Publications
A complete list of publications from my group can be found at this link: https://pubmed.ncbi.nlm.nih.gov/?term=peden+aa&sort=date
Select publications from the lab
We started this work at the height of the pandemic before there were readily available tests for following infection. We realised that the constructs we had been using to study SARS-CoV-2 biology could also be used for performing serological tests by fluorescence microscopy. Using this approach we have been able to show that there is a significant immune response to the M protein of SARS-CoV-2 which may be useful for performing large sero surveys. More importantly, this approach can be used to rapidly idenetify the most immunogenic componets of emerging pathagoens so poteitally help with pandemic preparedness.
In this study we worked as part of a large international consortium to identify host proteins and pathways hijacked by SARS-CoV-2 and other related coronaviruses with the aim to identify novel druggable targets. The main contribution we made to this study was analysing the intracellular localisation of the tagged viral proteins (CoV-1, CoV-2 and MERS). In addition, we also validated all the CoV-2 antibodies which were used to identify where the viral proteins were localised during infection. Our final contribution to the paper was the identification that tagged Orf9B from CoV-1 and 2 causes a reduction in the levels of TOM70 a key player in the host interferon response. One aspect of this project we would like to explore further is how the biosynthetic pathway is remolded by the viral proteins to promote viral egress.
In this study we made use of our pharmacologically regulated secretary reporter to determine which R-SNAREs are required for constitutive secretion. To overcome the issues we faced in mammalian cells we performed the experiments in drosophila S2 cells which have a fewer number of post-Golgi SNAREs. Through our functional genomic experiments we identified a strong genetic interaction between VAMP3 and Ykt6. One possible interpretation of this data is that there are two parallel pathways to the cell surface which may help explain why it has been so difficult to identify machinery which acts in this part of the pathway.
In this paper we characterised the mechanism by which STX19 is targeted to post-Golgi membranes. In this study we showed that STX19 is S-acylated (palmitoylated) at a cysteine rich domain at its C-terminus. As we are not experts in this field so we collaborated with the labs of Mark Collins and Luke Chamberlain to perform all the work related to S-acylation. One of the key findings is that the cysteine rich domain of STX19 is very efficiently targeted to tubular recycling endosomes which are defined by Rab8 and MICAL-L1. In addition, we also found that palmitoylation plays a key role in regulating the stability of STX19.
This project was performed in collaboration with the Davletov lab. In this study we developed a simple ELISA based method for measuring the activity of BoNT/B. This platform measures all the key steps of Botulinum intoxication so potentially can be used to replace animal testing for Botulinum toxins. We have continued this work supported from a grant from the NC3Rs are in the process of exploring the commercial potential of this work.
As a PhD student I was involved in cloning and characterising the function of the AP-3 complex in the lab of Scottie Robinson. This project directly builds on the expertise I developed in studying the biology of the AP-3 complex as a PhD student. This work was a joint collaboration between myself, Paul Luzio and David Owen. My lab performed all the cell biology for this paper and showed that in the absence of AP-3, VAMP7 fails to be targeted to late endosomes/lysosomes and becomes miss-localised to TGN.
This work was performed in collaboration with Scottie Robinson and David Owen. This study we directly builds on the EMBO Reports paper and uses biochemical and structural approaches to provide a molecular and mechanistic explanation of how CALM (PICALM) packages VAMPs 2, 3 and 8 into clathrin coated vesicles. The biochemical studies show that the R-SNAREs can only interact with CALM when they are not in a SNARE complex. Interestingly, the localisation of SNAREs which are unable to form SNAREs complexes is somewhat different to the wild type proteins suggesting that SNARE complex formation plays some role in regulating the trafficking of SNAREs.
