酶，其在从血流细胞分化为前循环细胞期间参与变体表面糖蛋白（VSG）的脱落。进一步的证据使我们假设TbMSP-A具有独特的蛋白水解功能，这在前循环细胞中是不需要的，尽管这种功能的确切性质尚未确定。在本申请中，我们建议使用免疫定位，亚细胞分级分离，代谢标记，蛋白酶表征和基因突变的技术，以确定TbMSP-A和TbMSP-B家族的亚细胞位置，以确定其生物学功能，并阐明调节其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.