The structure and function of SGTA, a key regulator of protein quality control

Lead Research Organisation: King's College London
Department Name: Chemistry

Abstract

Proteins are essential to life, providing important building blocks for our cells and performing numerous complex roles that maintain animal and human health and combat disease. Proteins are rather complex macromolecules, and this in turn means that making them is a complicated process made up of several stages, each of which can potentially go wrong resulting in the production of faulty or defective proteins. There are many reasons why faulty proteins might be made, including different kinds of physiological stress and mutations to the genes that encode them, but importantly the cell has developed a mechanism for checking its newly made proteins to make sure they are made correctly. This process is normally referred to as "protein quality control" and when it is operating correctly it acts to recognise and remove faulty proteins. This normal cellular quality control process is vitally important, since if faulty proteins are not dealt with quickly they have a tendency to stick to each other and clump together to form aggregates. In many cases these aggregates are toxic and can prevent cells from working properly, or even cause them to die. The misfolding and aggregation of proteins lies at the root of the prion diseases that are suffered by many animal species, including BSE and scrapie. Likewise, protein aggregates are a feature of human prion diseases, such as Creutzfeldt-Jakob disease, and neurological disorders, most notably Alzheimer's disease and Parkinson's disease. It is also suggested that one of the consequences of ageing is a reduced capacity for cellular protein quality control, and that this in turn might impact on ageing related diseases.

Misfolded membrane proteins have a strong tendency to aggregate, and mammalian cells like ours seem to have developed a fast track system to recognise this class of proteins and deal with them quickly, thereby avoiding any potential problems they might cause. We have identified a protein called SGTA which acts as a quality control factor and plays a very important role in the cells ability to deal with mislocalised membrane proteins that are misfolded because they have ended up in the wrong location with a cell. There is also evidence that SGTA is linked to certain cancers and viral infections. However, although there is a strong case that SGTA is a critical component of cellular quality control pathways, we really know very little detail about how it works, and it is precisely this question that we will answer during the course of this project. In particular, we will find out how SGTA specifically recognises mislocalised membrane proteins, and establish what it does with these aberrant proteins once they are bound, focussing on its ability to inhibit their destruction and looking directly at its role in protein aggregation. The purpose of our research is to understand the normal workings of SGTA in a healthy cell that is capable of efficiently removing mislocalised membrane proteins and avoiding a build up of protein aggregates.

Technical Summary

I am well placed to accede the overwhelming focus of protein targeting to the ER has been the fidelity and elegance of signal recognition particle dependent delivery. In contrast, efficiency was not a major concern, and hence the idea that a significant proportion of proteins destined for the ER can mislocalise to the mammalian cytosol is new. Nevertheless, the evidence for, and importance of, mislocalised proteins (MLPs) are now compelling, and a key component of the cellular pathway that deals with them is the focus of this project. MLPs are membrane and secretory protein precursors that fail to reach their intended destination and default to the cytosol. It is now apparent that MLPs are dealt with by a specific branch of the global cellular quality control network, and that SGTA plays a pivotal role in dictating their fate. SGTA acts in tandem with a second component, the BAG6 complex, forming a cycle: SGTA directs its MLP substrates towards deubiquitination and hence stabilisation; BAG6 directs them towards polyubiquitination and proteasomal degradation. Key to this pivotal role of SGTA is its capacity to recognise and bind to MLPs, yet we know little about the nature or extent of its substrate-binding region and have no detailed structural information whatsoever. Likewise the functional role of its novel N-terminal interaction motif is completely unexplored. Our major goals are therefore to obtain a complete molecular understanding of the interaction between SGTA and membrane proteins, and to define the importance of its UBL- and substrate- binding domains to the cellular quality control of MLPs. These studies will build on our recent successful studies of SGTA structure and function, and exploit a range of established tools, reagents and validated model MLPs, combined with robust in vitro and in vivo functional readouts.

