Enabling tools for proteomics
Our laboratory improves interaction proteomics through the development of experimental and computational approaches.
Sample quality is key to interaction proteomics success, and the choice of the expression system, epitope tag, and purification protocol can greatly influence outcome. Our lab has developed robust protocols for the purification of interactors using epitope-tagged approaches (some of the protocols are specific to specific classes of proteins such as chromatin-associated proteins). We also implemented the BioID in vivo biotinylation approach first introduced by Kyle Roux which enables us to probe interactions which were previously very difficult to detect (e.g. with membrane-associated proteins). Please visit our "resources" section for more information, detailed protocols and lists of plasmids.
A key factor in the success of interaction proteomics experiments is the ability to discriminate true interaction partners from background contaminants. Many excellent methods exist which utilize high stringency purification procedures to limit background, or employ isotope-based quantitative proteomics approaches to assist in the identification of true interactors. However, such approaches may not always be feasible or desirable and we perform single affinity purification coupled to mass spectrometry from unlabeled cells. Since background contaminants are generally more abundant in these cases, we are developing computational tools to allow us to identify true interaction partners. Together with Mike Tyers, we released the software ProHits to help other groups managing their interaction proteomics data. We also developed with Alexey Nesvizhskii and Hyungwon Choi the SAINT (Significance Analysis of INTeractome) series of algorithms that provide confidence scores for each detected interaction (see Resources), and recently introduced the Contaminant Repository for Affinity Purification to enable researchers worldwide to filter their data. We recently introduced a web-tool facilitating visualization of SAINT-filtered data. While we deposit our own raw mass spectrometry data at MassIVE, and our filtered interaction proteomics data both at IntAct and in the BioGRID, we have also introduced a data-rich interaction repository for our own data, available at prohits-web.
Together with Steve Tate and his group at AB-SCIEX, we adapted the quantitative approach SWATH to interaction proteomics (see our publication in Nature Methods) and are now using this strategy to systematically investigate the consequences on interactions, of the introduction of disease-associated mutations in the coding sequence of a protein.
While we are probably best known for our work on serine/threonine phosphatases (see next section), our research group has grown a lot recently, and new our members are interested in several regulatory mechanisms, which we are investigating systematically, in large part through interaction proteomics.
Postdoctoral fellow Amber Couzens has been using a variety of interaction proteomics approaches to systematically map the interactome of the Hippo signaling pathway in mammalian cells, leading her to uncover several phospho-recognition events.
Lab technician Zhen-Yuan Lin and mass spec coordinator Brett Larsen recently teamed up with postdoctoral fellow Mikko Taipale, from the Lindquist group, to determine how the human HSP90 machinery acquires specificity through its co-chaperones (the manuscript just came out in Cell).
Postdoctoral fellow Jean-Philippe Lambert (currently supported by a TD bank fellowship) is systematically investigating the interactome of bromodomain-associated proteins, in collaboration with Panagis Filippakopoulos and Stefan Knapp at the Structural Genomics Consortium in Oxford.
Postdoctoral fellow Payman Samavarchi-Tehrani is interested in the interactome of transcription factors during differentiation, and actively collaborates with the group of Michael Wilson at SickKids.
Postdoctoral Geoff Hesketh (whose first postdoc paper just came out in Dev Cell!) is using in vivo biotinylation coupled to mass spectrometry to unravel the endosomal trafficking pathway.
Lab technician Wade Dunham is the main force behind our studies of p bodies and stress granules, a project which just got funded through NSERC.
And last but not least, PhD student Chris Go is generating a physical map of a human cell using BioID-MS, in collaboration with the Raught group at the Princess Margaret Cancer Center.
While the activities of kinases and phosphatases are both required for controlling cell growth and proliferation, the study of phosphatases has generally lagged behind that of kinases, resulting in a lopsided view of signal transduction.
Our lab has undertaken systematic approaches to study the roughly 150 human phosphatases through systematic mapping of their interactions and through functional screenings (RNA interference and microscopy). We are also performing detailed (high density) mapping of the interactions for several serine/threonine phosphatases. These studies which combine physical interactions with functional outcomes are led by CIHR-sponsored postdoctoral fellow Nicole St-Denis in close collaboration with the Pelletier lab.
Genes encoding serine/threonine phosphatase catalytic subunits are ten times less abundant than genes corresponding to the serine/threonine kinases. Phosphatase specificity is conferred by the association of catalytic subunits with other proteins that provide regulatory or targeting functions. Cataloging these associating proteins is key to deciphering the function of the enzymes. Postdoctoral fellows Amber Couzens and Linda McBroom-Cerajewski are particularly interested in studying the PP2A-type enzymes (PP2A, PP4 and PP6), whose misregulation has been linked to several pathological conditions, including cancer. MSc student Yiwang Zhou is using affinity purification coupled to SWATH mass spectrometry for probing the function of these enzymes in health and disease by systematically monitoring the interactions formed by the wild type proteins and those associated with the disease variants.
PP2A, STRIPAK and cerebral cavernous malformations
In 2008-2009, lab manager Marilyn Goudreault reported on the discovery of a novel large protein complex, which we termed STRIPAK, for STRiatin Interacting Phosphatase And Kinase, which contains both the PP2A phosphatase (and regulatory subunit striatin) and a Ste20 kinase. Importantly, we also found that the protein CCM3 is a component of STRIPAK.
Composition of a STRIPAK complex. The previously-identified PP2A holoenzyme is shown in green. Kinases of the GCK-III family of serine/threonine kinases are part of STRIPAK, which also contains CCM3, and several less well-characterized proteins (see Goudreault et al., Mol Cell Proteomics 2009, for details).
CCM3 is one of three genes mutated in familial cases of cerebral cavernous malformations. CCMs are brain "caverns", in which blood accumulates due to leaky capillaries. CCMs can be asymptomatic, or patients may present with a wide array of symptoms, ranging from headaches to seizures and cerebral
hemorrhage. While there are treatment options to reduce the symptoms (such as epilepsy), there is currently no known cure for this disease. More information on CCMs can be found at the Angioma Alliance.
Imaging of a patient brain showing a large CCM. Image courtesy of Dr. M. Gunel.
Postdoctoral fellow James Knight aims to better understand the molecular function of CCM3 within STRIPAK, and to better characterize the molecular causes of cerebral cavernous malformations, in collaboration with several of our Toronto colleagues:
Brent Derry uses the worm C. elegans as a genetic model for CCM disease.
Frank Sicheri is a structural biologist with expertise in signaling complexes, and is working on understanding how STRIPAK is assembled.
Ian Scott uses zebrafish as a model to determine how cardiovascular development is modulated by CCM proteins and STRIPAK.
Sources of support
We are grateful to the following agencies for supporting the projects in the Gingras lab and the LTRI proteomics group:
Canadian Institutes of Health Research
Natural Sciences and Engineering Research Council
Canadian Cancer Society Research Institute
Cancer Research Society
Ontario Genomics Institute
Canada Foundation for Innovation
National Institutes of Health
CIHR Institute of Genetics
Canada Research Chair