Tuberculosis (TB) is currently the world’s leading cause of mortality due to a single infectious agent. It has been estimated that approximately one-third of the world’s population is infected with Mycobacterium tuberculosis, the bacteria that causes TB. Approximately two million people die of TB annually, and about eight million new cases arise each year. In addition to the emergence of multi-drug resistant strains of the disease, TB develops much more readily in people with HIV infection, and is a leading cause of AIDS-related death. There is an urgent need for novel therapeutics and drug targets in order to control the global spread of TB. In order to evade attack by the host immune system, M. tuberculosis secretes a protein called Protein tyrosine phosphatase A (PtpA). PtpA interacts with multiple proteins in the host that are normally essential for the destruction of bacterial pathogens. However, the exact role of these interactions in relation to the survival of M. tuberculosis within cells is not yet completely understood. Dennis Wong is defining the role of TB-Host interactions and identifying the molecular events that are disrupted by PtpA to promote TB infection. Understanding the mechanisms by which PtpA promotes the survival of M. tuberculosis will provide important insights regarding the pathogenesis of TB and the response of the host immune system to infections. As PtpA is a potential drug target, the new knowledge may contribute to the development of novel therapeutics against one of the deadliest diseases in the world.
Research Pillar: Biomedical
The mechanism of U4/U6 Di-small nuclear ribonucleoprotein formation: a modification/interference analysis
Proteins, the molecules that carry out many cellular functions, are synthesized according to information contained in DNA sequences. Converting information from DNA into a protein requires an intermediate step in which the DNA sequence is copied into a molecule called RNA. In humans there is an essential biochemical process called RNA splicing, in which non-coding portions of the sequence are removed and the remaining protein-coding portions are joined together to form a template for protein synthesis. Ninety percent of human genes are subject to splicing, so it is not surprising that errors in this process have been linked to a wide array of diseases, including retinitis pigmentosa, spinal muscular atrophy, cystic fibrosis, myotonic dystrophy, Alzheimer’s disease and cancer. Splicing is catalyzed by the spliceosome, a large and dynamic complex that consists primarily of five small nuclear ribonucleoproteins (snRNPs) designated U1, U2, U4, U5, and U6. During spliceosome assembly, the snRNPs interact with each other in a step-wise, ordered way. One of the first steps in assembly involves U4 and U6 pairing to form a particle called the U4/U6 di-snRNP. Although the di-snRNP complex is essential for spliceosome assembly and function, the mechanism by which it forms is poorly understood. Tara Wong is investigating the process by which U4 and U6 undergo essential conformational changes necessary for spliceosome assembly. She is using chemical modification/interference experiments to determine how free U4 and free U6 snRNPs interact to form the U4/U6 di-snRNP. This knowledge will be fundamental to understanding spliceosome assembly and function, and should ultimately lead to a better understanding, and treatment of splicing related diseases.
The immunomodulatory effects of host defence peptides on dendritic cells
Modern day vaccines are effective at preventing infections such as tetanus, influenza, polio and many others. To ensure full protection from illness, some vaccines require more than one immunization. This is commonly known as a booster shot. In developed countries, getting vaccinated usually means nothing more than going to the clinic. In developing countries the process is not so straight forward. Limited access to, and availability of vaccines makes widespread immunization a difficult process. The fact that people may have to return for a booster shot only compounds the problem. For all of the above reasons, there is clearly a need for improved vaccines in developing countries. Our laboratory is studying ways to create effective single-dose neonatal vaccines for developing countries. This means the vaccine would be given shortly after birth, and there is no need for a booster shot to ensure complete protection. Such a vaccine would alleviate the previously described difficulties. Specifically, our lab is developing more effective vaccine adjuvants. An adjuvant is simply any component added to a vaccine that will interact with the immune system to improve protection. We believe that a class of proteins known as host defence peptides (HDPs) will act as effective vaccine adjuvants. HDPs are short proteins, found almost ubiquitously in nature (microorganisms, insects, plants and mammals for example). Historically, the function of HDPs has been primarily to kill invading bacteria and viruses. Recent research conclusively shows that some HDPs are capable of altering the way in which immune system responds to an infection. My research will focus on how HDPs interact with and important type of immune cell known as a dendritic cell. Dendritic cells (DCs) circulate in the body in an “”immature”” form. When they encounter anything foreign (for example, bacteria or viruses), they become “”activated,”” capture the invader, and alert the immune system so it can mount a full response. They are now said to be “”mature.”” For this reason, DCs are a very unique type of cell. They are part of the front line of defence, yet they are also critical in generating the full immune response, which develops shortly after. We believe that HDPs will