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SUMMARY OF INDIVIDUAL PROJECTS |
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Developmental Regulation of Heat Shock Protein Expression in Oysters Developmental regulation of heat shock protein (HSP) expression has been reported for insect, plant, teleost, avian, and mammalian species. In many cases, significant changes in expression occur during metamorphosis and may be linked to cellular differentiation during this process. HSPs are molecular chaperones that aid in the proper folding of proteins and are known to play a role in normal cell processes and in response to environmental stress. Two types of HSPs have been identified, constitutively expressed forms which may support translocation of newly synthesized polypeptides and stress inducible forms which correct misfolded proteins in the cells. Despite reports from diverse taxonomic groups, little is known about developmental regulation of HSPs in marine invertebrates. The proposed study will examine variation in expression of HSPs during larval and spat development in eastern oyster, Crassostrea virginica as well as the impact of environmental stress on these expression patterns. This is part of a larger project associated with the Alabama Oyster Reef Restoration Program. The REU student funded on this project would gain both field and laboratory experience. The primary techniques used include gel electrophoresis and immunoassays. |
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| Proteomic analysis of
host-pathogen interactions in seagrass “wasting disease” Tim Sherman, Ph.D. Department of Biological Sciences We are examining factors affecting susceptibility of two North American seagrass species to the “wasting disease” caused by the protist Labyrinthula. We have taken two approaches in that work. First is a “biochemical” approach to the study of the infection process. The focus of that work is on characterization of factors that regulate the synthesis of phenolics, biomolecules that have been suggested to influence susceptibility of the young seagrass leaves to attack by the protist. We have taken a second approach that utilizes transmission electron, light, and fluorescence microscopy supplemented with immunological and cytochemical techniques to examine the infection process at high spatial resolution. These two approaches have produced insights into the process by which seagrasses are infected and respond to that infection. It is clear from our work that the plant responds to the attack, but it is unclear as to whether the response is one of general stress, one of specific to the pathogen, or (more likely) some combination of the two. Currently we are expanding upon our previous work to look into host response to pathogen attack, as well as other sorts of stresses, at the level of gene expression via analysis of plant and pathogen proteomes. |
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Characterization of the starvation-stress response (SSR) stimulon of Salmonella enterica |
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Abnormal collagen
accumulation in scleroderma Scleroderma is a complex and devastating disease that initially affects the skin but later also involves internal organs such as the kidney, lungs, gastrointestinal tract and cardiovascular system. The key morphological features of this disease are (a) structural and functional vascular abnormalities, (b) chronic inflammation, and (c) excessive deposition of newly synthesized matrix. The morbidity of scleroderma largely depends upon the third facet of the disease, fibrosis. Recent studies have focused on identifying the complex interactions of different cellular components that culminate in the major vascular and fibrotic pathology of scleroderma. However, despite many hypotheses and decades of studies, the mechanisms by which these changes occur remain elusive. Our laboratory is interested in unraveling the pathogenesis of vasculopathy and abnormal collagen accumulation associated with scleroderma. We utilize injury models to identify the proteins or key components involved in the development of the disease. |
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X-Ray Crystallography and Molecular Modeling Structure-Function Studies of cGMP Regulatory Domains Andrzej Wierzbicki, Ph.D. Department of Chemistry Achieving selective inhibition for individual gene families of cyclic nucleotide phosphodiesterases (CNPDE) isoenzymes has become an important pharmacological goal now that the diversity of these hydrolases has been recognized. Selective Apoptotic Antineoplastic Drugs (SAANDs) induce apoptosis in neoplastic cells and not in normal cells by a mechanism involving inhibition of overexpressed PDEs that hydrolyze cyclic GMP. SAANDs, though not selective, show a preference for PDE5 and PDE10. However, highly selective PDE5 inhibitors do