PEPTIDE EFFECTORS OF ENTERIC HOST DEFENSE

描述（由申请人提供）：证据表明Paneth细胞a-防御素在体内影响先天免疫。小鼠a-防御素在低浓度时具有广泛的杀菌作用，并以浓度释放。因此，确定Paneth细胞a-防御素及其前体的结构决定因素、其生物合成机制以及新的防御素相关Paneth细胞基因产物在肠道免疫中的作用具有很强的理论基础。在Aim #1中，将通过确定其膜破坏特性、膜模拟环境中的构象变化以及表征Crp4突变肽的杀菌亚片段来研究Crp4二硫变体的杀菌机制。在Arg7、Glu15和Gly19（所有a-防御素中保守的残基位置）上突变Crp4对杀菌活性、肽重折叠和结构以及膜破坏行为的影响。我们将在快速翻滚的磷脂单胞体存在下测定Crp4和Crp4位点定向突变体的核磁共振结构，以验证与磷脂双层相互作用的特定残基位置是杀菌肽活性的决定因素的假设。在Aim #2中，我们将突变proCrp4前区的Asp和Glu残基，并测量突变对proCrp4杀菌活性、膜破坏行为和构象的影响。将在Crp4和proCrp4的所有位置发生Arg到Lys突变，并将对突变对杀菌活性、构象变化和肽折叠的机制的影响进行表征。通过核磁共振光谱测定天然和突变体proCrp4分子的溶液结构，研究proCrp4失活的结构基础。Aim #3的重点是通过确定CRS1C和CRS4C的杀菌活性、膜破坏特性和二级结构来表征新的Paneth细胞防御相关基因产物。鉴定小鼠Paneth细胞中proccrsic和proccrs4c的加工和激活机制，并通过核磁共振光谱测定CRS4C和CRS1C在溶液和小束中的结构。从这些研究中，对肠道先天免疫这些关键生化成分的功能决定因素的理解将在机制和分子方面以及在结构水平上获得。了解这些机制对于增强先天防御和确保新疗法与宿主防御的内源性效应物的相容性至关重要。

DESCRIPTION (provided by applicant): 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.