DRUG DELIVERY VIA RECEPTOR-MEDIATED ENDOCYTOSIS

受体介导的内吞作用是细胞膜内陷的过程 细胞表面受体被选择性地内化为酸性 细胞内区室 这是主要的细胞摄取途径， 大分子药物，如抗体-药物缀合物和脂质体 胶囊药物 通过该途径将药物递送到细胞中， 由于其螯合作用，药物的作用具有更大的特异性 在细胞内。 此外，药物的代谢可能会受到影响 通过暴露于亚细胞区室中的酸性条件。 因此RME 是一个重要的细胞过程， 大分子药物的药代动力学及其分子调控 应该被理解。 RME是网格蛋白聚合的结果 分子在质膜的细胞质表面上。 聚合 网格蛋白形成蛋白质的多面体网络， 受体，并导致它们与结合的配体一起沿着内化。 的 网格蛋白网络的大小似乎影响颗粒的大小 内在化，并可以解释观察到的脂质体摄取的限制， 不同的细胞类型 该提案的目的是确定 控制和调节细胞凋亡的网格蛋白分子的结构特征 聚合反应 一种实验方法涉及表达 网格蛋白重链亚基和完整轻链的片段 细菌的亚单位。 个体光的表达与突变 链亚基将允许映射功能区域，如钙 影响网格蛋白装配的结合位点。 网格蛋白的共表达 轻链和重链片段将用于研究 网格蛋白亚基的相互作用。 这种方法也可能导致 表达和纯化大量的轻链， 重链片段来尝试晶体学研究。 主要 网格蛋白分子的晶体学分析的障碍是 轻链亚基的异质性。 在另一种方法中， 结构分析，网格蛋白将从具有以下结构的细胞中纯化： 最近在实验室中产生的，只表达一种形式的 网格蛋白轻链。 这种网格蛋白的同质群体也将是 分析组件属性。 最后，细胞将被转染 网格蛋白片段和突变的亚基，其表达水平 与内源性蛋白质竞争，从而研究这些蛋白质的作用。 分子对网格蛋白组装和脂质体原位摄取的影响。 理解 网格蛋白聚合的分子基础将提供对 控制大分子药物摄取的过程。

Receptor-mediated endocytosis (RME) is the process of membrane invagination by which cell surface receptors are selectively internalized into acidic intracellular compartments. This is the major cellular route for uptake of macromolecular drugs, such as antibody-drug conjugates and liposome encapsulated drugs. Delivery of drugs into cells by this pathway confers greater specificity of action of the drug by virtue of its sequestration inside the cell. Furthermore, the metabolism of the drug can be affected by exposure to the acidic conditions in subcellular compartments. thus RME is an important cellular process that influences both the pharmacodynamics and pharmacokinetics of macromolecular drugs and its molecular control should be understood. RME occurs as a result of polymerization of clathrin molecules on the cytoplasmic face of the plasma membrane. The polymerized clathrin forms a polyhedral network of protein that traps cell surface receptors and causes their internalization along with bound ligands. the size of the clathrin network appears to influence the size of the particles internalized and may explain the limits observed for liposome uptake in different cell types. the aims of this proposal are to define the structural features of the clathrin molecule that control and polymerization reaction. One experimental approach involves expressing fragments of the clathrin heavy chain subunit and the complete light chain subunits in bacteria. Expression and mutation of the individual light chain subunits will allow mapping functional regions, such as the calcium binding site that influences clathrin assembly. Co-expression of clathrin light chains and heavy chain fragments will be used to study the interaction of the clathrin subunits. This approach could also lead to the expression and purification of large amounts of light chains assembled with heavy chain fragments to attempt crystallographic studies. The main obstacle to crystallographic analysis of the clathrin molecule has been the heterogeneity of the light chain subunits. In another approach to structural analysis, clathrin will be purified from cells that have recently been produced in the laboratory that express only one form of clathrin light chain. This homogeneous population of clathrin will also be analyzed for assembly properties. Finally, cells will be transfected with clathrin fragments and mutated subunits that will be expressed in levels to compete with endogenous proteins and thereby study the effect of these molecules on clathrin assembly and liposome uptake in situ. Understanding the molecular basis for clathrin polymerization will provide insight into processes that control the uptake of macromolecular drugs.