A common genetic system for functional studies of pitrilysin and related M16A proteases

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.

Anion Activation Site of Insulin-degrading Enzyme

“…IDE Activity Assay in Yeast-Wild type IDE and IDE K898A,K899A,S901A activities were compared in Saccharomyces cerevisiae using both a serial dilution mating test and a spot halo assay as described previously (37,38). The assays take advantage of the ability of IDE, in lieu of the yeast M16 metallopeptidases Axl1p and Ste23p, to participate in the proteolytic (40)).…”

“…Effect of ATP-binding Site Mutation on Intracellular Activity of IDE-An established yeast system for functional studies of IDE (37,38) was used to assess the significance of anion activation on IDE activity in a cellular environment. The assay is based on the ability of IDE to support production of mature a-factor mating pheromone in the absence of yeast metallopeptidases (i.e.…”

Yeast Ste23p shares functional similarities with mammalian insulin‐degrading enzymes

“…Ste23p and Axl1p are not functionally equivalent in terms of their ability to promote mature a -factor production. Ste23p is responsible for at most 5% of all a -factor production, yet it is expressed at steady-state levels that are at least 10 times higher than that observed for Axl1p (Adames et al , 1995; Alper et al , 2006). These observations suggest an alternative biological function of Ste23p that has yet to be identified.…”

“…M16A enzymes have a quaternary structure that is similar to a clamshell in appearance, with N- and C-terminal bowl-shaped domains of approximately 50 kDa connected by a short linker region of 20–30 residues. Both the N- and C-terminal domains, as well as individual residues from highly conserved sequences within each domain, are required for the catalytic function of M16A enzymes (Alper et al , 2006; Becker and Roth, 1993; Ding et al , 1992; Kim et al , 2005; Li et al , 2006; Perlman et al , 1993). Recent studies of the human insulin-degrading enzyme ( Hs IDE) and bacterial pitrilysin ( Ec Ptr) have demonstrated that M16A enzymes can adopt either an ‘open’ conformation, which is hypothesized to permit substrate access to a central catalytic compartment, or a ‘closed’ conformation, which is presumed to be necessary for proteolysis, yet likely prohibits entrance and exit of substrates from the catalytic site (Maskos, 2005; Shen et al , 2006).…”

The metalloendopeptidase gene Pitrm1 is regulated by hedgehog signaling in the developing mouse limb and is expressed in muscle progenitors

“…This family is characterised by an inverted zinc-binding motif HXXEH(X) 76 E (Rawlings and Barrett, 1995) and a number of its members are responsible for degrading short peptides by encapsulating substrates in a catalytic chamber (Malito et al, 2008). In vertebrates, there are several well-characterised M16 proteins that share structural similarity with Pitrm1, including Insulin Degrading Enzyme (IDE), NRD convertase, and Mitochondrial Processing Peptidase (MPP) (Fumagalli et al, 1998;Alper et al, 2006). Of these, IDE has attracted the most research attention due to its ability to hydrolyse insulin (Kirschner and Goldberg, 1983), IGF-I, IGF-II (Misbin and Almira, 1989), and the Amyloid b-protein (Ab) (McDermott and Gibson, 1997).…”