Planned Impact

Protein secretion is a fundamental cellular process that is relevant in a number of spheres including biology, industry and medicine. The early stages of secretory protein production are intimately linked to the function of the endoplasmic reticulum, and our goal is to define the molecular basis of a novel pathway for the entry of proteins into the mammalian secretory pathway. The most direct economic impact of this work will be on the use of cultured mammalian cells for the production of high-value therapeutic proteins or biopharmaceuticals. The biological complexity of such components, combined with the physiological requirements of their targets, makes the use of mammalian cells the most viable platform for their production. The high cost and modest yields of such systems means that companies like Novo Nordisk have a substantial interest in trying to exploit current and emerging basic knowledge in order to enhance and improve output. Such approaches include strategies to enhance the early stages of secretory protein biogenesis at the endoplasmic reticulum by the rational engineering of cell line, and our work is directly relevant to these aspirations. In addition, there is anecdotal evidence that "premature" quality control may also reduce the yields of selected biopharmaceuticals, and our work addresses this issue directly. Pharmaceutical companies, including Novartis, have previously developed small molecules that directly inhibit the translocation of selected secretory proteins into the endoplasmic reticulum and explored their use as potential therapeutics. The outcomes of this project will provide the basis for complementary approaches that selectively inhibit the production of short secretory proteins. Our membership of the research council funded Manchester Chemical Biology Network gives us informal access to pharmaceutical companies (including AstraZeneca, GSK, Pfizer and Syngenta), providing a direct opportunity to explore and refine this strategy.

Both applicants are strongly committed to engaging with the general public about the importance of Science and the specific goals of their research. We actively participate in University and Faculty open days, and are proactive in our interactions with local schools. Hence, we have contributed to a variety of science-based activities in schools and provided mentoring to a nearby six-form college. We strongly encourage our staff and students to take part in raising the profile and understanding of science in the wider society, and in recent years our groups have participated in public events hosted by the University (Darwin Day at the Manchester Museum) and the Faculty of Life Sciences (Science Stars Days for Secondary School pupils).

Publications

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Description In this grant Professor Stephen High (Manchester University) and I (Rivka Isaacson, King's College London) were each awarded funds for PDRA, consumables and travel and have two separate grant numbers (mine is BB/L006952/1 and his is BB/L006510/1). Mine ran for 36 months and terminated at the end of February, 2017. His PDRA took a maternity break so the grant was extended for six months. These grants represent the only funding we have ever been awarded to work together so far. My lab is skilled in a range of biophysics techniques for studying protein structure and interactions (including NMR, X-ray crystallography, circular dichroism, isothermal titration calorimetry & microscale thermophoresis). The High lab has developed a vast range of in vitro and cell-based assays for identifying and characterising protein interactions and biological effects. Our symbiotic relationship feeds ideas and results into our respective work yielding a fruitful collaboration.

Our goals for the grant were to explore the ways in which cells control the quality of their crowded environment. We have determined the exact molecular shapes of protein machinery involved in this process, thereby shedding light on their mechanisms of action. Thanks to the BBSRC we have jointly authored six papers as listed on Researchfish and are currently preparing further publications. In our published work we have solved the novel structures, interactions and mechanisms of proteins that collaborate in the sorting of rogue hydrophobic substrates to their respective fates. In May 2016, our paper, in which we solved the structure of an E3 ligase interacting with quality control machinery, came out, and it has been viewed almost five thousand times and already cited nine times. It offers the first structural insight into the rubbish-labelling process for hydrophobic substrates designated for recycling. In our latest publication we have identified and characterised a pair of molecular tweezers that can grab unusable molecules in the cell environment and target them towards repair or recycling.