influence DCs in such a way that they will promote an efficient immune response in the context of vaccination. I hypothesize that HDPs impact DC function, activation, and maturation by altering specific genes and proteins important to DCs. This hypothesis has lead me to develop five goals to guide my research. I will provide an overview of these goals: 1) Bioinformatics. My preliminary experiments have tracked how HDPs influence the expression of 16,000 genes in mouse DCs. Such a large amount of data needs to be handled by a computer. Using specially designed programs, I am able to sort through the vast amounts of data and determine the broad trends occurring in response to HDPs. Furthermore, I am able to look at how small groups of genes behave in the context of their larger gene families; 2) IRAK-4. Results show that one peptide altered the behaviour of an important protein called IRAK-4. IRAK-4 is known to be important for specific immune responses. I will further analyze how this protein functions in the presence and absence of HDPs and other immune stimuli in DCs. I will also determine how proteins related to, and dependent on IRAK-4 will behave in response to HDPs; 3) Lyn Kinase. Another interesting finding was the altered production of Lyn, another protein important for proper DC function. I will continue analyzing the behaviour of Lyn in DCs in response to HDPs. I will also study the consequences of Lyn deficiency and determine its effects on HDP function. 4) DC Type. There are different types of DCs depending on where in the body you look, each performing similar, yet distinct functions. Currently it is not known how different types of DCs respond to HDPs. A lot of DC research is done with mouse DCs because they are relatively easy to generate compared to their human counterparts. The comparative responses of human and mouse DCs to HDPs are not well understood. For these reasons, I will be experimenting in multiple DC types, and in both human and mouse DCs. 5) In vivo peptide effects. Using the previously described experiments as a guide, I will examine how HDPs affect whole mice. We have access to mice deficient in all of the genes listed above, and this will be useful in determining the role of specific genes on the scale of a whole animal. At the completion of this project, I will have gained a comprehensive understanding of how HDPs influence DCs, with the goal of using this information to provide better vaccine adjuvant candidates aimed at developing countries.
Vascular dysfunction of the arteries in a mouse model of Marfan syndrome
Marfan syndrome is an inherited disorder of the connective tissue that causes abnormalities of the eyes, cardiovascular system, and musculoskeletal system. Its most serious and deadly complication is ballooning and rupture of the aorta, the major blood vessel that carries blood from the heart to the arteries and organs. The syndrome is caused by a defect in the gene that makes fibrillin-1 protein. Fibrillin-1 is essential in the formation of elastic fibres in arteries and in maintaining the functional and structural integrity of blood vessels’ endothelial and smooth muscle cells. Defects in this gene result in abnormalities in the way vessels contract and relax, increasing the susceptibility to ballooning and rupture of the aorta. Huei-Hsin Clarice Yang is studying the effect of Marfan syndrome on endothelial and smooth muscle cells in the aorta and the small arteries. She is expanding on previous research that found that smooth muscle in the Marfan-affected aortas is unable to relax normally. Her work focuses on the mechanisms that contribute to this dysfunction within smooth muscle cells and in the epilethial cells that regulate vascular contraction and relaxation. Yang’s work will provide valuable insight into how Marfan syndrome causes decreased contracting and relaxing abilities of the arteries. Ultimately, this knowledge could lead to innovative therapies to prevent or treat aortic rupture and to halt the vascular deterioration process in patients with Marfan syndrome
Structural characterization of Propionibacterium acnes virulence factors
Acne is the most common skin disorder worldwide, affecting approximately 80 per cent of individuals at some point in their lives. How the skin develops this inflammatory condition is not entirely understood, nor is there a cure for severe, persistent cases of acne that often result in permanent scarring. Antibiotics are often prescribed as a first-line treatment, but the most effective antibiotic (Accutane) is known to have serious side effects, including birth defects and depression. In addition, antibiotic resistance is a growing problem. Propionibacterium acnes is present on most people’s skin and is the principal microorganism associated with acne. It can behave as an opportunistic pathogen under certain circumstances, expressing genes that lead to symptoms of acne. The genome of the bacterium has been sequenced and research has shown several genes that can generate enzymes for degrading skin, and proteins that may activate the immune system, leading to the initiation of acne, its development into inflammatory lesions and scarring. Angel Yu is focusing on O-sialoglycoprotein endopeptidase, a skin tissue-degrading enzyme. In order to understand how this protease works and how it recognizes its protein targets, she is growing crystals of the enzyme and using X-ray crystallography to study its structure at the atomic level. She will conduct studies that confirm the enzyme’s biological function and identify associated amino acid residues. Ultimately, Yu hopes her findings will provide insight into the molecular mechanism of this inflammatory skin disorder and identify new leads for the treatment of acne.
