This proposal focuses on using proinsulin as a model substrate to address two fundamental questions regarding peptide-converting enzymes (PCE)-mediated processing. First, is the processing specificity determined by structural elements within the PCEs? Second, what specific structural elements of the PCEs are involved in the precise recognition of propeptide cleavage sites? The inial goal of this project is to obtain sufficient proinsulin for use in an in vitro enzyme assay. Products of the in vitro assay will be subjected to structural characterization to conclusively define the sites of PCE-mediated cleavage. This assay will then be used to test the effects of microdomain swapping between PC1 and PC2 in an attempt to define which structural regions of an individual furin/PC member is responsible for substrate recognition. Finally the proinsulin substrate will be manipulated to determine whether its three dimensional structure affects processing specificity. Results from these studies should provide valuable information regarding the tertiary structural interactions between the PCEs and their propeptide substrates. A number of cases of hyperproinsulinemia, hyperproglucanemia, insulin resistance and at least two cases of hemophilia have been shown to be caused by mutations affecting the endoproteolytic cleavage site in propeptides. These observations raise the possibility that patients could be treated by administering a modified version of the PCE via gene therapy.
| Yang, Yanwu; Hua, Qing-Xin; Liu, Jin et al. (2010) Solution structure of proinsulin: connecting domain flexibility and prohormone processing. J Biol Chem 285:7847-51 |
| Liu, Ming; Wan, Zhu-Li; Chu, Ying-Chi et al. (2009) Crystal structure of a ""nonfoldable"" insulin: impaired folding efficiency despite native activity. J Biol Chem 284:35259-72 |
