A novel substrate-trapping proteomics approach to elucidate the role of viral adaptors of the ubiquitin system

人类的免疫系统对入侵的病原体和恶性细胞提供了重要的防御。免疫反应不足，如过度的免疫刺激，可导致或促成自身免疫病理、炎症和过敏。牛皮癣、多发性硬化症、炎症性肠病和哮喘等疾病都有很强的炎症成分，通常需要免疫抑制治疗。移植受者还需要免疫抑制，而免疫刺激是治疗感染和癌症的一种相关方法。病毒是细胞内的病原体，它感染细胞，并利用其机制繁殖，从而感染新细胞。它们进化出多种独特的策略来调节宿主免疫系统，特别是在宿主传感器检测到它们之后的炎症环境。了解病毒是如何实现这种免疫调节的，不仅教会了我们关于病毒生命周期的知识，还教会了细胞如何启动抗病毒反应并触发相关的炎症反应。这些信息在对抗病原体方面是必不可少的，并可用于开发新的免疫抑制药物和疗法，模仿自然发生的微生物策略。痘病毒是一类病毒，可感染包括人类在内的多种哺乳动物，并将其基因组的30%-50%用于合成影响宿主免疫反应的分子。因此，它们是生物上强大分子的独特来源，它们的研究已经产生了关于病毒生命周期和宿主对它们发起的免疫反应的大量知识。在其独特的特性中，痘病毒是唯一编码泛素系统适配子的病毒。这些病毒接头的组织结构类似于它们的细胞等价物，因此我们认为它们的工作方式类似。也就是说，它们识别目标蛋白，并诱导一种名为泛素的小蛋白附着在目标蛋白上。这种泛素标记的蛋白质现在总是被细胞识别为“待销毁”的信号。这种优雅的机制被病毒利用来触发细胞蛋白质的降解以实现自身的利益。尽管有这些信息，但目前关于痘病毒适配器作用的知识有限，被降解的细胞底物的身份仍然难以捉摸。这主要是由于缺乏适当的技术，以及目标泛素化后立即降解。这项研究旨在使用一种基于底物捕获的新方法来发现这种底物。这种方法使用了不同版本的病毒适配器，这些适配器被设计成保留识别底物的能力，但失去了泛化它们的能力。这样的工程版本不会降解底物，而是将它们捕获并稳定在细胞内，使我们能够通过超灵敏的蛋白质组学技术来识别它们。我们的目标是发现这些底物，并描述它们在细胞中的作用。随后，我们将演示这些底物如何影响痘病毒的生命周期，以及病毒适配器如何进化以通过导致它们的降解来抵消这种影响。最后，我们将展示病毒适配器如何在哺乳动物宿主中赋予病毒生物学优势。鉴定由痘病毒泛素适配器控制的细胞底物对于理解这一蛋白质家族的作用至关重要，但更重要的是对细胞如何对抗病毒感染有新的见解。这项提案中产生的数据将扩大我们对Ub系统对免疫的贡献的知识，并将在抗病毒免疫反应中提供新的细胞参与者。这一知识可能反映在具有免疫调节特性的小肽或多肽模拟物的产生中。

The human immune system provides vital defences against invading pathogens and malignant cells. Inadequate immune responses such as excessive immune stimulation can lead or contribute to autoimmune pathologies, inflammation and allergies. Conditions such as psoriasis, multiple sclerosis, inflammatory bowel disease and asthma have strong inflammatory components and usually require immunosuppressive treatments. Transplant recipients also require immunosuppression, whereas immunostimulation is a relevant approach for infections and cancer.Viruses are intracellular pathogens that infect cells and utilise their machinery to multiply and so infect new cells. They have evolved multiple and unique strategies to modulate the host immune system, in particular the inflammatory environment that follows their detection by host sensors. Understanding how viruses achieve such immunomodulation not only teaches us about the virus life cycle, but also about how cells mount an anti-viral response and trigger associated inflammatory responses. Such information is essential in the fight against the pathogen and can be used in the development of new immunosuppressive drugs and therapies that mimic naturally-occurring microbial strategies.Poxviruses are a family of viruses that infect a wide range of mammalian species including humans, and dedicate 30-50% of their genome to synthesize molecules that affect the host immune response. As such they are a unique source of biologically powerful molecules and their study has already generated immense knowledge about the life cycle of viruses and the immune responses launched against them by the host. Amongst their unique properties, poxviruses are the only viruses encoding adaptors of the ubiquitin system. These viral adaptors have a similar organisation to their cellular equivalents and as such we believe they work in a similar manner. That is, they recognise the target protein and induce the attachment of a small protein known as ubiquitin onto it. This ubiquitin-tagged protein is now invariably recognised by the cell as a 'to-be-destroyed' signal. Such elegant mechanism is exploited by the virus to trigger degradation of cellular proteins for its own benefit.Despite this information, current knowledge regarding the role of poxvirus adaptors is limited and the identity of the cellular substrates that are degraded remains elusive. This is mainly due to the lack of appropriate techniques and the immediate degradation that follows ubiquitination of the target. This study aims at discovering such substrates using a novel methodology based on substrate trapping. This methodology uses versions of the viral adaptors that are engineered to retain the ability to recognise substrates but to lose the ability to ubiquitinate them. Such engineered versions do not degrade substrates, and rather trap and stabilise them inside cells, allowing us to identify them by ultrasensitive proteomics techniques. We aim to discover these substrates and characterise their roles in cells. Subsequently, we will demonstrate how these substrates affect the life cycle of poxviruses and how viral adaptors have evolved to counteract this by causing their degradation. Finally, we will show how viral adaptors confer a biological advantage to the virus in a mammalian host.Identification of the cellular substrates controlled by poxvirus ubiquitin adaptors is crucial to understand the role of this family of proteins, but more importantly to gain new insight into how cells fight viral infections. Data generated in this proposal will expand our knowledge on the contribution of the Ub system to immunity and will provide new cellular players in the anti-viral immune response. This knowledge might reflect in the generation of small peptide or peptidomimetics with immunomodulatory properties.