FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1972693783> ?p ?o ?g. }

• W1972693783 endingPage "52963" @default.
• W1972693783 startingPage "52953" @default.
• W1972693783 abstract "Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated. Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated. The N-terminal methionine excision (NME) 1The abbreviations used are: NMEN-terminal methionine excisionCDcatalytic domainDAPI4′-6-diamidino-2-phenylindoleFoN-formylGFPgreen fluorescent proteinHEKhuman embryonic kidneyEcE. coliHsH. sapiensMAPmethionine aminopeptidaseORFopen reading framePDFpeptide deformylasePDFIpeptide deformylase inhibitorspNAp-nitroanilideRFPred fluorescent proteinTCEPTris(2-carboxyethyl)-phosphineAtA. thaliana.1The abbreviations used are: NMEN-terminal methionine excisionCDcatalytic domainDAPI4′-6-diamidino-2-phenylindoleFoN-formylGFPgreen fluorescent proteinHEKhuman embryonic kidneyEcE. coliHsH. sapiensMAPmethionine aminopeptidaseORFopen reading framePDFpeptide deformylasePDFIpeptide deformylase inhibitorspNAp-nitroanilideRFPred fluorescent proteinTCEPTris(2-carboxyethyl)-phosphineAtA. thaliana. pathway is an essential pathway that removes the initial Met from two-thirds of the proteins of any proteome (1Meinnel T. Mechulam Y. Blanquet S. Biochimie (Paris). 1993; 75: 1061-1075Crossref PubMed Scopus (211) Google Scholar). Methionine aminopeptidase (MAP; EC 3.4.11.18) has been shown to be involved in this process in all organisms studied, and this activity facilitates subsequent protein modification in Eukaryotes (2Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (348) Google Scholar, 3Boisson B. Giglione C. Meinnel T. J. Biol. Chem. 2003; 278: 43418-43429Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In Eubacteria, plastids, and mitochondria, the N-terminal Met residue of nascent polypeptides carries an N-formyl (Fo) group whereas in Eukaryotes and Archaea, nascent proteins synthesized in the cytoplasm start with a free Met (4Kozak M. Microbiol. Reviews. 1983; 47: 1-45Crossref PubMed Google Scholar). The N-terminal Met must be exposed to allow MAP activity in Eubacteria, and this is achieved by systematic removal of the Fo group by peptide deformylase (PDF; EC 3.5.1.88). PDF activity was long considered to be unique to this kingdom (5Mazel D. Pochet S. Marliere P. EMBO J. 1994; 13: 914-923Crossref PubMed Scopus (220) Google Scholar). Recent studies have demonstrated that PDF orthologs are produced in most eukaryotes, including animals, plants, and many unicellular organisms (6Giglione C. Pierre M. Meinnel T. Mol. Microbiol. 2000; 36: 1197-1205Crossref PubMed Scopus (173) Google Scholar, 7Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (181) Google Scholar, 8Meinnel T. Parasitol. Today. 2000; 16: 165-168Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These orthologs have been shown to display peptide deformylase activity in plants, both in vitro and in vivo. All the eukaryotic orthologs have an extended N-terminal domain. The N-terminal domain targets the proteins to the plastids and mitochondria in plants (7Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (181) Google Scholar, 9Giglione C. Meinnel T. Trends Plant Sci. 2001; 6: 566-572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 10Giglione C. Vallon O. Meinnel T. EMBO J. 2003; 22: 13-23Crossref PubMed Scopus (117) Google Scholar, 11Serero A. Giglione C. Meinnel T. J. Mol. Biol. 2001; 314: 695-708Crossref PubMed Scopus (72) Google Scholar). Similarly, it has been suggested that the PDF orthologs of Apicomplexan parasites are targeted to the apicoplast, an essential plastid (8Meinnel T. Parasitol. Today. 