Homologues of insulinase, a new superfamily of metalloendopeptidases

Rawlings, Neil D.; Barrett, Alan J.

doi:10.1042/bj2750389

Cited by 102 publications

(51 citation statements)

References 35 publications

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“…The identified metalloprotease exhibits high overall homology with proteases of the pitrilysin subfamily. Members of the pitrilysin family are oligopeptidases of 100 kDa, such as insulinase from mammals and its homologue in bacteria, protease III (64). In the cell, these peptidases degrade small peptides of the similar size as mitochondrial presequences (65).…”

Section: Discussionmentioning

confidence: 99%

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

Ståhl¹,

Moberg²,

Ytterberg³

et al. 2002

Journal of Biological Chemistry

115

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Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form ␣-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F 1 ␤ subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Twodimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.

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Section: Discussionmentioning

confidence: 99%

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

Ståhl¹,

Moberg²,

Ytterberg³

et al. 2002

Journal of Biological Chemistry

115

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“…The Zn-MPs belong to the pitrilysin subfamily A. This subfamily contains oligopeptidases such as the human Insulin Degrading Enzyme (IDE) and the bacterial homologue protease III (Rawlings and Barrett, 1991). Site-directed mutagenesis of the zinc-binding motif in IDE and protease III has revealed different functions for the conserved residues (Becker and Roth, 1993;Perlman et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts

et al. 2003

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Both authors contributed equally to this work. SummaryWe have recently isolated and identi®ed a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli. We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate speci®city and intracellular localization. Mutagenesis studies of the residues involved in metal binding identi®ed the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate speci®city studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se. Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro. In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria ± degradation of targeting peptides.

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“…1), that is part of a conserved motif present in several other zinc-metalloendopeptidases (see below; for review, see ref. 30).…”

Section: -Rglelangikvllisd------------------------pttdk-ssaaldvhigsmentioning

confidence: 99%

“…The consensus sequence (see Fig. 3) reveals that the insulinase signature, G( (30), which is an extended zinc binding motif, is perfectly conserved in NRD convertase. From the multiple alignment, it appears that the NRD convertase acidic stretch is flanked by two short anchors of 12 and 7 residues and, surprisingly, is inserted in the most conserved region between all the members ofthe family, =25 residues upstream the HXXEH zinc binding site.…”

Section: -Rtvtlpgglqatlvhq------------------------pqadr-aaalarvaagshmentioning

confidence: 99%

“…31), the rat MPP a subunit (32), and the Bacillus subtilis processing protease, a putative gene flanking the diaminopimelate operon (33). This search for related protein sequences did not select 12 additional species that could be classified in the M16 family, among which a majority exhibit the insulinase signature (30). A multiple alignment of NRD convertase with the selected protein sequences in the region containing the zinc binding motif (Fig.…”

Section: -Rtvtlpgglqatlvhq------------------------pqadr-aaalarvaagshmentioning

confidence: 99%

See 1 more Smart Citation

N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes.

Pierotti

Prat

Chesneau

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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N-Arg dibasic convertase is a me tidase from rat brain cortex and testis that cleaves peptide substrates on the N terminu ofArg residues in dibasic s s.By using both an oinu ide and antibodies to screen a rat testis cDNA library, a fill-length cDNA (4,5). On the basis of its similarity to subtilisin and to furin (6), a human homolog of the Kex2 protein, a family of prohormone convertases (PCs) has been identified by PCR techniques. Their involvement in processing of a number of propeptides and proproteins was inferred mainly from cotransfection experiments (7-9), and for PC1, by the use of antisense mRNA (10).Characterization of putative processing endoproteases by classical biochemical techniques has led to the identification of a number of activities, selective for basic residues in precursors, that belong to the four classes of proteases (metallo-, serine, aspartyl, and thiol enzymes; for review, see ref. 11). This suggested that more than one processing endoprotease family could exist (12). To our knowledge, none of these basic-residue-specific enzymes has been cloned.Recently, a metalloendopeptidase was completely purified from rat testis and shown to cleave a number of peptide substrates on the N terminus of Arg residues in dibasic moieties (13). This enzyme was also present in rat brain cortex and its functional properties appeared undistinguishable from those of the somatostatin-28 convertase activity previously identified in this tissue (14,15). By using microsequencing of tryptic fragments of the purified enzyme to design an oligonucleotide probe and polyclonal antibodies raised against the purified protein (13) (20)].The in vitro highly restricted specificity ofNRD convertase for Arg residues in dibasic processing signals and its belonging to the M16 family, which contains other enzymes involved in maturation, suggest that this enzyme is the prototype of a distinct family of processing endoproteases. MATERIALS AND METHODSIsolatin and Cherizatin of cDNA Cones E d NRD Convertae. Four tryptic fragments were sequenced after digestion of previously purified NRD convertase following native PAGE. One fragment, GMQLIYLPPSPLLAE, was used to design the following degenerate inosine-containing oligonucleotide: 5'-GGIGGIAG(A/G)TAIATIA(G/A)(T/ C)TGCATICC-3'. Two additional peptides were obtained by endolysine C treatment (13) autoradiography of the filters, positive phage plaques were isolated and rescreened to obtain single purified phage isolates. A similar aliquot ofthe library was plated onto NZ-amine medium plates for screening using polyclonal antibodies raised Abbreviations: NRD convertase, N-arginine dibasic convertase; IDE, insulin degrading enzyme; MPP, mitochondrial matrixprocessing peptidase.

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Homologues of insulinase, a new superfamily of metalloendopeptidases

Cited by 102 publications

References 35 publications

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts

N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes.

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