Development of a pipeline for the analysis of flow cytometry data
Flow cytometry (FCM) is a method of sorting and measuring types of cells by fluorescent labelling of markers on the surface of the cells. It plays a critical role in basic research and clinical therapy in the areas of cancer, HIV and stem cell manipulation. For example, it can be used to diagnose some types of cancer, based on which labelled antibodies bind to a particular cell’s surface. It is widely recognized that one of the main stumbling blocks for FCM analysis is in data processing and interpretation, which heavily relies on manual processes to identify particular cell populations and to find correlations between these cell populations and their clinical diagnosis and outcome (e.g. survival). Manual analysis of FCM data is a process that is highly tedious, time-consuming (to the level of impracticality for some datasets), subjective and based on intuition rather than standardized statistical inference. Dr. Ali Bashashati has developed a “pipeline” for automatic analysis of FCM data – a computational platform that can identify cell populations, find biomarkers that correlate with clinical outcomes, and label the samples as normal or diseased. Preliminary evaluations of this pipeline have shown accuracy levels of more than 90 per cent in identifying some sub-types of lymphoma. Moreover, a biomarker that contributes to a more aggressive behaviour of a specific sub-type of lymphoma has been discovered. Bashashati is now testing and refining the platform to improve its analytical power and applicability to a range of FCM data, testing its performance across a number of ongoing FCM studies in BC. Ultimately, he hopes to provide an accurate, powerful computational platform to increase the efficiency of using FCM for research and clinical purposes.
CD34 in development of lung inflammatory diseases
Ever since its discovery more than 20 years ago, the CD34 antigen has been widely used as a marker to identify stem cells, precursor cells that give rise to all types of specialized cells. However, the exact function of CD34 expression on hematopoietic precursors and mature cells is still not well understood. Dr. Marie-Renée Blanchet and colleagues have uncovered some fascinating details about the role of CD34 in allergy and asthma. The team recently demonstrated that CD34 is expressed on mature mast cells and eosinophils – two types of cell that respond to injury during inflammation of the body’s tissues – and that the CD34 antigen is involved in their recruitment to the lung and peritoneum. They showed that mice without the CD34 antigen are protected against development of airway hyper-responsiveness and airway inflammation, which are two major hallmarks of allergic asthma. Finally, in preliminary experiments, these mice also showed protection in hypersensitivity pneumonitis, another model of lung inflammation. Now, Blanchet is working to better understand the mechanisms behind these recent findings. Many cell types involved in asthma and hypersensitivity pneumonitis express CD34, some in which the role of this protein remains unknown (eg. fibrocytes and dendritic cells). She plans to use models to elucidate the role of CD34 expression in these cells. Ultimately, she hopes her studies will reveal potential targets for treatment of allergy and inflammation.
