Pathways and itinerary of clathrin-independent endocytosis

不依赖网格蛋白的内吞作用的途径和行程

• 批准号: 8939840
• 负责人: Julie G Donaldson
• 金额: $ 85.66万
• 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/8939840
• 关键词: Alpha Interleukin 2 Receptor; Amino Acid Sequence; Back; Biological Models; CD44 gene; Cell Adhesion; Cell Surface Proteins; Cell membrane; Cell physiology; Cell surface; Cells; Clathrin; Coupled; Cytoplasmic Tail; Employee Strikes; Endocytosis; Endosomes; Goals; Half-Life; Human; Immune system; Laboratories; Life; Link; Lysosomes; Major Histocompatibility Complex; Malignant Bone Neoplasm; Mediating; Membrane; Membrane Proteins; Mind; Modeling; Modification; Morphogenesis; Neoplasm Metastasis; Nutrient; Pathway interactions; Play; Process; Proteins; Recycling; Regulation; Role; Route; S-nitro-N-acetylpenicillamine; Sorting - Cell Movement; System; Transferrin Receptor; Travel; USP6 gene; Ubiquitin; Ubiquitination; Variant; Vascular Endothelial Cell; cancer cell; cell motility; glycosylation; interest; late endosome; protein transport; receptor; trafficking; ubiquitin isopeptidase; ubiquitin-protein ligase; ubiquitin-specific protease; uptake; vasculogenesis

This project is focused on understanding clathrin-independent forms of endocytosis (CIE). Endocytosis that occurs without clathrin coats occurs in all cells but is poorly understood. We are interested in studying the cargo proteins that enter cells by this mechanism, their intracellular itinerary once they have been internalized and whether they contain amino acid sequences that allow for specialized sorting within cells. We have been identifying new cargo proteins and found that a subset of these proteins take alternative traffic routes once they have entered cells. The major histocompatibility complex Class I protein (MHCI), is a prototypical clathrin-indepenent cargo protein and after internalization it reaches endosomes that contain cargo proteins such as the transferrin receptor that enter via clathrin-depenent endocytosis. From there, MHCI travels either to late endosomes and lysosomes where it is degraded or on to recycling tubules that return MHCI back to the cell surface. CD44, CD98, and CD147, however, show an altered itinerary in many cells where they traffic directly into the recycling tubules and avoid trafficking to lysosomes. Consistent with this altered itinerary, CD44, CD98 and CD147 are long-lived proteins and are not degraded like MHCI, which is routed to lysosomes. One of the functions of CIE may be to accumulate select group of cargo proteins in endosomes for specific functions. We recently showed that during vasculogenesis, using a model of lumen formation in human vascular endothelial cells, that CIE cargo proteins are delivered to these forming lumens, suggesting that CIE endosomal membranes may contribute to cell hollowing during vessel morphogenesis (Porat-Shliom and Donaldson, 2014). The altered trafficking followed by CD44, CD98 and CD147 allows these proteins to avoid transfer to lysosomal compartments and thus avoid degradation. Other CIE cargo proteins, such as MHCI, traffic to lysosomes and we have been interested in the sorting of cargo at the sorting endosome and understand how MHCI is tagged for degradation. With that goal in mind we set up a system for following the turnover of model cell surface proteins using the interleukin 2 receptor alpha subunit (Tac) coupled to the SNAP tag as our model system (Cole and Donaldson 2012). The turnover of various SNAP-Tac variants demonstrated that the half-life of the proteins was determined by their mode of entry (clathrin-mediated vs clathrin-independent), orientation in the cell membrane (i.e. integral membrane vs. glycosylphosphatiylinositol-anchored proteins) and the capacity for ubiquitination of residues on the cytoplasmic tail (Karabasheva et al 2014). Additionally, a striking and efficient shedding of SNAP-Tac into the media was observed when O-linked glycosylation was inhibited, demonstrating that ectodomain shedding may play a significant role in turnover of cell surface proteins. We are interested in the role that ubiquitin modification of cargo proteins plays in regulating the sorting of cargo and routing of cargo to lysosomes for degradation. We previously showed that over expression of the MARCH8 E3 ubiquitin ligase leads to ubiquitination and degradation of CD98, normally a long-lived protein (Eyster et al 2011). We now show that expression of a de-ubiquitinase (DUB) or ubiquitin-specific protease (USP) can counteract the effect of expression of MARCH8. This DUB is called TRE17/USP6 and was previously shown to be unregulated in ABC bone cancer. Expression of TRE17 specifically de-ubiquitinates CD98 and returns its trafficking to the normal pathway (i.e. avoidance of lysosomes) (Funakoshi et al, 2014). However TRE17 did not rescue the effect of MARCH8 on degradation of the transferrin receptor.

