The long term objective of the proposed research is to enhance our understanding of the molecular mechanisms of lymphocyte chemotaxis. This is a process critical to the normal functioning of the immune system, but also implicated in a variety of diseases such as atherosclerosis and autoimmune disease where chemotactic signaling becomes unbalanced. A growing family of small soluble chemokines has recently been discovered whose primary role is to direct the migration of specific lymphocytes to areas of physiological insult, and to activate the lymphocytes for destruction of the antigen. This occurs by the interaction of these chemokines with receptors on macrophages, neutrophils and T-cells which initiates a signal transduction cascade coordinating the inflammatory response. Understanding the detailed molecular structures of the chemokines, points of contact with the receptor binding sites, and regions of the proteins critical for signal transduction will advance our general understanding of lymphocyte chemotaxis. Detailed comparison of the structures of several related chemokines will also forward our understanding of protein folding and the energetic interactions which allow different primary sequences to adopt similar 3-dimensional structures. Accordingly, the specific aims of the project are to: 1) determine the dynamics and high resolution NMR structures of several homologous beta-chemokines (MCP-1, MIP-1alpha and I-3090 involved in macrophage-mediated chemotaxis and chronic inflammation. 2) evaluate the aggregation states and stabilities of MCP-1, MIP-1alpha and I-309 under a variety of solution conditions. 3) map the regions of the chemokines (MCP-1 and MIP-1alpha) important for receptor binding and signal transduction; this will be accomplished using mutagenesis and assays of functional biological activity (binding, chemotaxis, and Ca+2 influx).
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