In this study we describe the development and application of a completely novel assay for measuring constitutive secretion. The assay uses a pharmacologically regulated secretory reporter that can be followed optically. In this assay secretion is simply measure by measuring the fluorescence intensity of the cells before and after secretion. Thus, the assay is very easy to use and does not require complex normalisations for cell number. One of the really surprising results from this study was that depletion of VAMPs 3, 4, 7 and 8 in conjunction does not block constitutive secretion. It is possible that the lack of a phenotype simply reflects a technical limitation f RNAi when used to study SNAREs. However, we feel that this is unlikely as we observed very potent block in secretion when we depleted SNAREs which function between the ER and Golgi. At the time we speculated that YKT6 maybe playing an unexpected role in this process.
This was the first paper from the lab. In this study we developed a system which allowed the endocytic trafficking of SNAREs to be followed by microscopy and flow cytometry. This project was built on the observation that VAMP4 can still be interanlised even when its dileucine motif has been disrupted. Our initial interpretation of our results was incorrect and the endocytosis of VAMPs 3 and 8 is predominantly driven through an interaction with PICALM and AP180 (see Cell reference) . However, the observations and approaches developed in this paper underpin the work we published in Cell with David Owen and Scottie Robinson.
Select publications from my PhD and postdoc
When I left Scotties lab to do my postdoc it was still unclear where the AP-3 complex was localised in cells. It was unclear whether it was budding from the TGN or endosomes. In Richard's lab it was quite common place to make monoclonal antibodies so I decided to make the first monoclonal antibodies against the delta subit of the AP-3 complex. In collaboration with Judith Kluperman we used these antibodies to localise the AP-3 complex in non-neuronal cells via immuno-EM. We found the AP-3 complex was localised to tubular structures which emanate from endosomes. In addition, I also showed that there was significantly more endocytic recycling of LAMP1 and CD63 in AP-3 deficient cells.
When I was a PhD student in Scotties lab I thought it would be cool to try and understand how SNARE proteins are packaged into vesicles. Through the support of the Wellcome trust I had the opportunity to work on this idea in Richard Scheller's lab. This paper is a direct output from my Wellcome funding and was the first paper to idetify the mechanism by which a post-Golgi SNARE is packaged into a vesicle. Interestingly, it turns out that most post-Golgi SNAREs don't used classical sorting signals but use cargo specif adaptor proteins such as PICALM. At present it is unclear why VAMP4 uses a classical sorting signal which competes with regular cargo.
This was my first paper from Richard Sheller's lab at Stanford University. In this paper we used basic bioinformatic tools to try and identify all of the rabs, SNAREs and coat proteins encoded in the first draft of the human genome. At the time we felt that most of the coat machinery had been identified. However, this turned out not to be the case as more sophisticated structure based alignment tools were developed that allowed the identification of the AP-5 adaptor complex!
After we published the cDNA sequences for AP-3, Scottie was contacted by two mouse geneticists who were working on mouse models of Hermansky-Pudlak syndrome a disorder characterised by pigmentation defects and prolonged bleeding (Dick Swank and Margit Burmeister). In collaboration with these labs we showed that mocha mouse was caused by mutation in the delta subunit and the pearl mouse was caused by mutations in beta3A. Around the same time Esteban Dell 'Angelica in Juan Bonifacino's group showed that loss of AP-3 perturbs the trafficking of lysosomal proteins. In addition, they also went to identify patients who have mutations in the beta3A subunit of the complex.
This was my first paper from my PhD in Scottie Robinson's lab. This work was done in collaboration with Fiona Simpson who was also a PhD student in the group. At the time we were in strong competition with several labs who were also working to characterise the function of the complex. In the end, I became good friends with several of the scientists from the other labs and ended up collaborating with them on future projects. In this paper we showed that the delta and beta3A subunits are part of the AP-3 complex and loss of the delta subunit in flies leads to a pigmentation defect. My main contribution to this project was cloning the beta3A and delta subunits. I also showed that these proteins form a complex with mu3A and sigma3A/B. I don't think any of the people involved in this work had any idea of the impact of this work would have in the future.