Amanda Chase - MUSM Microbiology, Macon
Dengue Virus Capsid-Mediated Inhibition of Immune Function
Infection by dengue virus (DENV), a member of the Flaviviridae family, may result in benign courses such as dengue fever or life-threatening presentations defined as dengue hemorrhagic fever and dengue shock syndrome. The general mechanisms that are involved in disease pathogenesis and severity during acute illness are not well understood. DENV infects and replicates in human dendritic cells (DCs). DCs serve as sentinels of the immune system, the main cell type that alerts the human body to foreign invaders. The central goal of our research is to understand how the capsid (C) protein of DENV mediates immune dysfunction at the level of DC infection. Viral C proteins, responsible for protecting the viral nucleic acid, have recently captured the attention of virologists due to their immune evasion properties. Our preliminary data suggests that DENV C protein inhibits the innate signaling pathway in DCs resulting in a significantly reduced amount of IL-12 secretion. IL-12 is an important co-stimulatory cytokine that drives the development of an antiviral T helper cell type I (Th1) response. We determined that Th1 cells that fail to receive the IL-12 co-stimulatory signal from bystander, DENV-infected DCs do not develop into antiviral effector cells. We aim to dissect the mechanism by which DENV C protein antagonizes DC priming capacity. Defining the mechanism of DENV C-mediated inhibition of DC function will help us to better understand the immune dysfunction that is associated with dengue infection.
Garland Crawfod - Department of Chemistry, Macon
O-GlcNAcylated Protein Purification Using Mutant Enzyme
Proteins are polymers composed of amino acids that show significant diversity in function and structure. Whether responding to the external environment, carrying out basic metabolism, or dividing, a cell performs these fundamental tasks using proteins. In the cell, these actions are coordinated to prevent the wasting of resources and the duplication of function. To orchestrate all of these activities, it is important that cells communicate between signaling pathways. One method of intracellular communication is the covalent modification of amino acid side chains by signal-regulating and signal-integrating proteins. The addition or removal of these post-translational modifications is critical because they may lead to a significant change in protein function.
One of these modifications is known as O-GlcNAcylation and it involves the addition of a single β-N-acetylglucosamine sugar to a serine or threonine residue of a target protein. This modification has been shown to alter the activity of proteins in a number of different signaling pathways. In some cases it has been shown to directly compete with other post-translational modification. O-GlcNAcylated proteins have been shown to play a role in a wide range of diseases including diabetes, heart disease and several neurodegenerative diseases.
While over 200 proteins have been suggested to be modified, far fewer have been confirmed due to experimental limitations that include a lack of stability of O-GlcNAc during mass spectrometry, cross-reactivity during the purification process, and a general lack of specificity using traditional affinity chromatographic methods. In an effort to increase the number of O-GlcNAcylated proteins that have been identified, new techniques must be developed that will allow for the selective isolation and identification of modified proteins with a minimum of cross-reactivity.
While other protein post-translational modifications are regulated by numerous proteins, O-GlcNAcylation is controlled by the action of two enzymes. It is added to target proteins by a glycosyltransferase termed O-GlcNAc Transferase (OGT) and is removed by the enzyme O-GlcNAc Hydrolase (OGA). The specificity of the enzyme OGA enzyme for this particular modification is the foundation of this area of research. While the structure for OGA has not been obtained, structures of similar enzymes show a high degree of similarity. The key catalytic residues responsible for the hydrolysis of the O-GlcNAc modification by OGA have been identified as aspartate 174 (D174) and aspartate 175 (D175). By altering these amino acids, the function of OGA can be altered in hopes of generating an OGA enzyme that will bind substrate but not hydrolyze the linkage.
Over the course of the summer, this project will focus on developing a series of techniques that will allow for the selective isolation of O-GlcNAcaylated proteins using mutant OGA enzymes as bait in affinity chromatography methods. Techniques will include cloning, site-directed mutagenesis, protein expression and purification, enzymology and assay development.
