Congratulations to our NSERC Award Recipients!
15 April 2025
Dr. Xing-Zhen Chen
Transient receptor potential (TRP) channels: shared pore gate architecture and regulation by interacting partners
The transient receptor potential (TRP) superfamily of cation channels has 28 mammalian members that are grouped into six subfamilies and involved in various sensory functions with stimuli including light, force, temperature and chemicals etc. TRP proteins share similar membrane organization, eg, possessing six transmembrane domains with intracellularly localized N- and C-tails and tetrameric assembly. Despite of available structural information, the functional significance of many structural features remains unclear. Further, while protein-protein interactions are important ways of mediating functional regulations, how TRPs are functionally regulated by binding partners are not well known. In particular, little is known about the reason why 1) the hydrophobic pore gate residue is ‘sandwiched’ by two hydrophilic residues that are conserved in almost all TRPs and 2) a binding partner stimulates the function of a TRP while it inhibits the function of a homologous TRP.
Based on our preliminary data, the OBJECTIVE of this program is to study in selected TRPs 1) a shared mechanism underlying how the two conserved hydrophilic residues that ‘sandwich’ the hydrophobic gate ensure proper execution of pore opening/closing, and 2) how their channel function is distinctly regulated by binding partners, through which HQP will be trained. For this, we will carry out experiments in three Projects.
Project #1, elucidation of a shared mechanism that accounts for why the ‘sandwich’ gate architecture, namely, the hydrophobic gate sandwiched by two hydrophilic residues, is essential for proper execution of the gate tasks in TRPP2, -P3, -M8 and -V6.
Project #2, distinct physical and functional interaction of TRPP2 and TRPP3 with binding protein called Rassf4.
Project #3, mechanism underlying how TRPV6 is regulated by membrane protein TMEM59 through physical binding and effect of the binding on autophagy regulation. My graduate and undergraduate students will perform the experiments through collaborations and using interdisciplinary approaches.
METHODOLOGY. Functional effects of changes in the hydrophobicity and size of residues around the TRP pore gate will be studied by electrophysiology using frog oocytes and mammalian cells, mutagenesis, homology modeling and molecular dynamics simulation. Surface biotinylation and immunofluorescence will be used to assess protein expression and localization. Physical and functional interaction of a TRP with a binding partner will be assessed by co-immunoprecipitation, GST pull-down and bimolecular fluorescence complementation, electrophysiology, and blocking peptide strategy. Autophagy will be induced by starvation and assessed by LC3 and p62 marker levels.
This program will allow gaining novel insights into functional role of a shared ‘sandwich’ pore gate architecture in TRPs and mechanisms underlying the distinct regulation by binding partners, which will eventually contribute to therapeutic interventions. It will provide dynamic and productive research environments for training HQP.
Dr. Robin Clugston
Biological determinants of hepatic vitamin A homeostasis
Vitamin A is an essential dietary micronutrient required to maintain a healthy state. Since its discovery over 100 years ago we have gained a good understanding of vitamin A biology; however, knowledge gaps remain. The liver is the most important organ in whole body vitamin A homeostasis. It acts as a warehouse, storing vitamin A in hepatic stellate cells [HSCs] and secreting it from hepatocytes to supply the rest of the body. The long-term goal of this program is to advance our understanding of the cellular and molecular processes governing hepatic vitamin A metabolism. In the short-term, we plan to leverage our established expertise, experience with mouse models, and innovative transcriptomic approaches to achieve three aims:
Aim 1: Hepatic retinoic acid target genes, their cellular expression, and role in hepatic vitamin A homeostasis: The active metabolite of vitamin A in the liver is retinoic acid. This aim will identify genetic targets of retinoic acid in the liver, define their expression within different liver cell types, and study their homeostatic regulation with varying vitamin A status.
Aim 2: Spatial organization of hepatic vitamin A metabolism: We aim to characterize how hepatic vitamin A metabolism is spatially distributed, testing the hypothesis that hepatocyte vitamin A metabolism is zonal. We will assess and validate the zonal expression of genes associated with hepatic vitamin A metabolism, and functionally characterize spatially distributed hepatocyte vitamin A metabolism.
Aim 3: Molecular identification of novel retinyl ester synthesizing enzymes in liver and white adipose: We aim to establish the presence of LRAT-independent retinyl ester synthesis and test the novel hypothesis that MGAT1 synthesizes retinyl esters in vivo.
This work will provide new insight into how retinoic acid regulates hepatic vitamin A homeostasis (Aim 1), the spatial distribution of hepatocyte vitamin A metabolism (Aim 2), and novel mechanisms of retinyl ester synthesis (Aim 3). Impactfully, this work will advance our understanding of basic vitamin A metabolism and can be leveraged to understand the pathophysiologic interplay between hepatic vitamin A metabolism and liver disease.
Dr. Elaine Leslie
Regulation of Multidrug Resistance Protein 1 (MRP1/ABCC1) by Phosphorylation and N-glycosylation
The multidrug resistance protein 1 (MRP1) is a plasma membrane protein and member of the large and ancient ATP-binding cassette (ABC) transporter superfamily, found in all domains of life. MRP1 is widely expressed in healthy tissues and plays diverse and important roles in the cellular export of endogenous molecules including reduced and oxidized glutathione (GSH and GSSG, respectively), mediators of inflammation, and conjugated steroids. Furthermore, MRP1 is crucial for the export of many drugs, carcinogens, toxicants, and their metabolites. Included in this latter role, MRP1 is an established transporter of arsenic metabolites including arsenic triglutathione (As(GS)3). While MRP1 has been studied for decades, very little is known about its regulation by post-translational modifications. Using MRP1-enriched membrane vesicles we discovered two amino acids modified by phosphorylation that regulate transport of As(GS)3. Furthermore, the stability of these phosphorylation sites was influence by sugar groups at the amino terminus of MRP1. The sugar groups were different between multiple cell lines and this subsequently influenced the phosphorylation status of MRP1. Experiments proposed in this application are designed to provide mechanistic insight into how MRP1 can be modulated by phosphorylation and sugars, and how this may contribute to the structurally diverse array of solutes transported by MRP1. The specific objectives are as follows:
Objective A: To characterize MRP1 phosphorylation in multiple cytosolic loops and how this influences the transport of physiological compounds and arsenic metabolites. Putative phosphorylation sites will be mutated to phospho- and dephospho-mimicking amino acids and characterized for their influence on MRP1 transport of a panel of substrates (physiological and arsenic metabolites).
Objective B: To determine how sugar groups attached to MRP1 differ between cell lines, and how these differences influence MRP1 transport function. We will determine if the same number of sugar groups are attached to MRP1 in different cell lines. We will also characterize the sugar content and we will manipulate enzyme pathways important for removing and adding sugar groups to proteins to test the influence of specific sugar components on MRP1 transport function.
Objective C: To determine the molecular mechanisms of cross-talk between MRP1 sugar modifications and phosphorylation. We will determine if different sugar groups change the structure of MRP1. We will also determine if the sugar groups attached to MRP1 influence the cellular proteins that MRP1 interacts with. Ultimately, this work will contribute to fundamental knowledge about how MRP1 is modified post-translationally and how sugar groups and phosphorylation influence MRP1 function. This work has implications for understanding not only how phosphorylation and sugar groups influence MRP1 but also other transporters and membrane proteins.