Evidence shows that Paneth cell a-defensins affect in innate immunity in vivo. Mouse a-defensins are broadly bactericidal at low ?M levels and are released at mM quantities. Thus, there is strong rationale for determining structural determinants of Paneth cell a-defensins and their precursors, mechanisms of their biosynthesis, and the role of novel defensin-related Paneth cell gene products in enteric immunity.
In Aim #1, the bactericidal mechanisms of Crp4 disulfide variants will be studied by determining their membrane disruptive properties, conformational changes in membrane mimetic environments, and by characterizing bactericidal subfragments of Crp4 mutant peptides. The effects of Crp4 mutagenesis at Arg7, Glu15, and Gly19, residue positions conserved in all a-defensins, on bactericidal activities, peptide refolding and structure, and membrane disruptive behavior. We will determine the NMR structures of Crp4 and site-directed mutants of Crp4 in the presence of rapidly-tumbling phospholipid bicelles to test the hypothesis that specific residue positions that interact with phospholipid bilayers are determinants of bactericidal peptide activity.
In Aim #2, we will mutate Asp and Glu residues in the proCrp4 proregion and measure effects of mutagenesis on proCrp4 bactericidal activity, membrane disruptive behavior, and conformation. Arg to Lys mutations will be made in Crp4 and proCrp4 at all positions, and the effects of mutagenesis on mechanisms of bactericidal activity, conformational changes, and peptide folding will be characterized. The structural basis for proCrp4 inactivity will be studied by determining the solution structure of native and mutant proCrp4 molecules by NMR spectroscopy.
Aim #3 is focused on characterizing new Paneth cell defensin-related gene products by determining the bactericidal activities, membrane disruptive properties, and secondary structures of CRS1C and CRS4C. The processing and activation mechanisms of proCRSIC and proCRS4C in mouse Paneth cells will be identified, and the structures of CRS4C and CRS1C in solution and in bicelles will be determined by NMR spectroscopy. From these studies, an understanding of the functional determinants of these key biochemical components of enteric innate immunity will be gained in mechanistic and molecular terms and at the structural level. Knowledge of such mechanisms is essential to augment innate defenses and to insure compatibility of new therapeutics with endogenous effectors of host defense.
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