2000; 16: 165-168Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Bracchi-Ricard V. Nguyen K.T. Zhou Y. Rajagopalan P.T. Chakrabarti D. Pei D. Arch. Biochem. Biophys. 2001; 396: 162-170Crossref PubMed Scopus (59) Google Scholar, 13Giglione C. Meinnel T. Emerg. Therap. Targets. 2001; 5: 41-57Crossref PubMed Scopus (33) Google Scholar). Actinonin, a natural antibiotic that specifically inhibits PDF, targets both bacterial and plant PDFs (10Giglione C. Vallon O. Meinnel T. EMBO J. 2003; 22: 13-23Crossref PubMed Scopus (117) Google Scholar, 14Chen D.Z. Patel D.V. Hackbarth C.J. Wang W. Dreyer G. Young D.C. Margolis P.S. Wu C. Ni Z.J. Trias J. White R.J. Yuan Z. Biochemistry. 2000; 39: 1256-1262Crossref PubMed Scopus (277) Google Scholar). Studies carried out in vivo with this drug have shown that PDF is essential in bacteria and plastids (11Serero A. Giglione C. Meinnel T. J. Mol. Biol. 2001; 314: 695-708Crossref PubMed Scopus (72) Google Scholar, 14Chen D.Z. Patel D.V. Hackbarth C.J. Wang W. Dreyer G. Young D.C. Margolis P.S. Wu C. Ni Z.J. Trias J. White R.J. Yuan Z. Biochemistry. 2000; 39: 1256-1262Crossref PubMed Scopus (277) Google Scholar, 15Wiesner J. Sanderbrand S. Beck E. Jomaa H. Trends Parasitol. 2001; 17: 7Abstract Full Text Full Text PDF PubMed Google Scholar). In plants plastids, NME plays a crucial role in controlling the half-life of a major organellar protein complex, photosystem II (10Giglione C. Vallon O. Meinnel T. EMBO J. 2003; 22: 13-23Crossref PubMed Scopus (117) Google Scholar). The effect on photosystem II stability was similar regardless of whether Met alone or Fo-Met was retained. N-terminal methionine excision catalytic domain 4′-6-diamidino-2-phenylindole N-formyl green fluorescent protein human embryonic kidney E. coli H. sapiens methionine aminopeptidase open reading frame peptide deformylase peptide deformylase inhibitors p-nitroanilide red fluorescent protein Tris(2-carboxyethyl)-phosphine A. thaliana. N-terminal methionine excision catalytic domain 4′-6-diamidino-2-phenylindole N-formyl green fluorescent protein human embryonic kidney E. coli H. sapiens methionine aminopeptidase open reading frame peptide deformylase peptide deformylase inhibitors p-nitroanilide red fluorescent protein Tris(2-carboxyethyl)-phosphine A. thaliana. MAPs are part of a large family of metallo-aminopeptidases (16Lowther W.T. Matthews B.W. Chem. Rev. 2002; 102: 4581-4608Crossref PubMed Scopus (277) Google Scholar). Two types of MAPs have been described to date (17Bradshaw R.A. Brickey W.W. Walker K.W. Trends Biochem. Sci. 1998; 23: 263-267Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). MAP2s occur in Archaea and in the cytoplasm of Eukaryotes, whereas MAP1s have been found in Eubacteria, in the cytoplasm of Eukaryotes (MAP1A) and in the organelles of plants and Apicomplexa (MAP1D, Ref. 7Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (181) Google Scholar). The sequences of MAP2 and MAP1 have weak similarity but these two enzymes display similar active site folding patterns. Organellar MAP1s have a cleavable N-terminal extension that is not found in bacterial MAP1s. This extension targets the catalytic domain to the correct cell compartment. Cytoplasmic MAP1A enzymes also have an extension including a conserved zinc finger. Although not involved in catalytic activity, this additional domain is not removed from the mature form and is essential for cell function, possibly allowing interaction with ribosomes (18Vetro J.A. Chang Y.H. J. Cell Biochem. 