SMITH LABORATORY
cancer signalling & structural biology
Funding
OUR RESEARCH
Philosophy
Our research philosophy is inclined towards data-driven approaches that lead to directed analyses of key, disease-driven questions. We aim to advance novel biophysical, structural and cell-based approaches to quantify and characterize molecular mechanisms and integrated signalling causative of human disease, with particular emphasis on cellular transformation, metastasis and the discovery of new therapeutic targets for modulation by small molecules.
Tools
We take a very multidisciplinary approach to our work, and this also forms the core of the IRIC research model. "The emergence of highly sophisticated technologies and new scientific disciplines (genomics, proteomics, bioinformatics, etc.) has rapidly accelerated biomedical research and led to the birth of integrated systems biology." Using such tools, we now consider all components of a biological process concurrently rather than as single components in isolation. In the Smith lab, we integrate modern experimental approaches in structural biology, biophysics, evolutionary bioinformatics, and cell biology to achieve this goal (click images below for further descriptions). We further aim to be highly collaborative with biologists, clinicians, geneticists and others to gain comprehensive insights to the systems we study. If you are interested in collaboration please don’t hesitate to send us a message: smithlabiric@gmail.com
We can use NMR to observe labelled proteins in physiological mixtures or even in cells. This allows real-time studies of protein structure, dynamics or enzymatic activity in environments such as cell extracts.
We define protein-protein interaction sites using a variety of tools, including binding to small peptide libraries derived from longer protein sequences.
We study interactions that typically occur on cellular membranes using nanodiscs, small discoidal lipoprotein complexes (Image: M. Mazhab-Jafari).
We use a variety of biophysical techniques, such as isothermal titration calorimetry (ITC), to study detailed properties of protein-ligand interactions.
We strongly believe in the power of bioinformatics and genomics to uncover meaningful relationships between protein partners and networks.
We explore the impact of disease mutations at a mechanistic level by correlating changes in protein structure, dynamics, or ligand-binding with cellular assays.
We determine protein structures using either crystallography or NMR, and use these data in a variety of follow-up studies.
We exploit the power of NMR to observe labelled proteins in complex mixtures, allowing real-time observations of enzymatic and regulatory activities.
Epigenetic Activation and Leukemia
Several rearrangements of the mixed-lineage leukemia (MLL) gene result in acute leukemia’s in both children and adults, cancers which currently lack therapeutic options. The RAS effector AF6 (also MLLT4, afadin or canoe) is one of six common protein partners (inset), and efficacy of its fusion with the epigenetic regulator MLL was narrowed to its N-terminal RBD domain. Using a crystal structure of this domain, along with biophysical, proteomic, cell-based, and genomic data we can provide a remarkable picture of how this fusion protein functions in leukemogenic transformation. Our work reveals an evolutionarily conserved mechanism exhibiting commonality with many other MLL fusion partners at a protein function level. Our subsequent studies look to characterize MLL structure-function related to its epigenetic activation by fusion partners, and further exploration of shared binding partners discovered using cutting-edge proteomic techniques (in collaboration with Dr. Anne-Claude Gingras). This has proven an excellent example of how a discovery-based approach provides an opportunity to study specific disease-associated anomalies.
Quantitative Signalling and Rewiring
Our current understanding of complex signalling networks is limited by a shortage of quantitative data describing outputs to numerous pathways. We thus developed an NMR ‘competition’ approach to study multiple protein binding partners in parallel, providing a new and powerful tool to dissect biological interactions in a more quantitative fashion. Using such an approach, we have defined a
‘hierarchy’ of RAS binding domains (inset), and revealed that RAS G12V mutants signal differentially to downstream effectors. Thus, quantitative data expose pathways to serve as new therapeutic targets. We are now exploiting this competition-based design to rewire signalling, by coupling NMR assays with computation, mutagenesis, cell biology, and eventually compound screening. For specific RAS mutants having distinct properties in tumourigenesis (via purported differential activation of downstream signalling pathways), this strategy represents a patient-specific therapeutic approach that is much needed to improve overall survival rates. We also intend to expand this approach to the study of other signalling lacking quantitation (i.e. phospho-sites, nucleic acids binding).
RAS Effector Proteins and Adhesion
RAS stimulates a variety of effector proteins including RAF kinases, PI3-kinases, PLCε, RIN1, and TIAM1. In the absence of RAS-specific drugs, the most valuable therapies currently available for RAS-driven cancers are targeted toward effectors (i.e. Vemurafenib (RAF) or Idelalisib (PI3K)). Several of the most conserved RAS effectors, however, are not currently drug targets, and remain poorly understood in general. This includes effectors such as AF6/afadin, RALGEFs and RASSF proteins. We are aiming to gain a systematic understanding of how these effector proteins function in cells, the role of their modular components (including RAS binding), and their contribution to RAS-mediated oncogenesis. These effectors are demonstrating function in very different cellular processes than RAF and PI3K, including cell adhesion (inset) and apoptosis. Their conserved nature and that of their signalling networks will allow use of various model organisms, in addition to our own work with proteomics, cell biology, structure and biophysics.​
Activation and Resistance in Cancer Cells
NMR allows us to monitor biological activities in a virtually continuous manner, at atomic resolution, and in the complex environments of native cell and membrane extracts, tissue homogenates, biopsy samples, even in intact
cells. We can compare and contrast activities from normal vs. diseased cells, including defects in GTPase regulation, PTMs, and others. We believe that NMR and other biophysical approaches have yet to be fully applied as tools to explore system-level properties of signalling networks and their disease correlation. We are working to exploit gene expression and proteomic datasets to identify novel signalling abnormalities relevant to human cancers, and adapt NMR assays that will directly ‘fingerprint’ signal defects in physiological environments (i.e. cell and tissue extracts). Direct observations of protein function in this manner will provide novel, systems-level insight to protein networks, comprehensive analysis of disease mutations, and as we’ve recently demonstrated with RASopathies (inset), tools to delineate molecular pathology. Our integrative, multidisciplinary expertise allows us to combine cell biology and biophysical approaches in these types of novel and innovative ways.
