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

Lead Research Organisation: University of Surrey
Department Name: Microbial & Cellular Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Description This research grant concerns how viruses escape immune responses. The work uses two model viruses of the same family: ectromelia virus, the mousepox causative agent, and vaccinia virus, the smallpox vaccine. Both are DNA viruses that have evolved multiple mechanisms to antagonise the host immune response. First, my research team has discovered that poxviruses block the host immune response that results from the recognition of the viral DNA. This knowledge is important because it may provide clues on why some viruses are capable of causing disease whereas others (ie. vaccines) are not. Secondly, my group has discovered a new strategy used by poxviruses to exploit the cellular machinery to its advantage. This strategy may reveal key insights into how our cells operate and open new avenues for therapeutical interventions that prevent viral replication.
Exploitation Route Our discoveries may apply to other pathogens. Our results and methodologies are, or will be, published so other researchers can apply the same approaches to study other pathogenic microorganisms. Upon further work to understand the details of our discoveries, our work may lead to the development of therapeutic strategies aimed at boosting immune responses.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Asthma UK Innovation Grants
Amount £48,074 (GBP)
Funding ID AUK-IG-2016-353 
Organisation Asthma UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 06/2018
 
Title DNA sensing knock-down monocytic cells 
Description THP-1 monocytic cells depleted for the key cytosolic DNA sensing molecules cGAS and STING 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact These THP-1 cell lines allow to determine whether any inflammatory response is mediated by the STING-dependent cytosolic DNA sensing pathway that involves cGAS and STING. 
 
Title Inducible cell lines for mousepox proteins 
Description Cell lines expressing mousepox genes inducibly. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? No  
Impact These tools allow the study of the actions of specific mousepox proteins in a controlled manner since the expression of the gene remains silent until an induced is added by the operator. 
 
Description African Swine Fever Virus - INIA (Spain) 
Organisation National Institute for Agricultural and Food Research and Technology
Country Spain 
Sector Public 
PI Contribution A collaboration has been established between the group of Dr. Covadonga Alonso (INIA) and mine to study immune evasion by African Swine Fever Virus (ASFV) the causative agent of African Swine Fever. My laboratory is hosting a PhD student from INIA for a period of 14 weeks to characterise the role of one of ASFV proteins in innate immunity. My laboratory is contributing the intellectual guidance and research expertise as well as providing the infrastructure and laboratory space for conducting the work.
Collaborator Contribution The partner laboratory in INIA is contributing the manpower and the knowledge and expertise in ASFV. The PhD student is sponsored by a PhD fellowship funded by the Spanish Ministry of Education, which allowed her to successfully apply for a travel grant to work in the United Kingdom
Impact The partnership has resulted in the successful application for a Travel Grant from the Spanish Ministry of Education for the PhD student to travel to the United Kingdom and work in my laboratory for over 14 weeks.
Start Year 2016
 
Description Southern 4 Proteomics Consortium 
Organisation University of Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution We have contributed protocols and samples to perform triple SILAC (stable isotype labelling by amino acids in cell culture) proteomics, and will promote the use of the equipment available in the Proteomics Consortium to other researchers in the University of Surrey and the Pirbright Institute.
Collaborator Contribution Besides the University of Surrey, the Consortium is formed by the University of Southampton, the University of Reading and the University of Portsmouth. Equipment is based at the University of Southampton. All partners have contributed knowledge and expertise to make sure that a top notch mass spectrometer can be used at its full capacity by users from all collaborating Institutions. The University of Southampton is also contributing a research assistant providing help to new and existing users.
Impact This is a technological collaboration. Multiple scientist are benefiting from the acquired mass spectrometry capability for their respective projects in diverse disciplines such as RNA translation, plant science, cancer biology, metabolic diseases as well as innate immune activation and inflammation.
Start Year 2015
 
Description Viral evasion of DNA sensing 
Organisation University College London
Department Division of Infection and Immunity
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team has validated key reagents and tools generated by our collaborators in UCL to measure the extent of inflammation that is produced by macrophages that recognise foreign invading microorganisms via their DNA genome. We have established standard operating procedures that allow to use these tools in poxvirus and herpesvirus research
Collaborator Contribution The partner research team has created tools to measure the extent of inflammation that is produced by macrophages that recognise foreign invading microorganisms via their DNA genome. These tools were originally developed to study retroviruses, but can now be applied to multiple microorganisms.
Impact This collaboration has been generated a publication in the Journal of Virology (Georgana et al., Journal of Virology 2018 doi: 10.1128/JVI.02145-17).
Start Year 2017
 
Description Invited Seminar (FUSC, Brazil) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation on poxvirus, immunity and infection at the level of undergraduate, postgraduate and doctoral students (~70-80) as well as researchers and principal investigators. Plenty of discussion after the talk in the conference hall and later individually with the students and PIs.
Year(s) Of Engagement Activity 2017