| Title: | A common genetic system for functional studies of pitrilysin and related M16A proteases |
| Author(s): | Alper BJ; Nienow TE; Schmidt WK; |
| Address: | "Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, GA 30602, USA" |
| ISSN/ISBN: | 1470-8728 (Electronic) 0264-6021 (Print) 0264-6021 (Linking) |
| Abstract: | "Pitrilysin is a bacterial protease that is similar to the mammalian insulin-degrading enzyme, which is hypothesized to protect against the onset of Alzheimer's disease, and the yeast enzymes Axl1p and Ste23p, which are responsible for production of the a-factor mating pheromone in Saccharomyces cerevisiae. The lack of a phenotype associated with pitrilysin deficiency has hindered studies of this enzyme. Herein, we report that pitrilysin can be heterologously expressed in yeast such that it functionally substitutes for the shared roles of Axl1p and Ste23p in pheromone production, resulting in a readily observable phenotype. We have exploited this phenotype to conduct structure-function analyses of pitrilysin and report that residues within four sequence motifs that are highly conserved among M16A enzymes are essential for its activity. These motifs include the extended metalloprotease motif, a second motif that has been hypothesized to be important for the function of M16A enzymes, and two others not previously recognized as being important for pitrilysin function. We have also established that the two self-folding domains of pitrilysin are both required for its proteolytic activity. However, pitrilysin does not possess all the enzymatic properties of the yeast enzymes since it cannot substitute for the role of Axl1p in the repression of haploid invasive growth. These observations further support the utility of the yeast system for structure-function and comparative studies of M16A enzymes" |
| Keywords: | "Amino Acid Motifs Biological Assay/*methods DNA Mutational Analysis DNA, Fungal/genetics Gene Expression Genes, Mating Type, Fungal/genetics Haploidy Lipoproteins/biosynthesis/chemistry Mass Spectrometry Metalloendopeptidases/chemistry/classification/*gen;" |
| Notes: | "MedlineAlper, Benjamin J Nienow, Tatyana E Schmidt, Walter K eng Research Support, Non-U.S. Gov't England 2006/05/26 Biochem J. 2006 Aug 15; 398(1):145-52. doi: 10.1042/BJ20060311" |