not induce apoptosis. To study differences in PDE inhibitors, we have constructed homology models of the target catalytic domains of PDE5A and PDE10A based upon the X-ray crystal structure of the catalytic domain of PDE4B [1]. Molecular mechanics docking was used to define new SAANDs versus ineffective agents. We have investigated the binding of several selective inhibitors to PDE4, 5, and 10 isoforms using our molecular models and compared the resultant estimated binding energies with experimental IC50 values. These modeling studies have allowed us to identify some differing structural features of the catalytic pockets which may be helpful in the development of active and more selective drugs to treat tumors found in different tissues while maintaining minimal side effects. Recently a non-catalytic regulatory cGMP binding fragment of PDE5 protein has been expressed and made available [2] for both structural and binding studies. We propose to apply protein homology modeling to study the specificity of cGMP binding to this regulatory fragment of PDE5. We will use a recently crystallized PDE2 GAF domain [3] to build a homology model for the regulatory domain of PDE5 to study the high specificity of binding of cGMP to this domain. We will attempt to elucidate why known efficient inhibitors of the catalytic domain of PDE5 do not competitively inhibit this regulatory site [2]. Independently we will pursue crystallization and x-ray structure determination of this regulatory cGMP binding fragment of PDE5 protein alone, and in the presence of cGMP and some known competitive inhibitors of this domain such as 8-bromo cGMP, for example. Our preliminary attempts to crystallize this protein have already resulted in small plate-like crystals yielding approximately 6 Angstrom resolution x-ray data. We propose to continue our work on the purification and crystallization of this regulatory cGMP binding fragment of PDE5 protein alone and in the presence of cGMP and its competitive inhibitors in order to obtain an atomically resolved structure of this fragment. We believe that these studies will allow to better understand the complex structure, activity and phosphorylation regulation of PDE5. References: 1. E. A. Salter, A. Wierzbicki, G. Sperl, and W. J. Thompson Homology Models of the Catalytic Sites of Phosphodiesterase Types 5A and 10A and Molecular Docking of Selective Apoptotic Antineoplastic Drugs (SAANDs). Accepted in International Journal of Quantum Chemistry (2003). 2. Li Liu, Tashandra Underwood, Han Li, Rifat Pamukcu, W Joseph Thompson Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase (PDE5). Cellular signalling. (2002) 14 45-51 3. S.E. Martinez , A.Y. Wu, N.A. Glavas, X-B Tang, S. Turley, W.G.J. Tol and J.A. Beavo. The Two GAF Domains in Phosphodiesterase 2A have distinct roles in Dimerization and in cGMP Binding. PNAS (2002) 99, 13260-13265. |
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Targeting recombinant proteins to mitochondria through protein transduction Mikhail Alexeyev, Ph.D. Department of Cell Biology and Neurosciences Recently, several naturally occurring proteins were described to contain short regions rich in basic amino acids, so called protein transduction domains (PTDs). These regions, when fused to proteins, enable the translocation of such fusion through the cytoplasmic membrane in time-and concentration dependent fashion. The fusion between one such PTD derived from amino acids 58-60 of the HIV-1 Tat protein (TAT PTD) and bacterial ß-galactosidase, when injected intraperitoneally into mice, was found in all tissues, including the heart, lung and brain. Although intracellular protein delivery has few, if any, limitations and is well documented in literature, the subcellular targeting of transduced proteins has not been described so far. This proposal seeks to apply the protein transduction methodology for mitochondrial delivery of restriction endonucleases for the purpose of manipulating the mitochondrial DNA heteroplasmy. Synthetic DNA constructs encoding protein chimeras consisting of mitochondrial targeting sequence, TAT PTD and restriction endonuclease will be generated and expressed in E. coli. Purified recombinant proteins will be applied to cultured human cells heteroplasmic for T8993G transversion and the effect of these proteins on the state of heteroplasmy will be studied. |