David Goode - Department of Chemistry, Macon
Design, and Synthesis of Anti-viral CompoundsCentral to a successful mammalian host cell invasion by a virus is the ability to gain control of apoptosis, or programmed cell death. There are multiple systems by which viruses accomplish this, and most require the mimicking of host proteins in order to halt the death signal. The host Bcl-2 family of proteins creates a complex balance of both pro-apoptotic and anti-apoptotic signals—the scales tipping one way or the other based upon cellular conditions. Virus invasion usually tips this scale towards apoptosis, but viral mimics of anti-apoptotic Bcl-2 proteins (M11L and F1L) help to restore the balance and give the virus time to overtake cellular machinery—resulting in viral replication. These viral mimics have very different primary sequences from the native Bcl-2 proteins, and thus could be selectively targeted by small, organic molecules. Disruption of the viral proteins should allow apoptosis to proceed—killing the initially infected cell, but also halting viral replication and spread. Compounds have been previously discovered to bind the native anti-apoptotic Bcl-2 proteins and induce apoptosis. One such compound is ABT-737 and its derivatives (see the target structure below). Students are needed to synthesize ABT-737 derivatives, express and purify proteins, and/or synthesize BH3-domain peptides for biochemical assays. This work will lead to cellular culture and possibly animal studies with a graduate-level professor in the future.
Sinjae Hyun, Biomedical Engineering, Macon
Experimental and Computational Measurements of Inhaled Particle Deposition in Human Lung Airways
Evaluation of the health and safety risks of products using particles including nano-sized particles is critical as there are ever increasing applications and sources as diverse as cleaning agents, food stuff, cosmetics, device fabrication, chemical processes, drug delivery, and textile weaving, to name a few. In addition, air pollutions from natural sources to tobacco smoke and unclean combustion exhausts contribute to the inhalation of particles as well. Thus, the development of novel tools and approaches to determine the impact of an array of engineered particles as well as indoor/outdoor pollutants on biological systems and health outcomes is necessary to protect human health.
Employing experimental and computational approaches, the underlying hypothesis to be tested is that a realistic and accurate model can be developed to predict inhaled particle depositions in the upper respiratory regions. Subject-specific in vitro particle deposition experiments will provide new physical insights into complex fluid-particle-wall interaction mechanisms as well as thorough validation of the computer simulation model in the future. Thus, the specific aims are: (i) development of realistic human breathing conditions (i.e., cyclic flow behavior) for experimental study to measure detailed lung aerosol deposition data for widely-used materials of particulate matters; and (ii) in vitro measurements of inhaled aerosol transport and deposition in the realistic human airways created.
Building on a decade of experience in lung-aerosol dynamics modeling and simulation by the PI and his research team, the in vitro experimental work, focusing on sub-micron-sized aerosol particle depositions in physical models, will be carried out during summer months.
Concerning the first Specific Aim, newly developed subject-specific airway models will provide a better understanding of how inhaled sub-micron particle interactions in the oral cavity (i.e., soft palate) and glottis will affect particle deposition due to the cyclic flow condition. Concerning the second Specific Aim, lab measurements of inhaled particle deposition in subject-specific replica will provide new physical insight into the mechanics of inhaled lung-aerosol deposition and will generate sufficient data sets for computer model validation.
In summary, for the first time experimentally measured data in realistic, subject-specific human lung airway models would be available, which are accurate, comprehensive, and flexible. Thus, modern environmental (and medical) problems related to particle inhalation could be readily solved. Examples include: reliable particle-deposition data to analyze the impact of inhaled toxic particles (US EPA), the evaluation of the secondhand tobacco smoke inhalation (NIH), new aerosol inhaled drugs for pharmacokinetics modeling (US FDA), and evaluation of the effect of local airway obstruction, e.g., due to severe COPD, on air-particle flow and inhaled particle deposition.