LITAF: coupling ubiquitination to transport at the endosome

LITAF：将泛素化与内体运输相结合

• 批准号: BB/X001970/1
• 负责人: Philip Woodman
• 金额: $ 75.71万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX001970%2F1
• 关键词: LITAF; coupling; ubiquitination; transport; endosome

How cells respond to their environment is determined by hundreds of different membrane proteins that are expressed on the cell surface. Cells must constantly sample and modify this complement of surface membrane proteins, to remove proteins that are no longer required at the surface due to a changed environment, or to replace proteins which become damaged by day-to-day stresses. The process that performs this important 'quality control' is endocytosis, whereby surface membrane proteins are internalised ('endocytosed') within vesicles to reach an intracellular compartment, the endosome. From there, membrane proteins that are no longer required are packaged into internal vesicles within the endosome, and the resulting structure (the multivesicular body) is then transported towards a degradative compartment (the lysosome). Alternatively, surface membrane proteins that are to be re-used are packaged at the endosome into membrane tubules and return to the cell surface. Each step of the endosomal pathway involves machinery that selects cargo membrane proteins for different destinations, coupled to machinery that deforms the membrane to generate transport intermediates (i.e. vesicles and tubules). In summary, the endosomal pathway is characterised by an abundance of high-curvature membranes that enact transport reactions. The function of virtually all cellular proteins is regulated by post-translational modifications. One key such modification is ubiquitination (the covalent attachment of the small protein ubiquitin, or polyubiquitin chains). Ubiquitination is widespread, with cells expressing >1000 ubiquitinating enzymes (ubiquitin ligases), but is particularly important during endocytosis, where it performs several important tasks. First, membrane proteins that are targeted for lysosomal degradation are tagged by ubiquitin, which is recognised by endosomal ubiquitin receptors that select such cargo and engage membrane-deforming proteins to enact each transport step. Second, these endosomal ubiquitin receptors can themselves be ubiquitinated, resulting in their auto-inhibition and thus they are switched off. Third, many membrane-deforming proteins are also ubiquitinated, often resulting in their rapid and reversible inactivation. Their coordinated ubiquitination ensures that transport reactions within the endosomal pathway are processive and efficient.Many of these ubiquitination events are driven by a family of ubiquitin ligases called Rsp5s, of which ITCH is a notable member. These ligases are located throughout the cell, so they must be closely controlled by 'adaptor' proteins to ensure that they ubiquitinate the correct targets at the right time and place. We propose that a protein called LITAF is the crucial adaptor that activates ITCH (and other Rsp5 ligases) upon the high-curvature membranes within the endosomal pathway and hence promotes the range of ubiquitination events that underpin the proper functioning of the endosomal pathway. We base our hypothesis on our findings: i) LITAF can itself support membrane curvature and is important for endosomal transport; ii) LITAF activates ITCH; iii) LITAF binds to endosomal ubiquitin receptors; iv) Depletion of LITAF and ITCH generate similar defects in membrane curvature within the endosomal pathway.Our proposal has two aims. First, we seek to understand how LITAF activates ITCH, and hence behaves as a bona fide ITCH adaptor with the potential to activate ITCH on high-curvature membranes. Second, we will identify the proteins for which LITAF promotes ITCH-dependent ubiquitination. We believe that endosomal ubiquitin receptors may be a key class of such substrates, and we will test this. We will also perform a non-biased screen to look for other LITAF clients, looking out for important components that facilitate endosomal transport steps. For a small subset of these we will test the impact of their ubiquitination.

细胞对环境的反应由细胞表面表达的数百种不同的膜蛋白决定。细胞必须不断地取样和修饰表面膜蛋白的这种补充，以去除由于环境变化而在表面不再需要的蛋白质，或替换因日常应激而受损的蛋白质。进行这一重要“质量控制”的过程是内吞作用，其中表面膜蛋白在囊泡内被内化（“内吞”）以到达细胞内区室，即内体。从那里，不再需要的膜蛋白被包装到内体内的内部囊泡中，然后将所得结构（多囊泡体）转运到降解区室（溶酶体）。或者，待再利用的表面膜蛋白在内体处包装成膜小管并返回细胞表面。内体途径的每一步都涉及为不同目的地选择货物膜蛋白的机制，与使膜变形以产生运输中间体（即囊泡和小管）的机制偶联。总之，内体途径的特征在于大量的高曲率膜，其产生转运反应。几乎所有细胞蛋白质的功能都受到翻译后修饰的调控。一个关键的修饰是泛素化（小蛋白泛素或多聚泛素链的共价连接）。泛素化广泛存在，细胞表达>1000种泛素化酶（泛素连接酶），但在胞吞过程中特别重要，它执行几项重要任务。首先，靶向溶酶体降解的膜蛋白被泛素标记，泛素被内体泛素受体识别，内体泛素受体选择这样的货物并接合膜变形蛋白以制定每个运输步骤。其次，这些内体泛素受体本身可以被泛素化，导致其自身抑制，因此它们被关闭。第三，许多膜变形蛋白也是泛素化的，通常导致它们快速和可逆的失活。它们协调的泛素化确保了内体途径内的转运反应是进行性的和有效的。许多这些泛素化事件是由一个称为Rsp 5s的泛素连接酶家族驱动的，ITCH是其中的一个著名成员。这些连接酶位于整个细胞中，因此它们必须受到“接头”蛋白的密切控制，以确保它们在正确的时间和地点泛素化正确的靶标。我们提出，一种名为LITAF的蛋白质是激活ITCH（和其他Rsp 5连接酶）在内体通路内的高曲率膜上的关键适配器，因此促进了一系列泛素化事件，这些事件支持了内体通路的正常功能。我们的假设基于我们的发现：i）LITAF本身可以支持膜曲率，并且对于内体转运很重要; ii）LITAF激活ITCH; iii）LITAF结合内体泛素受体; iv）LITAF的耗尽和ITCH在内体途径内产生类似的膜曲率缺陷。首先，我们试图了解LITAF如何激活ITCH，因此表现为真正的ITCH适配器，具有激活高曲率膜上ITCH的潜力。其次，我们将确定LITAF促进ITCH依赖性泛素化的蛋白质。我们相信，内体泛素受体可能是这类底物的一个关键类别，我们将对此进行测试。我们还将进行无偏见的筛选，以寻找其他LITAF客户端，寻找促进内体转运步骤的重要组分。对于其中的一小部分，我们将测试其泛素化的影响。