The purpose of this research is to investigate the molecular mechanisms of action of biologically active proteins from arthropod disease vectors and pathogenic microorganisms. We use biological and physical techniques to characterize and understand the modes of action of pharmacologically active components from the saliva of blood-feeding vector insects and ticks, as well as immunomodulatory components secreted by parasitic organisms such as Toxoplasma and Schistosoma. Proteins and small molecules found in the saliva of vectors inhibit the host hemostatic responses and are essential for the successful completion of a blood meal. Most vector borne diseases are transmitted during feeding, so elucidation of the physiology and biochemistry of this process is necessary for understanding disease transmission. Saliva has also been shown to have pronounced effects on host inflammatory and immune responses which persist after feeding and can dramatically alter the environment for the pathogen after transmission. Determining the specific role of salivary molecules in these processes is essential for the understanding their importance to pathogen survival after transmission Over the past several years we have identified the functions of numerous salivary molecules involved primarily in overcoming host hemostatic defenses. The raw material for these studies comes from the analyses of salivary transcriptomes produced in collaboration with Dr. Jose Ribeiro. Bioinformatic analysis of sequence data is used to predict function of salivary proteins. Candidate proteins are then expressed in bacterial or eukaryotic cell systems. The proteins are purified and assayed using a variety of methods. Functionally characterized proteins are then produced in larger quantity for structural and other biophysical studies. Over this same period we have collaborated with Dr. Alan Sher's laboratory to characterize a number of pathogen-produced proteins involved in immune responses to infection. These projects included: The isolation of a T cell antigen from a Helicobacter species that is involved in the induction of colitis in a mouse model, the characterization of a chemokine receptor ligand from Toxoplasma which was evaluated for potential as an anti-retroviral agent, the isolation of a toll-like receptor ligand from Toxoplasma, and the isolation of an apparent T cell polarizing factor from the eggs of Schistosoma. During the 2013 fiscal year we have 1) determined the structures of two new salivary proteins and applied structural information to determine the mechanism of action of these proteins, 2) continued to produce recombinant proteins for use in an experimental saliva-based leishmaniasis vaccine, 3) published the structure and ligand binding properties of a anti-platelet/anti-vasoconstrictor protein from Rhodnius prolixus 4)Completed our structural and functional analysis of a contact pathway inhibitor from Phlebotomous duboscqi saliva 5) Collaborated with Louis Miller on physical analysis of potential inhibitors of Plasmodium cellular invasion. 6) Produced a recombinant version of a salivary inhibitor of coagulation factor IX for a collaborative study on quality of commercially prepared factor VIII with Mikhail Ovanesov if the FDA.7) Began studies on an antiinflammatory binding protein from the saliva of Rhodnius prolixus with Willy Jablonka, a new post doc fellow in the laboratory. 1) We continue our work on the crystallization of salivary proteins in the laboratory and now almost exclusively use remote data collection from the SER-CAT beamlines at Argonne National Laboratory for collection of diffraction data. We have produced recombinant protein, crystallized and determined the structure of two new proteins over the last year. We are also currently analyzing diffraction data on several additional novel proteins. 2) Salivary components of vector sand flies have been shown to be useful as potential leishmaniasis vaccine components based on their ability to induce delayed hypersensitivity responses in hosst skin. As part of a vaccine development project directed by Jesus Valenzuela, I have continued to produce salivary antigens from the saliva of Phlebotomous duboscqi in a recombinant system. 3)Proteins in the lipocalin protein family are widely distributed in the saliva of disease vectors, but they are not functionally well characterized. We have published a crystal structure analysis and functional characterization of a serotonin-binding member of this family from the blood feeding bug Rhodnius prolixus. The protein inhibits platelet aggregation and vasoconstriction by removing serotonin and norepinephrine produced by platelets and nerve terminals. 4) We have characterized members of the """"""""SP-15"""""""" protein family from Phlebotomous duboscqi as inhibitors of the """"""""contact"""""""" pathway of coagulation. These are major proteins in the saliva that act by binding to glycosaminoglycans secreted from mast cells in the skin. These carbohydrates serve as an activating matrix for coagulation factor XII. Inihbition of factor XII activation prevents the production of bradykinin in the skin thereby limiting inflammation in the area of the insect bite. In addition to identifying the mode of action of these inhibitors, we have determined the crystal structures of two forms and identified structural possible structural determinants for these activities. During the past year we have shown that the protein also blocks contact pathway activation by polyphosphate, a key endogenous activator. 5) I have collaborated with Louis Miller and members of his laboratory to analyze the affinity of interactions between proteins of the invasion complex using surface plasmon resonance. 6)I have produced recombinant nitrophorin 2, an inhibitor of coagulation factor IX. This is being used in the evaluation of preparations of coagulation factor VIII that are used for treatment of hemophilia. 7)We have found salivary proteins specifically binding cysteinyl leukotrienes that belong to several different protein families and occur in a variety of blood feeding vector insects. Recently we discovered a member of the lipocalin protein that binds leukotriene C4 with very high affinity. We will attempt to determine the crystal structure of this molecule in complex with the ligand. These results, along with those from other studies indicate that removal of this proinflammatory mediator is particularly important for feeding. We will investigate the effects this protein using model animal systems.
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