2002; 85: 678-688Crossref PubMed Scopus (45) Google Scholar). PDFs constitute a growing family of hydrolytic enzymes related to the thermolysin-metzincin HEXXH motif-containing family of metalloproteases (6Giglione C. Pierre M. Meinnel T. Mol. Microbiol. 2000; 36: 1197-1205Crossref PubMed Scopus (173) Google Scholar). PDFs contain three distinct short stretches of amino acids (Motifs 1-3; Fig. 1) that constitute the active site (19Meinnel T. Lazennec C. Villoing S. Blanquet S. J. Mol. Biol. 1997; 267: 749-761Crossref PubMed Scopus (77) Google Scholar). Motif 3 contains the HEXXH motif (Fig. 1). In most PDFs, an Fe2+ cation is bound by three residues of motifs 2 and 3 and plays a crucial role in hydrolytic activity (20Rajagopalan P.T.R. Yu X.C. Pei D. J. Am. Chem. Soc. 1997; 119: 12418-12419Crossref Google Scholar, 21Groche D. Becker A. Schlichting I. Kabsch W. Schultz S. Wagner A.F. Biochem. Biophys. Res. Commun. 1998; 246: 342-346Crossref PubMed Scopus (111) Google Scholar). This ion is highly unstable, and several agents and procedures have been described that preserve deformylase activity (reviewed in Ref. 6Giglione C. Pierre M. Meinnel T. Mol. Microbiol. 2000; 36: 1197-1205Crossref PubMed Scopus (173) Google Scholar). The iron cation may be replaced by zinc, resulting in a stable but weakly active enzyme. Zinc PDFs have similar substrate specificities to their iron-coordinated counterparts, but lower catalytic constants (22Meinnel T. Blanquet S. J. Bacteriol. 1995; 177: 1883-1887Crossref PubMed Google Scholar, 23Ragusa S. Blanquet S. Meinnel T. J. Mol. Biol. 1998; 280: 515-523Crossref PubMed Scopus (110) Google Scholar). Three types of PDF have been identified on the basis of structural and large scale sequence analysis (6Giglione C. Pierre M. Meinnel T. Mol. Microbiol. 2000; 36: 1197-1205Crossref PubMed Scopus (173) Google Scholar, 13Giglione C. Meinnel T. Emerg. Therap. Targets. 2001; 5: 41-57Crossref PubMed Scopus (33) Google Scholar, 24Guilloteau J.P. Mathieu M. Giglione C. Blanc V. Dupuy A. Chevrier M. Gil P. Famechon A. Meinnel T. Mikol V. J. Mol. Biol. 2002; 320: 951-962Crossref PubMed Scopus (118) Google Scholar). The three classes differ in several parts of their three-dimensional structures but their active sites are conserved and entirely superimposable. Type 2 and type 3 PDF enzymes are found only in Gram-positive bacteria. In contrast to type 2, type 3 PDF orthologs have no associated deformylase activity, due to amino acid substitutions in motifs 1 to 3 (13Giglione C. Meinnel T. Emerg. Therap. Targets. 2001; 5: 41-57Crossref PubMed Scopus (33) Google Scholar, 25Margolis P. Hackbarth C. Lopez S. Maniar M. Wang W. Yuan Z. White R. Trias J. Antimicrob. Agents Chemother. 2001; 45: 2432-2435Crossref PubMed Scopus (69) Google Scholar). Each of the specific amino acids of the motifs appears to be required for enzyme activity and stability, as indicated by near-systematic site-directed mutagenesis analysis followed by in vivo and in vitro studies (19Meinnel T. Lazennec C. Villoing S. Blanquet S. J. Mol. Biol. 1997; 267: 749-761Crossref PubMed Scopus (77) Google Scholar, 26Meinnel T. Lazennec C. Blanquet S. J. Mol. Biol. 1995; 254: 175-183Crossref PubMed Scopus (73) Google Scholar, 27Meinnel T. Lazennec C. Dardel F. Schmitter J.M. Blanquet S. FEBS Lett. 1996; 385: 91-95Crossref PubMed Scopus (41) Google Scholar, 28Ragusa S. Mouchet P. Lazennec C. Dive V. Meinnel T. J. Mol. Biol. 1999; 289: 1445-1457Crossref PubMed Scopus (56) Google