PEPTIDE EFFECTORS OF ENTERIC HOST DEFENSE

描述（由申请人提供）：证据表明潘氏细胞α-防御素影响体内先天免疫。小鼠α-防御素在低μ M水平下具有广泛的杀菌作用，并以mM的量释放。因此，有很强的理由来确定潘氏细胞α-防御素及其前体的结构决定因素，其生物合成的机制，以及新型防御素相关的潘氏细胞基因产物在肠道免疫中的作用。在目标#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Crp 4突变肽的杀菌亚片段来研究Crp 4二硫键变体的杀菌机制。Crp 4在Arg 7、Glu 15和Gly 19（所有α-防御素中保守的残基位置）突变对杀菌活性、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双胞的存在下确定Crp 4和Crp 4定点突变体的NMR结构，以测试与磷脂双分子层相互作用的特定残基位置是杀菌肽活性决定因素的假设。在目标#2中，我们将突变proCrp 4前区中的Asp和Glu残基，并测量突变对proCrp 4杀菌活性、膜破坏行为和构象的影响。将在Crp 4和proCrp 4的所有位置进行Arg至Lys突变，并表征诱变对杀菌活性、构象变化和肽折叠机制的影响。将通过NMR光谱测定天然和突变proCrp 4分子的溶液结构，研究proCrp 4无活性的结构基础。目的#3通过确定CRS 1C和CRS 4C的杀菌活性、膜破坏性质和二级结构来表征新的潘氏细胞防御素相关基因产物。将鉴定proCRSIC和proCRS 4C在小鼠潘氏细胞中的加工和活化机制，并且将通过NMR光谱确定溶液和双胞中的CRS 4C和CRS 1C的结构。从这些研究中，了解肠道先天免疫的这些关键生化成分的功能决定因素，将获得在机制和分子方面，并在结构水平。这些机制的知识是必不可少的，以增加先天防御，并确保新的治疗与宿主防御的内源性效应物的兼容性。

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.