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

人体免疫系统对入侵的病原体和恶性细胞提供至关重要的防御。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有很强的炎症成分，通常需要免疫抑制治疗。移植受者也需要免疫抑制，而免疫刺激是治疗感染和癌症的相关方法。病毒是细胞内的病原体，它感染细胞并利用它们的机制进行繁殖，从而感染新细胞。它们已经进化出多种独特的策略来调节宿主免疫系统，特别是宿主传感器检测到它们后的炎症环境。了解病毒如何实现这种免疫调节，不仅能让我们了解病毒的生命周期，还能让我们了解细胞如何产生抗病毒反应并引发相关的炎症反应。这些信息在对抗病原体的斗争中是必不可少的，可以用于开发新的免疫抑制药物和模仿自然发生的微生物策略的疗法。痘病毒是一类病毒，可感染包括人类在内的多种哺乳动物物种，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，它们是生物学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。在其独特的性质中，痘病毒是唯一编码泛素系统适配器的病毒。这些病毒接头具有与其细胞等效物相似的组织，因此我们认为它们以类似的方式工作。也就是说，它们识别目标蛋白，并诱导一种被称为泛素的小蛋白附着在其上。这种泛素标记的蛋白质现在总是被细胞识别为“被破坏”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，以达到自己的目的。尽管有这些信息，目前关于痘病毒接头的作用的知识是有限的，并且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和目标泛素化后的立即降解。本研究旨在利用一种基于衬底捕获的新方法来发现这种衬底。这种方法使用的病毒接头的版本被设计为保留识别底物的能力，但失去泛素化它们的能力。这种工程版本不会降解底物，而是在细胞内捕获并稳定它们，使我们能够通过超灵敏的蛋白质组学技术识别它们。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，我们将展示这些底物如何影响痘病毒的生命周期，以及病毒接头如何通过引起它们的降解来进化以抵消这种影响。最后，我们将展示病毒接头如何在哺乳动物宿主中赋予病毒生物学优势。鉴定由痘病毒泛素接头控制的细胞底物对于理解该蛋白家族的作用至关重要，但更重要的是对细胞如何抵抗病毒感染获得新的见解。本提案中产生的数据将扩展我们对Ub系统对免疫的贡献的认识，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产生具有免疫调节特性的小肽或肽拟物。

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.