Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.56 (insulin-degrading enzyme)
 737 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 1242 base pair DNA fragment from Bacillus halodurans H4 isolated from alkaline sediments of Lake Bogoria (Kenya) coding for a potential protease was cloned and sequenced. The hexa-histidine-tagged enzyme was overexpressed in Escherichia coli and was purified in one step by immobilized-metal affinity chromatography (IMAC) on Ni-NTA resin. The protease (ppBH4) presents an inverted zincin motif, HXXEH, which defines the inverzincin family. It shares several biochemical and molecular properties with the clan ME family M16 metallopeptidases (pitrilysins), as well as with database hypothetical proteins that are potential M16 family enzymes. Thus, like insulysin and nardilysin, but contrary to bacterial pitrilysin, ppBH4 is inactivated by sulfhydryl alkylating agents. On the other hand, like bacterial pitrilysin, ppBH4 is sensitive to reducing agents. The enzymatic activity of ppBH4 is limited to substrates smaller than proteins. In contrast to insulin, dynorphin and insulin B-chain are very good substrates for ppBH4 and several cleavage sites are common with those observed with well-characterized pitrilysins. As deduced from amino acid sequence, as well as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis, ppBH4 is an active monomer of 46.5 kDa. This feature distinguishes ppBH4 from all other enzymes of the pitrilysin family so far described whose molecular masses range from 100 to 140 kDa.
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PMID:Cloning, expression and characterization of a 46.5-kDa metallopeptidase from Bacillus halodurans H4 sharing properties with the pitrilysin family. 1586 16

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).
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PMID:Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid beta-peptide cleaved from a recombinant fusion protein. 1623 26

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.
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PMID:A common genetic system for functional studies of pitrilysin and related M16A proteases. 1672 21


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