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Mechanical forces in lung cell biology Abu-Bakr Al-Mehdi , MD , Ph.D. Department of Molecular and Cellular Pharmacology Pulsatile blood flow evokes physical forces such as pressure, shear stress and stretch that act on the vascular wall. The cells in the blood vessel wall, particularly endothelial cells, transform these forces into electrical and biochemical signals, in a process called mechanotransduction. Although the precise mechanism(s) of mechanotransduction have not been yet elucidated, stretch and shear-stress responsive protein molecules on the plasma membrane, such as ion channels, may be involved. With cessation of blood flow in a variety of pathological conditions, channel mediated mechanotransduction could involve ionic shifts resulting in changes of membrane potential as an initiating event. Ionic shifts, such as rise in intracellular calcium, by themselves could initiate a series of metabolic responses. The changes in membrane potential could also lead to conformational changes of voltage-gated channel proteins or receptor proteins. As an alternate possibility, flow induced mechanical deformation of cytoskeletal elements (cellular structural proteins) associated with particular membrane proteins may modulate their activity independently of membrane potential changes. Therefore, the ability of the vascular endothelium to "sense" the presence or absence of blood flow may be related to specialized protein function. Our goal is to elucidate the nature of the flow-sensing proteins in endothelial cells. |
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Characterization of Nuclear Dehydratase/DCoH June Ayling, Ph.D. Department of Molecular and Cellular Pharmacology Dehydratase/DCoH is a bifunctional protein found in all mammals. It is composed of subunits of 12,000 Daltons. As a dehydratase, it is a tetramer involved in the regeneration of tetrahydrobiopterin, a co?]factor for catecholamine neurotransmitter biosynthesis. As DCoH, it is a dimer functioning as a dimerization co?]factor of the transcription factor, hepatic nuclear factor 1α (HNF1α). The relationship between these two functions is currently not understood. A similar protein has been found in the plant, Arabidopsis thaliana. Plants do not synthesize catecholamine neurotransmitters, nor do they contain tetrahydrobiopterin. A study of this protein in plants may reveal a broader spectrum of activities than previously recognized, and ultimately may help to clarify the connection between the two known functions in mammals. |
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Role of protein phosphorylation in host-parasite interactions Sailen Barik, Ph.D. Department of Biochemistry and Molecular Biology Our laboratory is focused on two major pathogens: Respiratory Syncytial Virus (RSV) and Toxoplasma gondii (T. gondii). While RSV is a negative-stranded RNA virus of the Paramyxoviridae family, T. gondii is an Apicomplexan parasite. Together, they cause devastating diseases that claim millions of human lives throughout the world, including the US. In the last few years, through a combination of cell biology, molecular genetics and RNA interference, we made major progress in understanding how these human pathogens interact with their host cells and cause the characteristic immunopathology. Our studies put special emphasis on protein phosphorylation and on the structure and function of protein phosphatases in particular. An aspiring NSF student can choose from a number of projects, the major ones being: (i) Identify the major phosphatases and their substrates in the various developmental stages of T. gondii, (ii) Determine the role of these phosphatases in host-parasite interaction, using phosphatase inhibitors, synchronous parasite cultures, and infectivity assays; (iii) Determine the function of cytoskeletal proteins in RSV morphogenesis and host-RSV signalling; (iv) Characterize the structure and function of cellular chaperons in pathogenesis. |
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Analysis of proteins associated with cancer Lewis Pannell, Ph.D. Cancer Research Institute and Department of Biochemistry and Molecular Biology The Cancer Research Institute, as part of the University of South Alabama, has established a proteomics facility headed by Prof. Lewis Pannell. While the genome is constant throughout and organism, the proteome varies depending on the function of the cells, their age and their actual state. The proteome is the complement of the proteins in a cell and their actual state. Thus, most proteins are altered after expression by posttranslational modifications, the least of which is removal of the signal sequence. New approaches to the analyses of proteins are concentrated on mass spectrometry which allows the analysis of the sequence of the peptides derived from proteins after enzymatic digestion. Posttranslationally modified peptides are not as easily characterized as non-modified peptides. Research in the proteomics facility is centered on the development of new methods to probe these modifications, especially glycosylation which involves the addition of sugar chains to proteins. These proteins are derived from various sources related to cancer such as cell lines which are metatsatic compared to those which are not metastatic. Unlike genes, proteins vary significantly in abundance and methods for protein enrichment are