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

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.

Protein ubiquitination is a crucial post-translational modification performed by hundreds of E3 ubiquitin (Ub) ligases. Cullin-RING Ub ligases (CRL) are a group of E3 ligases that utilise specific substrate adaptors (SAs) to select proteins for ubiquitination. Such specificity makes CRLs a very attractive target for viral manipulation and indeed this has been reported for various viral families. One of these families, the poxvirus family, has evolved a unique method to use cellular ubiquitination to their advantage by encoding adaptors of CRLs. These viral SAs are analogous to their cellular counterparts: they recognise substrates using dedicated protein-protein interaction domains and bridge them to CRLs via N-terminal F-box domains. Despite experimental evidence proving that viral SAs bind cellular CRLs, their functions remain unknown.

To understand the role of poxvirus SAs, one needs to identify the substrates that they recruit for ubiquitination. Presently this constitutes a major challenge due to the immediate degradation that follows recognition and the necessity of ultrasensitive proteomics techniques. I have recently developed a novel substrate-trapping proteomics approach that has the capacity of identifying substrates controlled by viral SAs. This system is based on unbiased stable isotope labelling in cell culture (SILAC) proteomics to compare the interactomes of wild-type vs mutant SAs that lack the F-box domain. These mutants still bind their cognate substrates but fail to engage CRL complexes, thus causing substrate accumulation and allowing mass spectrometry identification. The goal of this project is to identify the substrates controlled by viral SAs, characterise their anti-viral roles in cells and understand how poxviruses have evolved to target them using specific viral SAs. Such information will be instrumental to understand this unique mechanism of cellular manipulation and to discover novel anti-viral strategies employed by cells.

This study will have an overarching benefit on public health, as well as direct benefits for the private sector in the following ways -
(1) Smallpox and poxvirus zoonosis. Bioterrorism is a concern in western countries. The National Institute of Allergy and Infectious Diseases (NIAID) receives about $1.6 billion each year for biodefence research. Variola virus (VARV), the causative agent of smallpox, is considered a Category A Biothreat Agent. There are no vaccination programs in place after the eradication of smallpox and a vast majority of the population has never encountered VARV or vaccinia virus. This means that a potential bioterrorist attack could have catastrophic consequences given the VARV mortality rate. At the same time, given the successful eradication, no data is available for smallpox infections in humans and for their therapeutic treatment. Acknowledging this problem, the Food and Drug Administration approved in 2002 the Animal Efficacy Rule. This rule allows the use of well-controlled animal efficacy data to support the licensure of drugs and biologicals to treat or prevent smallpox. Our work with ectromelia virus, the most appropriate model for human smallpox and monkeypox, will thus contribute to our understanding of smallpox pathogenesis and of poxvirus host range and the emergence of zoonotic outbreaks.
(2) Infectious diseases. Cellular targets of viral proteins discovered in this project can have anti-viral properties against other viral diseases. Therapies enhancing the activity of these novel cellular components represent potential tools to counteract infectious diseases, particularly those where the development of potent vaccines is difficult. For instance, of the 8 human herpes viruses, an effective vaccine is currently available only for varicella zoster virus, the cause of chickenpox and shingles. Similarly, no vaccine exists for respiratory syncytial virus, an agent responsible for 18,000-75,000 hospitalisations and 90-1,900 deaths in US annually.
(3) Immunomodulation. The global market for immunomodulators was worth $80.8 billion in 2011, where the North American and European market were worth $40 and $21 billion respectively. Our work has the potential to discover new proteins that have roles in the immune system and have immunomodulatory capacity, and therefore represent novel potential drug to target worldwide pathologies such as autoimmune and inflammatory disorders. Traditional treatments for these disorders are inadequate. For instance, glucocorticoids are very potent and widely used drugs for inflammatory disorders that have estimated 1-2% prevalence worldwide such as rheumatoid arthritis, inflammatory bowel disease or asthma. However, they mediate complex side-effects that include diabetes, glaucoma and osteoporosis. This is particularly disturbing for these lifelong diseases that usually require long-term medication. There is therefore a medical need for innovative drugs.
(4) Cancer. The pharmaceutical industry is highly interested in the development of new therapeutics affecting ubiquitination to treat cancer conditions. For instance, bortemozib (VelcadeTM), a pan-inhibitor of the proteasome, has been approved and successfully applied for multiple myeloma and relapsed mantle-cell lymphoma; and MLN4924, the first-in-class small-molecule inhibitor of neddylation, holds promise as a treatment of non-Hodgkin lymphoma or elapsed and/or refractory multiple myeloma. Despite these efforts, the currently available anti-cancer drugs that target the proteasome display excessive toxicity caused by general inhibition of protein degradation. It is envisaged in the field that specific inhibitors of ubiquitin ligases can overcome this problem as they will only cause dysregulation of specific substrates controlled by that particular ligase. Our studies with viral adaptors of the ubiquitin system will serve as a model for this hypothesis and may provide data applicable to their cellular homologs.