Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

• 批准号: 7154181
• 负责人: rosa puertollano
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7154181
• 关键词: Golgi apparatus; clathrin; growth factor receptors; hepatocyte growth factor; intracellular transport; membrane proteins; molecular pathology; mucopolysaccharidosis; protein transport; small interfering RNA; synaptophysin; ubiquitin; vesicle /vacuole; yeast two hybrid system

In the last several years an increasing number of genes associated with different human diseases have been identified. Interestingly, many of these genes have been demonstrated to encode components of the intracellular sorting machinery that mediates the selective trafficking of lipids and proteins in the secretory and endocytic pathways. Two different projects in my laboratory try to address how defects in intracellular trafficking might lead to human diseases: Project 1. Mucolipidosis type IV (MLIV) is an autosomal recessive lysosome storage disorder characterized by severe psychomotor retardation and ophthalmologic abnormalities, including corneal opacity, retinal degeneration, and strabismus. Unlike the situation in other lysosomal disorders, the accumulation of heterogeneous storage material observed in MLIV does not result from the block in the catabolic pathways, but is due to a transport defect in the late steps of endocytosis. MCOLN1, the gene mutated in MLIV patients, encodes a protein called mucolipin-1 that might function as a Ca2+ permeable channel and has been implicated in the biogenesis of lysosomes. To gain information on the mechanisms underlying this pathology we are trying to identify proteins that interact with or regulate the function of mucolipin-1 by using a combination of pull-down, yeast two hybrid screening, and siRNA techniques. In addition we are seeking to identify the sorting motifs that regulate trafficking of mucolipin-1 within the cell. So far we have found that mucolipin-1 can reach lysosomes through both a direct (from Golgi to lysosomes) and an indirect (through the plasma membrane) route. The direct route appears to be dependent on a dileucine motif located at the N-terminal cytosolic tail of the protein that mediates interaction with the clathrin adaptors AP-1 and AP-3. In contrast, the indirect pathway is dependent on an internalization motif positioned at the end of the mucolipin-1 C-terminal cytosolic tail. This sequence binds AP-2 and promotes the endocytosis of the protein from the plasma membrane through clathrin-coated vesicles. Interestingly, palmitoylation of three cysteine residues seems to regulate the efficiency of mucolipin-1 internalization. Project 2. Growth factors and their transmembrane receptor tyrosine kinases (RTK) play important roles during embryonic development and in the regulation of several cellular processes including proliferation, survival, migration and differentiation. Binding of growth factors to their receptors activates a myriad of signaling pathways that permit cells to respond to changes in the environment. In many cases the termination of these signaling events is mediated by receptor internalization and degradation. Defects in receptor down-regulation might lead to sustained signaling and transformation. The epithelial growth factor receptor (EGFR) is considered the prototypal member of the RTK family and its activation by EGF and trafficking has been exhaustively characterized. However, there are still some aspects that remain to be addressed in more detail. One of these aspects is the role played by kinases in the regulation of EGFR trafficking. Our results show that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNA (siRNA), abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. These results suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. While further studies will be required to assess the specific mechanisms used by p38 to regulate EGFR internalization, our results strengthen the idea that there is a clear intercommunication between signaling and intracellular traffic and suggest that p38 is a key player in this process. Another protein that might coordinate the connection between trafficking and signaling is Tom-like1 (Tom1L1). Tom1L1, and related proteins Tom1 (Target of Myb1) and Tom1L2 (Tom1-like2), constitute a new family of proteins that are characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion followed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 bind ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). We have identified a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepatocyte growth factor?regulated tyrosine kinase substrate), while a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for the binding to TSG101 (tumor susceptibility gene 101). Myc-epitope tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In addition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after EGF internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together we propose that Tom1L1 could act as an intermediary between signaling and degradative pathways.