Epigenetic mechanisms regulating the acquisition and extinction of conditioned fear: exploring the neurobiology of relapse
A major obstacle in the treatment of fear-related anxiety disorders is their likelihood for relapse. Fear-related behaviour can be inhibited with extinction therapy (repeated exposure to specific fear-inducing cues). This is, however, a temporary fix because fear often returns after exposure to cues associated with the original learning. In the case of post-traumatic stress disorder, fear can also “incubate” or sensitize over time and further exacerbating symptoms of the disorder. These phenomena likely reflect long-term neural adaptation that occurs during learning – changes that may be based on lasting epigenetic modification of genes responsible for maintaining fear memories. Epigenetic modifications influence the way a gene functions without altering the underlying DNA sequence- processes now recognized to participate in the regulation of gene expression in the adult brain. Rapidly emerging evidence suggests that epigenetic mechanisms play an important role in psychiatric disease and in disorders of learning and memory. Dr. Timothy Bredy is employing state-of-the-art technologies to investigate the fundamental epigenetic mechanisms of associative fear memory. He is using a genome-wide approach to examine epigenetic machinery involved in regulating critical gene targets during the acquisition and extinction of conditioned fear. Dr. Bredy hopes his findings will provide insight into the molecular basis of relapse and its prevention and that this research will ultimately contribute to the design of novel pharmacotherapeutic treatment approaches for fear-related anxiety disorders.
The role of H2AX in non-Hodgkin lymphoma
Non-Hodgkin lymphoma (NHL) is a specific type of cancer where an abnormal growth of immune cells produces what is known as a lymphoid tumour. Since the 1970s, NHL has become increasingly common, indicating that lifestyle and environment are likely causative factors. However, certain individuals may also have a genetic make-up that makes them more susceptible. NHL tumours often show a type of DNA damage called a translocation, where two chromosomes are incorrectly joined together. In NHL tumours, translocations are generally found near genes that are important for the development of immune cells. They cause changes in how these genes are regulated (turned on or off), that result in abnormal cell growth. Certain genes are responsible for repairing damaged DNA. If these genes are not functioning properly, DNA breaks will not be repaired and harmful translocations may occur. Previous studies have found that a common DNA sequence change at one of these DNA repair genes, called H2AX, was much more frequent among the NHL patients than unaffected individuals. Individuals who carry this gene variant have twice the risk of NHL as those who do not carry it. Dr. Karla Bretherick is interested in how common genetic variants influence risk for complex diseases. MSFHR has previously funded her graduate training, which involved studying the genetic factors that contribute to premature menopause. Now, she is looking at why individuals with the H2AX gene variant have increased risk of NHL. She will look at how this DNA sequence change affects H2AX gene regulation, modifies protein binding, and affects the ability of the cell to repair DNA damage. Ways to understand, prevent, and avoid NHL and other cancers are of increasing importance for the Canadian healthcare system. Understanding how and why this specific gene variant increases risk for NHL will lead to a better knowledge of how this cancer develops. This information will eventually be useful for identifying new drug targets and therapies for NHL, and may also provide insight into the development of cancers in general.
Degradation of tumour suppressor ING3: Pathway and its role in cell cycle progression
Cutaneous malignant melanoma is a life-threatening skin cancer that is very resistant to conventional radio- and chemotherapy and has a low survival rate. Thus, it is important to understand the molecular changes underlying the onset and progression of the disease. The novel tumour suppressor ING3 acts to inhibit cell growth. A number of previous studies have demonstrated that ING3 switches on and off during normal cell division, and that it enhances cell death in melanoma cells when they are exposed to UV-light. Dr. Guangdi Chen has identified that the expression of ING3 degrades (or decreases) much faster in melanoma cells than in regular melanocytes (healthy melanin-producing cells) during the cell cycle. This rapid degradation may be an important cause of aberrant ING3 expression and the loss of its tumour suppressing function. However, the mechanism of ING3 protein degradation and its role in cell cycle progression remain unclear. Chen is investigating the pathway of ING3 protein degradation and assessing its role in cell cycle progression. By understanding the molecular mechanisms of ING3 tumour suppressive functions in cell cycle progression, he hopes his work could help in the design of novel strategies for cancer prevention and treatment. Chen’s post-doctoral fellowship is jointly funded by MSFHR and the VGH & UBC Hospital Foundation.