Overall our joint BBSRC grant has greatly facilitated our successful collaboration. We have answered many questions and are at the forefront of discovery in the chaperone-mediated processing of mislocalised hydrophobic proteins in the cell cytoplasm. Prof. Stephen High is the now the recipient of a Wellcome Trust Investigator Award, entitled 'The quality control of mislocalised membrane and secretory proteins' which is a direct result of the data we were able to gather in this grant. This means that the cell-biology side of our collaboration is funded for the next five years. In order to continue our productive collaboration, and drive forward molecular level insights into protein quality control, I have applied for several grants, none of which have yet been funded, but I am hopeful that our recent publication, plus those in process will enable us to raise further funding to take this project forward from the biophysics/structure perspective.
Exploitation Route The structures we have solved might be used in rational drug design or to predict other structures or to design mutations that will test functional hypotheses.
Sectors Chemicals,Education

 
Description project grant
Amount £349,039 (GBP)
Funding ID BB/N006267/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2016 
End 06/2019
 
Description Collaboration with Prof. Blanche Schwappach, Göttingen University, Germany 
Organisation University of Göttingen
Department Medical School
Country Germany 
Sector Academic/University 
PI Contribution We have jointly embarked on studies of steroid hormone receptor interactions with SGTA - we perform the structural and biophysics experiments.
Collaborator Contribution Her lab performs in-cell assays such as measuring effects of mutations on a transcriptional reporter.
Impact We are currently preparing a manuscript and have written a grant application to the Medical Research Council to take this research further.
Start Year 2016
 
Description TA Protein Insertion 
Organisation University of Manchester
Department Faculty of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have solved two protein structures (one by NMR and one by X-ray crystallography) which have led to the design of functional mutants. UPDATE 2014 - many further structural and biophysical studies are underway UPDATE 2016 - we have had numerous meetings and submitted a further manuscript which is currently in the review process - we are also starting to plan our next steps with regard to pursuing further funding to continue our work
Collaborator Contribution Biological testing of structure-driven functional hypotheses - this is interdisciplinary as it uses in vivo and in vitro techniques to which we do not have access.
Impact We are currently writing up two papers which combine our structural expertise with their functional testing which was driven by our results. We originally cloned our proteins using their reagents and have fed back our results for design of mutants. This is multidisciplinary in combining our biophysics skills with their skills in yeast genetics and cell biology. UPDATE in 2013: These papers are now published in PNAS and PLoS One. UPDATE in 2014 - We now have a third paper accepted to PLoS ONE which is due for publication on 21/11/14 - this collaboration has also resulted in our current joint award from the BBSRC UPDATE 2016: We have a new publication with another in review and several others in preparation.
Start Year 2010
 
Description Viewing the Invisible 
Organisation London Fine Art Studios
Country United Kingdom 
Sector Private 
PI Contribution The collaboration is at an early stage but we have recently secured funding from the BBSRC to go forward with the project. It will explore and demythologise common methodology/ways of thinking between Arts and Sciences and produce a touring multimedia exhibition involving videos and portraits of scientists.
Collaborator Contribution Our partners are portrait painters trained in traditional methods. They bring this expertise along with a wealth of experience in curating exhibitions, working with partners in other fields etc.
Impact None yet - we have had several meetings - the project is still in planning stages
Start Year 2015
 
Description Christmas Lecture 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Talk prompted interesting questions and discussion and the event was recorded and has been watched by several hundred extra people via the internet.

We believe this event encouraged school children to choose science subjects.
Year(s) Of Engagement Activity 2013
URL https://www.youtube.com/watch?v=Hu6utI-9H-Y
 
Description Interview on The Science Show 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was interviewed about my research by Robyn Williams for The Science Show which is broadcast to a large listening public by the Australian Broadcasting Corporation (ABC)
Year(s) Of Engagement Activity 2017
URL http://www.abc.net.au/radionational/programs/scienceshow/misshaped-proteins-the-cause-of-many-diseas...
 
Description K+ career speed-networking 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I took part in a speed-networking careers event as part of K+ which is the KCL widening-participation programme for BAME and/or underprivileged local sixth-formers.
Year(s) Of Engagement Activity 2016
URL http://www.kcl.ac.uk/study/widening-participation/our-activities/k-plus/index.aspx
 
Description Living Streets careers panel 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I was the 'scientist' in a careers panel discussion for primary school children who had won awards for designing badges for Walk to Work day - a campaign from the charity, Living streets. There was lively discussion about the pros and cons of being a scientist and many of the children expressed interest in scientific careers.

