The overall goal of the proposed collaboration is to provide information about how structural changes within motile cells such as macrophages, platelets and lymphocytes are achieved when these cells respond to extracellular signals. Cell motility is necessary for such physiologic processes as phagocytosis, platelet response during blood coagulation, wound healing, and neurologic development. Understanding the link between cell signalling and motility is important for understanding the defects that causes impaired or uncontrolled movements and proliferation. This work focuses on the actin filament-severing proteins gelsolin, villin, and other actin-binding proteins such as profilin that are regulated by polyphosphoinositides (PPI's). Polyphosphoinositides are ubiquitous phospholipid components of the cell membrane whose phosphorylation or hydrolysis by several different enzymes is involved in many signal transduction pathways. The parent project on which this collaboration is based involves biochemical and biophysical studies of actin and other protein networks, designed to determine how such networks are remodeled and how their remodeling may be involved in producing motion. This extension will allow detailed analysis of the site on proteins to which polyphosphoinositides bind and of the structural changes within the protein caused by this binding.
The specific aim of this proposal is to examine interactions of polyphosphoinositides with synthetic peptides based on the sequences within proteins implicated as potential PPI-binding sites by studies of proteolytically-derived protein fragments or mutants. The advantage of studying small synthetic peptides is that their structures can be studied more rigorously than intact proteins, and in some cases their solution structures can be completely determined by nmr and other methods as discussed in the parent grant. Peptide synthesis requires specialized skills and equipment that are very different from those required to study cell biology or the effects of the peptides on, for example, gelsolin/PPI interactions, and therefore the parent grant made no provision for peptide synthesis. The opportunity to collaborate with a laboratory of organic synthesis that specializes in peptides expands the range of experiments that can be applied to study the regulation of actin-binding proteins and will increase the precision with which the sites necessary for protein regulation by PPI's can be defined. The latter possibility could potentially be relevant to the design of pharmacologic agents that modulate PPI-metabolism or cytoskeletal remodeling in vivo.
