Genome-wide functional screen of lysosome biogenesis

基于RNA干扰的全基因组筛选现已成为鉴定调节高真核细胞特定细胞功能的基因网络的有力工具。溶酶体的生物发生是调节细胞稳态的关键细胞功能之一，超过50种溶酶体贮积障碍和相关疾病证明了这一点。溶酶体的生物发生依赖于几个主要途径的协调作用：首先是内吞作用，它介导了注定要在溶酶体中消化的细胞外成分的内化；其次是生物合成运输，它带来了新合成的可溶性水解酶，以消化内吞物质和新合成的膜蛋白，这些膜蛋白是构成溶酶体膜成分的一部分。二，非冗余途径介导溶酶体的生物合成转运：AP-1途径介导溶酶体酶受体（甘露糖6-磷酸受体，MPRs）及其结合配体的分选，AP-3途径介导溶酶体相关膜糖蛋白的分选。为了获得对溶酶体的生物合成转运和人类溶酶体的生物发生及其调控手段的全面了解，我们希望进行基于RNA干扰的全基因组功能筛选，以识别调节AP-1和ap -3依赖途径的基因。RNA干扰在HeLa细胞系统中得到了很好的证实。作为进行初级筛选的读出，我们将使用HeLa细胞稳定表达gfp标记的溶酶体酶受体作为ap -1依赖途径的标记物，然后通过荧光显微镜观察其稳态分布的变化来评估调节该途径的基因。我们还将使用基于荧光的检测来监测Lamp运输的变化，以评估调节AP-3途径的基因。我们将首先说明人类基因组中含有的600种激酶和100种磷酸酶在基于RNA干扰的筛选中的作用，作为建立原理证明所需的第一步，然后实现整个人类基因组的筛选，以建立调节溶酶体生物发生的基因网络的功能图谱。在我们之前对AP-1和ap -3依赖通路的研究过程中，我们已经开发了许多定量分析和试剂，可用于进行二次筛选和进一步表征调节这些通路的单个基因产物。因此，本提案中描述的研究将提供调节AP-1和ap -3依赖途径的基因网络的完整功能表征，从而提供溶酶体生物发生所需的生物合成运输中涉及的核心机制和调节机制的全面功能图谱。

Genome-wide screens based on RNA interference have now emerged as one powerful tool for identifying gene networks that regulate specific cellular functions of high eukaryotic cells. Lysosome biogenesis is one of the key cellular functions regulating cell homeostasis, as illustrated by more than 50 lysosomal storage disorders and related diseases in humans. Lysosome biogenesis relies on the coordinated action of several major pathways: first endocytosis, which mediates the internalization of extracellular components destined to be digested in lysosomes and, second the biosynthetic transport, which brings newly synthesized, soluble hydrolytic enzymes in order to digest endocytosed material and newly synthesized membrane proteins as part of the components constituting lysosomal membranes. Two, nonredundant pathways mediate biosynthetic transport to lysosomes: the AP-1 pathway mediates the sorting of the lysosomal enzyme receptors (the mannose 6-phosphate receptors, MPRs) and their bound-ligands whereas the AP-3 pathway mediates the sorting of the lysosomeassociated membrane glycoproteins (Lamps). In order to obtain a large, comprehensive understanding of biosynthetic transport to lysosomes and lysosome biogenesis in humans as well as its means of regulation, we wish to perform a genome-wide functional screen based on RNA interference to identify genes that regulate both the AP-1 and AP-3-dependent pathways. RNA interference is well established in the HeLa cell system used. As a read-out for performing the primary screen, we will use HeLa cells stably expressing a GFP-tagged lysosomal enzyme receptor as a marker of the AP-1-dependent pathway and follow by fluorescence microscopy changes of its steady state distribution to evaluate the genes regulating this pathway. We will also use an assay based on fluorescence to monitor changes in Lamp trafficking in order to evaluate the genes regulating the AP-3 pathway. We will first illustrate the role of ¿600 kinases and ¿100 phosphatases contained in the human genome in a RNA interference-based screen, as a first step required for establishing proofs of principal, and then achieve a screen of the entire human genome in order to establish a functional map of gene networks regulating lysosome biogenesis. During the course of our previous studies on the AP-1- and AP-3-depndent pathways, we have developed a number of quantitative assays and reagents that can be used to perform secondary screens and a further characterization of individual gene products regulating these pathways. Thus, the research described in this proposal will provide a complete functional characterization of the gene networks regulating both the AP-1- and AP-3-dependent pathways and, therefore a comprehensive functional map of core machineries and regulatory mechanisms involved in the biosynthetic transport required for lysosome biogenesis.