Endocytic Trafficking and Human Diseases

在过去的几年里，越来越多的与不同人类疾病有关的基因被发现。有趣的是，这些基因中的许多已经被证明编码细胞内分选机制的组件，该分选机制介导了脂类和蛋白质在分泌和内吞途径中的选择性运输。 我实验室中的两个不同项目试图解决细胞内交易缺陷可能导致人类疾病的问题： 项目1.IV型黏脂沉积症(MLIV)是一种常染色体隐性遗传性溶酶体储存障碍，以严重的精神运动迟缓和眼科异常为特征，包括角膜混浊、视网膜变性和斜视。与其他溶酶体疾病不同的是，MLIV中观察到的异质储存物质的积累不是由于分解代谢途径的阻塞，而是由于内吞作用后期的运输缺陷。MCOLN1基因在MLIV患者中发生突变，它编码一种名为粘蛋白-1的蛋白质，该蛋白质可能具有钙离子渗透通道的功能，并与溶酶体的生物发生有关。为了获得关于这种病理的潜在机制的信息，我们试图通过组合使用下拉、酵母双杂交筛选和siRNA技术来鉴定与黏脂蛋白-1相互作用或调节其功能的蛋白质。此外，我们正在寻求确定调节细胞内黏脂-1运输的分选基序。到目前为止，我们已经发现粘蛋白-1可以通过直接途径(从高尔基体到溶酶体)和间接途径(通过质膜)到达溶酶体。直接途径似乎依赖于位于蛋白质N端胞液尾部的二亮氨酸基序，该基序介导与笼状蛋白适配器AP-1和AP-3的相互作用。相反，间接途径依赖于位于粘脂素-1C末端胞浆尾部的内化基序。这一序列与AP-2结合，并通过包被网状蛋白的小泡促进质膜蛋白的内吞。有趣的是，三个半胱氨酸残基的棕榈酰化似乎调节了粘脂素-1的内化效率。 项目2.生长因子及其跨膜受体酪氨酸激酶(RTK)在胚胎发育过程中发挥重要作用，并在包括增殖、存活、迁移和分化在内的几个细胞过程的调节中发挥重要作用。生长因子与其受体的结合激活了无数的信号通路，使细胞能够对环境变化做出反应。在许多情况下，这些信号事件的终止是由受体内化和降解介导的。受体下调的缺陷可能导致持续的信号和转化。表皮生长因子受体(EGFR)被认为是RTK家族的原型成员，其被EGF激活和转运已被详尽地描述。然而，仍有一些方面需要更详细地讨论。其中一个方面是激酶在管制EGFR贩运方面所发挥的作用。我们的结果表明，苯甲酸对p38MAP的激活足以诱导EGFR的内化。茴香素和EGF使用不同的机制来促进EGFR的内吞作用，因为它诱导的内吞不需要酪氨酸激酶活性或受体的泛素化。与特定的p38抑制剂孵育，或通过小干扰RNA(SiRNA)耗尽内源性p38，可消除山奈素诱导的EGFR内化，而对转铁蛋白内吞作用没有影响。有趣的是，抑制p38的激活也取消了紫外线诱导的EGFR的内吞作用。这些结果表明，p38刺激EGFR内化可能是在应激条件下阻止增殖或抗凋亡信号产生的一般机制。虽然还需要进一步的研究来评估p38调节EGFR内化的具体机制，但我们的结果强化了这样的观点，即信号和细胞内流量之间存在明显的相互通信，并表明p38在这一过程中发挥关键作用。 另一种可能协调贩运和信号之间联系的蛋白质是Tom-like 1(Tom1L1)。Tom1L1和相关蛋白Tom1(Myb1的靶标)和Tom1L2(Tom1-like 2)组成了一个新的蛋白质家族，其特征是在N-末端存在VHS(Vps27p/Hars/Stam)结构域，紧接着是GAT(GGA和Tom)结构域。最近发现Tom1和Tom1L1的GAT结构域都与泛素结合，提示这些蛋白可能参与泛素化蛋白的多囊泡体(MVB)的分类。我们已经确定了Tom1L1和MVB分选机械成员之间的一种新的相互作用。具体来说，我们发现Tom1L1的VHS结构域与HRS(肝细胞生长因子调节的酪氨酸激酶底物)相互作用，而位于Tom1L1的VHS和GAT结构域之间的PTAP基序负责与TSG101(肿瘤易感基因101)的结合。MYC表位标记的Tom1L1呈胞质分布，但在HRs表达后被募集到内体。此外，Tom1L1在C-末端有几个酪氨酸基序，这些基序介导了与Src家族激酶成员以及其他信号蛋白如Grb2和P85的相互作用。我们发现，Fyn激酶的一部分定位于内体，并且这种分布在EGF内化后变得更加明显。此外，Fyn结构活性形式的表达也促进了Tom1L1向扩大的内体募集。综上所述，我们认为Tom1L1可能在信号转导和降解途径之间起中介作用。

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.