在过去的几年中，越来越多的与不同的人类疾病相关的基因被发现。有趣的是，其中许多基因已被证明编码细胞内分选机制的组分，该机制介导分泌和内吞途径中脂质和蛋白质的选择性运输。 我实验室的两个不同项目试图解决细胞内运输缺陷如何导致人类疾病： 项目1。IV型粘脂沉积症（MLIV）是一种常染色体隐性溶酶体贮积症，其特征为严重的精神发育迟滞和眼科异常，包括角膜混浊、视网膜变性和斜视。与其他溶酶体疾病中的情况不同，在MLIV中观察到的异质性储存物质的蓄积不是由于分解代谢途径的阻断，而是由于内吞作用后期步骤中的转运缺陷。MCOLN 1是MLIV患者中突变的基因，编码一种称为粘脂蛋白-1的蛋白质，其可能作为Ca 2+渗透通道发挥作用，并与溶酶体的生物发生有关。为了获得这种病理学机制的信息，我们试图通过使用下拉，酵母双杂交筛选和siRNA技术的组合来鉴定与粘脂-1相互作用或调节粘脂-1功能的蛋白质。此外，我们正在寻求确定的分选图案，调节运输的粘脂-1在细胞内。到目前为止，我们已经发现，粘脂-1可以通过直接（从高尔基体到溶酶体）和间接（通过质膜）途径到达溶酶体。直接途径似乎依赖于位于蛋白质的N-末端胞质尾部的双亮氨酸基序，其介导与网格蛋白衔接子AP-1和AP-3的相互作用。相反，间接途径依赖于位于粘脂-1 C-末端胞质尾部末端的内化基序。该序列结合AP-2并促进蛋白质从质膜通过网格蛋白包被的囊泡的内吞作用。有趣的是，三个半胱氨酸残基的棕榈酰化似乎调节粘脂-1内化的效率。 项目2.生长因子及其跨膜受体酪氨酸激酶（RTK）在胚胎发育过程中以及在包括增殖、存活、迁移和分化在内的若干细胞过程的调节中起重要作用。生长因子与其受体的结合激活了无数的信号通路，使细胞能够对环境的变化做出反应。在许多情况下，这些信号传导事件的终止由受体内化和降解介导。受体下调的缺陷可能导致持续的信号传导和转化。表皮生长因子受体（EGFR）被认为是RTK家族的原型成员，其被EGF激活和转运的特征已被详尽描述。然而，仍有一些方面有待更详细地处理。这些方面之一是激酶在调节EGFR运输中所起的作用。我们的研究结果表明，茴香霉素激活p38 MAP激酶足以诱导EGFR的内化。茴香霉素和EGF采用不同的机制来促进EGFR内吞作用，因为茴香霉素诱导的内化不需要受体的酪氨酸激酶活性或泛素化。孵育与p38的特异性抑制剂，或消耗内源性p38的小干扰RNA（siRNA），取消茴香霉素诱导的EGFR的内化，而转铁蛋白的内吞作用没有影响。有趣的是，抑制p38活化也消除了由UV辐射诱导的EGFR的内吞作用。这些结果表明，刺激EGFR内化的p38可能代表了一个一般的机制，以防止产生的增殖或抗凋亡信号的压力条件下。虽然需要进一步的研究来评估p38用于调节EGFR内化的具体机制，但我们的研究结果加强了信号传导和细胞内交通之间存在明确的相互通信的想法，并表明p38是这一过程中的关键参与者。 另一种可能协调运输和信号传导之间联系的蛋白质是Tom-like 1（Tom 1 L1）。Tom 1 L1和相关蛋白Tom 1（Myb 1的靶蛋白）和Tom 1 L2（Tom 1样蛋白2）构成了一个新的蛋白家族，其特征在于在N-末端部分存在VHS（Vps 27 p/Hrs/Stam）结构域，随后是GAT（GGA和Tom）结构域。最近的研究表明，Tom 1和Tom 1 L1的GAT结构域结合泛素，这表明这些蛋白可能参与了泛素化蛋白到多泡体（MVB）的分选。我们已经确定了Tom 1 L1和MVB分选机制成员之间的新相互作用。具体来说，我们发现Tom 1 L1的VHS结构域与Hrs（肝细胞生长因子？受调节的酪氨酸激酶底物），而位于Tom 1 L1的VHS和GAT结构域之间的PTAP基序负责与TSG 101（肿瘤易感基因101）结合。Myc-表位标记的Tom 1 L1表现出胞质分布，但被招募到内涵体后Hrs表达。此外，Tom 1 L1在C-末端区域具有几个酪氨酸基序，其介导与Src家族激酶和其他信号蛋白如Grb 2和p85的成员的相互作用。我们发现一部分Fyn激酶定位于内体，并且这种分布在EGF内化后变得更加明显。此外，Fyn的组成型活性形式的表达也促进了Tom 1 L1向扩大的内体的募集。综上所述，我们认为Tom 1 L1可以作为信号传导和降解途径之间的中介。