CIHR Project Grant Competition Funded Projects, Fall 2017

Congratulations to our LSI PIs who were funded in the Fall 2017 CIHR Project Grant Competition.

CD127 in the development and function of Type 2 Innate Lymphoid Cells (ILC2s)

Principal Investigator: Ninan Abraham
Co-Investigator: Fumio Takei
Amount awarded: $1,036,575

The protection of the airway lining is extremely important since it is a single cell layer thick and its integrity allows for oxygen exchange. Immune responses to infectious microbes and to innocuous environmental agents like pollen have to be balanced to clear serious challenges yet not over react to harmless ones. This balance in immune responses depends on several immune mediators called “cytokines”. Several cell types can make cytokines but one relatively new type of immune cells called innate lymphoid cells (ILCs), have;; been found to play important roles in recovering from infections by initiating repair; but ILCs are also capable of triggering allergic reactions that can lead to asthma. We work on a cytokine receptor molecule, CD127 or IL-7Ralpha that is a known surface protein on ILCs. We know it has diverse roles in other immune cells that display it but its unknown what its exact role in ILCs is. We will determine that, using selective gene targeted mouse models, gene profiling and large immune profiling approaches in 2 models of airway diseases: asthma and influenza. We expect that this study will shed light on how CD127 signals direct ILC development and function and how this is changed in the diseases above. This will allow identification of better targets to treat such diseases.

Mechanisms of tricellular junction formation and regulation

Principal Investigator: Vanessa Auld
Amount awarded: $719,100

Cells such as those found in the intestine, kidney or skin are used to form barriers between your body and the environment to ensure pathogens like bacteria and virsuses do not move into or through the body and to ensure that your blood or other body fluids are contained. Cells do this by creating junctions or barriers made of proteins between neighboring cells and when three cells meet at the corners cells also make a tricellular junction. It is clear how the permeability barrier forms at the boundary between two cells but less is known about how the corners of cells create the permeability barrier. To understand how this important junction forms and is regulated, we will identify and study those proteins that are uniquely located at this tricellular junction using the model system Drosophila (fruit fly). In fruit flies we can easily discover new proteins and test their functions.. If we understand how this tricellular junction is controlled we can then understand how it functions. We know this complex is highly conserved in humans but the function of these proteins in the tricellular junction has never been studied. Therefore we can take advantage of the ease of the genetics and cell biology capable in Drosophila to understand the function of this family of proteins in epithelial cells in all animals.

Ectosteric inhibitors of cathepsin K: their mechanism and in vivo efficacy

Principal Investigator: Dieter Brömme
Co-Investigator: Jean-Marie Delaisse
Amount awarded: $956,250

Worldwide, 1 in 3 women and 1 in 5 men over 50 years of age will experience osteoporotic fractures. Current osteoporosis treatments have various shortcomings such as poor bone quality and several skeletal and non-skeletal side effects. Therefore major investments have been made into the development of more effective drugs that are less prone to side effects. Due to efforts of ourselves and many others it has been discovered that bone-degrading cathepsin K (CatK) is a highly suitable drug target whose inhibition led to increased bone quality and reduced fracture rates in clinical osteoporosis trials. Although there is little doubt about the validity of CatK as a drug target, none of the CatK inhibitors have been approved because of various safety concerns. Based on our research, this might be due to the multifunctional character of the enzyme, where total inhibition leads to the observed side effects. However, the osteoporosis relevant activity of this enzyme is limited to its potent collagenase activity, which should be selectively blocked. Based on our previous studies on the mechanism of collagen degradation by CatK, we have developed a strategy to specifically block the collagen-degrading activity without affecting other biologically important actions of this protease. We termed this novel and innovative type of drugs “ectosteric inhibitors” and our proposed project is aimed at characterizing these substrate-specific drugs at their molecular, cellular and in vivo levels of action and efficacy as anti-resorptives. We have identified compounds that have a similar bone-preserving potency in cell assays and in an osteoporosis mouse model similar to the most advanced clinically evaluated CatK inhibitor without affecting non-bone-related pathways. This proposal introduces the novel concept of substrate-selective inhibitors that are superior to all previously developed protease drugs and aims to develop potent, efficacious, and side effect free osteoporosis drugs.

