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Signal epitopes in peptide precursor processing and sorting
180 SIGNAL EPITOPES IN PEPTIDE PRECURSOR PROCESSING AND SORTING
Thue W. Schwartz, Birgitte S.Wulff, Marianne Skak Pedersen, Teit E. Johansen, Charlotte K. Vogel, Susanne Tolstoy, Jannie Fuhlendorff, and Mairead M.T. O'Hare. Laboratory for Molecular Endocrinology, Rigshospitalet 6321, Copenhagen, Denmark. Regulatory peptides display epitopes which are recognized by their corresponding receptors and thereby delineates their biological signal. Likewise, precursors for peptides contain information for their intracellular sorting and for their processing and derivatization. This structural information is apparently not confined to the primary sequence of the precursors as such; e.g. some dibasic sequences are used as processing sites, whereas others, surrounded by apparently similar residues, are not processed even within the same precursor. Our basic hypothesis is, that signal epitopes of both the final, activated peptide and its precursor must be sought in their three-dimensional structure. However no peptide precursor has yet been truly characterized in respect of the three-dimensional structure. We have chosen to perform structure-function studies on peptides and their biosynthetic precursors in parallel. As model peptides we are using peptides, neuropeptide Y (NPY), pancreatic polypeptide, and insulin, which all have been shown to display a well ordered structure using Xray crystallography, and which all appear to hold this structure in aqueous solution as demonstrated by C.D. and/or NMR analysis. Because these peptides constitute a major part of their respective, relatively small precursors, it is assumed that the unknown structures of the precursors are built over the structures of the peptides. The protein engineering of the peptides is performed through the production of synthetic peptide analogs; whereas the structure of the peptide-precursors are modified through site directed mutagenesis of their cDNAs. The binding and biological activity of the peptides are studied in membrane preparations, cell cultures and in vivo. The sorting and processing of the precursors are studied in well differentiated neuroendocrine cells after transfection with constitutive and inducible eukaryotic expression vectors. The effect of the substitutions on the secondary structure of the peptides is characterized by e.g. circular dichroism. In order to be able to perform more drastic deletions, substitutions etc. of the peptide precursor we have also constructed hybrid molecules in which the pro-NPY is combined with a reporter protein, tile thiolprotease inhibitor cystatin-C. As alternative models in studies on the sorting of the precursors, we are also using e.g. pancreatic secretory trypsin inhibitor, a small secretory molecule which in contrast to the peptide precursors is structurally well characterized. The mutations and substitutions were primarily aimed at making major changes in the threedimensional structure of the peptides and the corresponding precursors (in order to test the basic hypothesis); and against changing e.g. known processing sites. One of the first, major results in this series of mutations is the demonstration that e.g. dibasic sequences which are found in alpha-helical segments are normally not accessible for processing; however, when we though different site-specific mutations alter the secondary structure of the protein processing occurs. The second series of mutations are directed towards alterations in the areas of the peptides which are known to be involved in the dimerization and further oligomerization of the peptides. These mutations are based on the hypothesis that oligomerization of peptides and or oligomerization of their precursors, occurring under the special physio-chemical conditions in particular compartments of the secretory pathway, is important for the sorting process.
Thue W. Schwartz, Birgitte S.Wulff, Marianne Skak Pedersen, Teit E. Johansen, Charlotte K. Vogel, Susanne Tolstoy, Jannie Fuhlendorff, and Mairead M.T. O'Hare. Laboratory for Molecular Endocrinology, Rigshospitalet 6321, Copenhagen, Denmark. Regulatory peptides display epitopes which are recognized by their corresponding receptors and thereby delineates their biological signal. Likewise, precursors for peptides contain information for their intracellular sorting and for their processing and derivatization. This structural information is apparently not confined to the primary sequence of the precursors as such; e.g. some dibasic sequences are used as processing sites, whereas others, surrounded by apparently similar residues, are not processed even within the same precursor. Our basic hypothesis is, that signal epitopes of both the final, activated peptide and its precursor must be sought in their three-dimensional structure. However no peptide precursor has yet been truly characterized in respect of the three-dimensional structure. We have chosen to perform structure-function studies on peptides and their biosynthetic precursors in parallel. As model peptides we are using peptides, neuropeptide Y (NPY), pancreatic polypeptide, and insulin, which all have been shown to display a well ordered structure using Xray crystallography, and which all appear to hold this structure in aqueous solution as demonstrated by C.D. and/or NMR analysis. Because these peptides constitute a major part of their respective, relatively small precursors, it is assumed that the unknown structures of the precursors are built over the structures of the peptides. The protein engineering of the peptides is performed through the production of synthetic peptide analogs; whereas the structure of the peptide-precursors are modified through site directed mutagenesis of their cDNAs. The binding and biological activity of the peptides are studied in membrane preparations, cell cultures and in vivo. The sorting and processing of the precursors are studied in well differentiated neuroendocrine cells after transfection with constitutive and inducible eukaryotic expression vectors. The effect of the substitutions on the secondary structure of the peptides is characterized by e.g. circular dichroism. In order to be able to perform more drastic deletions, substitutions etc. of the peptide precursor we have also constructed hybrid molecules in which the pro-NPY is combined with a reporter protein, tile thiolprotease inhibitor cystatin-C. As alternative models in studies on the sorting of the precursors, we are also using e.g. pancreatic secretory trypsin inhibitor, a small secretory molecule which in contrast to the peptide precursors is structurally well characterized. The mutations and substitutions were primarily aimed at making major changes in the threedimensional structure of the peptides and the corresponding precursors (in order to test the basic hypothesis); and against changing e.g. known processing sites. One of the first, major results in this series of mutations is the demonstration that e.g. dibasic sequences which are found in alpha-helical segments are normally not accessible for processing; however, when we though different site-specific mutations alter the secondary structure of the protein processing occurs. The second series of mutations are directed towards alterations in the areas of the peptides which are known to be involved in the dimerization and further oligomerization of the peptides. These mutations are based on the hypothesis that oligomerization of peptides and or oligomerization of their precursors, occurring under the special physio-chemical conditions in particular compartments of the secretory pathway, is important for the sorting process.