该项目的重点是理解非依赖性的内吞作用形式（CIE）。 无网层涂层发生的内吞作用发生在所有细胞中，但知之甚少。 我们有兴趣研究通过这种机制进入细胞的货物蛋白，一旦它们被内化，它们的细胞内行程以及它们是否包含允许在细胞内进行专门分类的氨基酸序列。 我们一直在识别新的货物蛋白，发现这些蛋白的子集一旦进入细胞，就会采用其他交通路线。 主要的组织相容性复合物I类蛋白（MHCI）是一种典型的网格蛋白 - 独立货物蛋白，在内部化后，它达到了含有货物蛋白的内体，例如通过网格蛋白 - 氧化激素 - 异常内吞作用进入的转铁蛋白受体。 从那里，MHCI可以在其降解的后期内体和溶酶体上行驶，或者在回收MHCI回到细胞表面的小管中回收小管。然而，CD44，CD98和CD147在许多细胞中显示出改变的行程，它们直接流入回收小管中，并避免运输到溶酶体。 与这种改变的行程相一致，CD44，CD98和CD147是长期寿命的蛋白质，并且不像MHCI那样降解，该蛋白被路由到溶酶体。 CIE的功能之一可能是在内体中积累精选的货物蛋白以进行特定功能。我们最近表明，在血管生成期间，使用人血管内皮细胞中的管腔形成模型，将CIE货物蛋白递送到这些形成的流明中，这表明CIE内体膜可能在血管形成过程中有助于细胞中空心（Porat-Shliom和Donaldson，2014年）。 随后发生的运输变化，CD44，CD98和CD147允许这些蛋白质避免转移到溶酶体室，从而避免降解。其他CIE货物蛋白，例如MHCI，流量到溶酶体，我们对排序内体的货物分类感兴趣，并了解如何将MHCI标记为降解。考虑到这个目标，我们建立了一个系统，用于使用白介素2受体α亚基（TAC）遵循模型细胞表面蛋白的营业额，并将其耦合到SNAP标签作为我们的模型系统（Cole and Donaldson 2012）。 The turnover of various SNAP-Tac variants demonstrated that the half-life of the proteins was determined by their mode of entry (clathrin-mediated vs clathrin-independent), orientation in the cell membrane (i.e. integral membrane vs. glycosylphosphatiylinositol-anchored proteins) and the capacity for ubiquitination of residues on the cytoplasmic tail （Karabasheva等，2014）。此外，当抑制O连接的糖基化时，观察到SNAP-TAC向培养基中的醒目和有效的脱落，这表明胞外域的脱落可能在细胞表面蛋白的周转中起重要作用。 我们对货物蛋白的泛素修饰在调节货物的分类以及货物与溶酶体降解的路线方面发挥作用的作用感兴趣。我们先前表明，三月E3泛素连接酶的表达过度会导致CD98的泛素化和降解，通常是长期寿命的蛋白质（Eyster等人，2011年）。现在，我们表明表达泛素酶（DUB）或泛素特异性蛋白酶（USP）可以抵消三月8的表达作用。该配音称为TRE17/USP6，以前被证明在ABC骨癌中不受监管。 TRE17的表达特异性去泛素化CD98并将其运输返回正常途径（即避免溶酶体）（Funakoshi等，2014）。但是，TRE17并未挽救3月8日对转铁蛋白受体降解的影响。