Role of XIST in human X-chromosome inactivation

Principal Investigator: Carolyn Brown
Amount awarded: $963,900

Females have two X chromosomes while males have only one X chromosome and the sex-determining Y chromosome. The X chromosome contains over 1000 genes that therefore have the potential to be expressed twice as much in females compared to males. To avoid this expression imbalance one X chromosome is silenced in females. We have discovered that the gene that initiates this silencing process is located on the X chromosome and named XIST for X-inactive-specific-transcripts, because, unlike all other genes, it is only expressed from the inactive or silent X chromosome. In addition, XIST, does not make a protein, rather it is the founding member of a class of genes who functions as a long RNA. We believe it does this by binding proteins and bringing them to the inactive X and in this work we will identify these proteins and how they act to silence the X. This will help us understand silencing, and also some of the sources of differences between males and females.

Biophysical properties and regulation of KCNQ1 and KCNE(x) ion channel complexes

Principal Investigator: David Fedida
Amount awarded: $627,300

In this study we will examine a complex protein that is able to conduct potassium and has a vital role in maintaining the normal resting activity of excitable tissues. This protein is made up of two or three subunits which greatly affect its behavior when they combine with it. The aims of the present work are to fully understand how the main protein subunit interacts with its partners to produce the activity that we observe in the human body. The approach proposed in this study is to express the individual genes that code for the human potassium ion channel, IKs, and its partner subunits and then study the complexes together in a cell-based system. The work will entail using special chemicals to bind the subunits together when they are exposed to ultraviolet light, and then subjecting them to rigorous analysis to find out the exact places where they bind together. Powerful mass spectrometry techniques combined with electrical analysis of the channel function will be used to identify and locate the exact sites in different parts of the ion channels that contribute to binding between the subunits and how those sites shift as the channel opens and closes. We will be the first group to use these kind of very detailed methods and specific binding method to understand how the partners in this channel complex work together. We expect to obtain completely new information about the relationships between these proteins that will allow us to construct a model of how they act in concert during the normal functioning of the channel complex.

Host cell protein substrates of enterovirus proteases

Principal Investigator: Eric Jan
Amount awarded: $726,750

Enterovirus infections are associated with several acute and chronic diseases worldwide and in Canada. Most enterovirus infections are cleared by the immune system, however, infection by certain enteroviral species and strains can be more severe. Poliovirus is the most well-known member which causes paralytic poliomyelitis. The non-poliovirus members are more common such as rhinovirus, which causes the common cold, and coxsackievirus and EV71, which are associated with more debilitating diseases such as viral myocarditis, Type I diabetes, and hand-foot-and-mouth disease, and in rare cases, meningitits. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. One of the signature properties of enteroviruses is that the virus expresses a protein, called a protease, which cleaves host proteins in the cell to facilitate virus infection. Currently, the extent of host proteins targeted by the viral protease is not known. In this study, we will utilize a novel unbiased proteomics approach to comprehensively identify all of the targets of the proteases from coxsackievirus. Unlike other methods, this new approach is sensitive and can identify the location of the cleavage site on the target protein. The identification of novel targets will provide a catalog of the host processes that viruses affect. Importantly, the knowledge gained will allow us to identify new targets for antiviral therapy. The development of this strategy can be exploited to identify the repertoire of host targets of proteases from other viruses.