key in proteomic analyses. Methods are being developed to assist the analyses of these lower level, yet critical, proteins. The source of the proteins varies but they may be those secreted from the cells or be present on the cell surface. Other research is related to the analysis of changes in proteins from body fluids which are associated with cancer. This research is designed to further our understanding of the cancer. Protein changes may also lead to the identification of biomarkers for the early detection of cancer or its response to therapy. The laboratory is well equipped with all the necessary equipment including the mass spectrometers. The applicant will be given their own research project and, with assistance and training, taught to prepare their own samples for analysis and to perform their own analyses of the data. |
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Protein interactions of Molecular Chaperones in Transcription regulation controlling Cancer
Rajeev Samant, Ph. D. USA-Cancer Research Institute and Department of Pharmacology We recently discovered that MRJ (a heat shock protein/chaperone with J domain) binds to BRMS1, a protein which suppresses metastasis (spread of cancer to distant sites) of human breast carcinoma. While unraveling the mechanism this suppression phenomenon, we found. “J” domain is implicated in regulation of the tumor suppressor gene retinoblastoma (RB). It stops RB function leading to uncontrolled cell division and formation of tumor. Since BRMS1 binds to MRJ, we propose that it stops MRJ from inhibiting RB function, thus allowing proper cell cycle control. This may activate the apoptosis (cell death) machinery, leading to destruction of cancer cells. Aim 2: Cells constitutively expressing MRJ will be compared with the parental lines for cell cycle using propidium iodide staining followed by flow cytometric analysis The apoptosis will also be evaluated by AnnexinV staining and TUNEL |
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1. Effect of autocrine growth factors on the proteome of Vibrio vulnificus The pathogen Vibrio vulnificus is a marine bacterium that causes a serious, often lethal, blood infection in susceptible humans. This project examines a unique chemical signaling system used by V. vulnificus to control its cell division. Discovering this system contradicts a central tenet of bacterial physiology that any microbe capable of growing in a given medium can do so starting as a single cell. E. coli, for instance, easily divides and grows in liquid broths inoculated with a single viable cell. However, we determined that V. vulnificus requires a minimum density of approximately one million cells per ml to grow in liquid media. When diluted to lower concentrations, V. vulnificus stops dividing– a process termed dilution growth arrest (DGA). To counter V. vulnificus DGA, V. vulnificus secretes small molecular weight, autocrine-like growth factors that stimulate cell division. Thus, growth of low cell density cultures can be delayed for days until enough of the secreted factor accumulates. DGA is a cell division control mechanism in which individual members of a bacterial community actively promote each other’s growth. The molecular features of this system will be examined in two ways: (1) identifying proteins required for growth factor synthesis using genetic cloning and mutagenic strategies; and (2) characterizing proteins whose levels are regulated by growth factor. The later will be determined through the use of two dimensional gel separation techniques coupled to mass spectral analysis. The results may alter the paradigm used to explain bacterial cell division. |
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2. Proteins required for acid resistance in Escherichia coli John W. Foster, Ph.D. Department of Microbiology and Immunology How microbes cope with acid stress remains a fundamental question of biology with important medical significance. All intestinal pathogens encounter extreme acid stress upon ingestion, moving from the safe, neutral pH of food or water into the harsh, pH 2 milieu of the stomach. This is a five-log increase in acidity that destroys most bacteria and thereby protects the host from infection. Yet commensal and pathogenic strains of E. coli have developed elegantly regulated acid resistance mechanisms that enable survival in stomach acid. This project will focus on identifying proteins involved in providing that acid resistance. Mutagenic procedures will be used to identify mutants defective in acid resistance and the proteins involved will be identified by cloning. The project will then focus on potential protein-protein and protein-DNA interactions required to achieve acid resistance. The outcome of these studies will provide insight into bacterial physiology under extreme environmental stress. |
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