I received positive feedback from the organisation who said the children had very much enjoyed talking to a real scientist and were surprised at how normal I seemed.
Year(s) Of Engagement Activity 2014
URL http://www.livingstreets.org.uk/walk-with-us/walk-to-school/primary-schools/walk-once-a-week/badge-c...
 
Description National Portrait Gallery Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact For International Women's Day, I delivered one of the National Portrait Gallery's lunchtime lectures. These occur every other Thursday and are ticketed events open to the general public. I looked at the pioneering work of female structural biologists in the Gallery's Collection including Rosalind Franklin, Dorothy Hodgkin, Dame Louise Johnson, Dame Kathleen Lonsdale and Olga Kennard, explaining my science as I went along. There was a large audience and an interesting question and answer session afterwards.
Year(s) Of Engagement Activity 2017
URL http://www.npg.org.uk/whatson/event-root/march/lunchtime-lecture-02032017#.WK-LgvRBaWk.facebook
 
Description Olga Kennard interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact I interviewed pioneering female X-ray crystallographer, Olga Kennard (who is 93) and wrote about it in Times Higher Education.
Year(s) Of Engagement Activity 2017
URL https://www.timeshighereducation.com/blog/international-womens-day-meeting-olga-kennard
 
Description PDB Europe Art Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I participated in a training event in which we planned projects with teachers to use protein structures in art classes.
Year(s) Of Engagement Activity 2018
 
Description STEM from the City Balloon Debate 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I was competing against 3 other speakers in a Balloon Debate defending the merits of biological research with examples from my own projects and experience. I won the debate i.e. I was the only one left in the metaphorical balloon and not voted off. The event was introduced by the Lord Mayor of London and then driven by the hundreds of 13 year-old school children who asked many questions and voted off my competitors. The event stimulated endless discussions about the career options that would be made possible by doing science A-levels.

The feedback from this event was unanimously positive and we have anecdotal evidence that the pupils (who were from inner city schools) expressed increased interest in pursuing science subjects.
Year(s) Of Engagement Activity 2014
URL http://www.sciencecouncil.org/content/london-schools-experience-diversity-science-and-engineering-fu...
 
Description School Visit (Seven Kings, Essex) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I visited Seven Kings School for their 'Women in Science' day and gave the same presentation three times to different groups of ~25 female sixth-formers about my career so far and work-life balance. I received positive feedback from students and teachers and my visit inspired the school to organise a trip to the HWB-NMR national facility in Birmingham.
Year(s) Of Engagement Activity 2016
 
Description Shadow a Scientist 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact My post-doc, Katherine Collins, and two Ph.D. students, Ewelina Krysztofinska and Nicola Evans, volunteered to introduce school age students to the lab with Scientist & co, Shadow a Scientist programme and also helped them with their CVs afterwards.
Year(s) Of Engagement Activity 2016
URL http://www.scientistsandco.org/projects/shadow-scientist/
 
Description Woman's Hour 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact I was invited to appear on BBC Radio 4 Woman's Hour as a 'game-changer' in science and technology. I was interviewed by Jane Garvey about my career. It was a thoroughly stimulating experience which prompted much discussion and positive feedback.

There were several positive repercussions from this interview all of which have had a positive impact on admissions to our department and encouraging people to engage with science: The head of Natural and Mathematical Sciences at King's blogged about me. I've been invited to give talks about my work at three universities and to examine someone's Ph.D. At the University of East Anglia. I was contacted by various scientists about potentially interesting collaborations, one using a new technique I've been keen to try. I am now co-applicant on a grant application to the BBSRC to obtain some new machines for our department. I have also gathered quite a few new twitter followers - which another way I spread the word about our research.
Year(s) Of Engagement Activity 2014
URL http://www.bbc.co.uk/programmes/b042cq8h