Targeting platelet-specific molecules in periodontal disease

Principal Investigator: Hugh Kim
Amount awarded: $856,800

Periodontitis, or gum disease, is a very common infection of the mouth that causes inflammation of the gum tissues and also destroys the connective tissues and bone that support the teeth. Unfortunately, periodontitis is detected only after the gums and teeth have already been affected (and often irreversibly) by disease. The tools currently available to dentists do not allow them to predict those patients (or teeth) who may be at high risk for future gum disease. In addition, the treatments for gum disease often involve costly and invasive gum surgical procedures that do not target specific molecules responsible for the disease process. During periodontal infection, white blood cells release signaling molecules known as cytokines, some of which can aggravate periodontal disease. In addition, platelets control blood coagulation (clotting) but also release cytokines that can promote inflammation. The goal of this grant application is to study platelet-specific cytokines and to determine whether shutting down the function of these molecules might help to prevent tissue damage caused by gum disease. This is a unique approach to studying this very common disease and may, over the long term, lead to improved, less invasive treatments that would benefit dental patients in Canada and worldwide.

Pannexin1 mediation of stroke recovery responses: Sex and sex steroid influences

Principal Investigators: Christian Naus, Victor Viau
Amount awarded: $692,325

Pannexin 1 (Panx1) is a protein found in the membrane of various cell types, including neurons, to assist in the passage or communication of very small molecules. Ischemic injuries that deprive neurons of oxygen and glucose (e.g. stroke) can enhance the activity of Panx1 to promote cell death (apoptosis). Men and women often show differences in stroke severity to suggest a potential role for the gonadal steroid hormones, estradiol and testosterone. Because Panx1 can mediate apoptosis and inflammatory signaling responses in injured neurons, we reason that it may come to explain sex differences in stroke outcome responses. Indeed, in our brain stroke model we have evidence to show that either the loss of the Panx1 gene or its pharmacological blockade significantly reduce the amount of neuronal damage in female, but not in male mice. This striking difference underscores Panx1 as an important focal point for unmasking underlying gonadal hormone and cellular mechanisms mediating sexual dimorphisms in stroke severity, as we propose to examine in the current application. The findings of this grant promise to provide novel frameworks for sex- and age-dependent differences in stroke vulnerability and rescue.

Regulation of Stem-Cell niche interactions in the hematopoietic system by occluding and gap junctions

Principal Investigator: Guy Tanentzapf
Amount awarded: $768,825

Stem cells are essential for animal development and allow the maintenance and regeneration of tissues. Stem cells retain the capability to divide and to develop into many types of adult cells throughout the lifetime of an organism. The blood stem cells in humans and animals are particularly important for fighting infections as they produce the immune cells required to fight infection. The work we propose seeks to understand the mechanisms that regulate the activation of blood stem cells such that they produce immune cells upon infection. Our work focuses on the role played by the blood stem cell’s environment in regulating this transition. In particular we will focus on the role of various cell junctions in shaping the signals that control stem cell behavior. We propose that in response to infection the stem cell environment changes in very specific ways and that this induces the activation of blood stem cells. Understanding the mechanisms that induce blood stem cell activation upon infection will help us gain insight into what happens in diseases that affect the blood stem cell niche such as leukemia and autoimmune diseases.

Caveolin-1 regulation of focal adhesion tension and the breast cancer cell response to matrix stiffness

Principal Investigator: Robert Nabi
Co-Investigators: Calvin Roskelley, Ghassan Hamarneh
Amount awarded: $100,000

Mammography detection of dense breast tissue is associated with a four-fold increased risk of breast carcinoma and a greater risk for invasive breast cancer. This is due to increased organization and stiffness of the extracellular matrix, primarily collagen, and matrix stiffness is associated with aggressive cancers in other tissue types, such as prostate cancer. Focal adhesions are cell surface adhesion structures responsible for cell attachment to the extracellular matrix that mediate the cancer cell response to matrix stiffness. We have identified a protein, caveolin-1, that regulates focal adhesion tension and that is also associated with breast and prostate cancer malignancy. We will now determine how caveolin-1 controls focal adhesion tension and whether its role as a regulator of focal adhesions mediates the breast cancer cell response to matrix stiffness. These studies will lead to a better understanding of the association of caveolin-1 with malignant breast cancers and may lead to the identification of specific biomarkers for the cancer cell response to matrix stiffness.