Scholar, 29Dardel F. Ragusa S. Lazennec C. Blanquet S. Meinnel T. J. Mol. Biol. 1998; 280: 501-513Crossref PubMed Scopus (85) Google Scholar). There are two classes of type 1 PDF. Class 1B (B for bacterial) PDF enzymes are found in Gram-negative bacteria, some Gram-positive bacteria and plants. Eukaryotic class 1B PDFs are targeted to both plastids and mitochondria (7Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (181) Google Scholar, 9Giglione C. Meinnel T. Trends Plant Sci. 2001; 6: 566-572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The three-dimensional structure and enzymatic properties of the class 1B PDFs of eukaryotes and bacteria are similar (11Serero A. Giglione C. Meinnel T. J. Mol. Biol. 2001; 314: 695-708Crossref PubMed Scopus (72) Google Scholar, 24Guilloteau J.P. Mathieu M. Giglione C. Blanc V. Dupuy A. Chevrier M. Gil P. Famechon A. Meinnel T. Mikol V. J. Mol. Biol. 2002; 320: 951-962Crossref PubMed Scopus (118) Google Scholar, 30Kumar A. Nguyen K.T. Srivathsan S. Ornstein B. Turley S. Hirsh I. Pei D. Hol W.G. Structure (Camb.). 2002; 10: 357-367Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Class 1A PDFs include plant mitochondrial PDFs and PDF orthologs from animals (9Giglione C. Meinnel T. Trends Plant Sci. 2001; 6: 566-572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Biochemical characterization of plant PDF1As has shown that they differ from most previously studied PDFs, including PDF1Bs and PDF2s, particularly in terms of the optimal metal cofactor: zinc rather than iron (11Serero A. Giglione C. Meinnel T. J. Mol. Biol. 2001; 314: 695-708Crossref PubMed Scopus (72) Google Scholar). In this work, we provide compelling evidence that the complete machinery, including specific and functional PDF and MAP1D, is expressed and exported to animal mitochondria. Site-directed mutagenesis data, combined with biochemical and structural analyses, showed that HsPDF differs considerably from previously characterized PDFs in terms of its properties. These differences could be used as the basis for chemical modifications for improving PDF inhibitors for use in the clinical treatment of bacterial infections in humans. All chemicals, including Tris(2-carboxyethyl)-phosphine (TCEP), and enzymes for protein analysis were purchased from Sigma. The peptides used have been described elsewhere (28Ragusa S. Mouchet P. Lazennec C. Dive V. Meinnel T. J. Mol. Biol. 1999; 289: 1445-1457Crossref PubMed Scopus (56) Google Scholar, 31Meinnel T. Patiny L. Ragusa S. Blanquet S. Biochemistry. 1999; 38: 4287-4295Crossref PubMed Scopus (79) Google Scholar). Enzymes for DNA manipulation were purchased from New England Biolabs. Oligonucleotides were synthesized by MWG-AG Biotech. Plasmid DNA was purified with mini- and mid-prep kits (Qiagen). For enzyme purification, we used plastic containers rather than glassware and all containers were thoroughly rinsed with metal-free water (see details in Ref. 23Ragusa S. Blanquet S. Meinnel T. J. Mol. Biol. 1998; 280: 515-523Crossref PubMed Scopus (110) Google Scholar). For the sake of clarity, all PDF sequences were numbered similar to the EcPDF sequence, as previously suggested (24Guilloteau J.P. Mathieu M. Giglione C. Blanc V. Dupuy A. Chevrier M. Gil P. Famechon A. Meinnel T. Mikol V. J. Mol. Biol. 2002; 320: 951-962Crossref PubMed Scopus (118) Google Scholar). A substitution of residue X in HsPDF or AtPDF1A indicates that the substitution concerns the residue corresponding to amino acid X in EcPDF, as shown in the alignment in Fig. 1. Residues upstream from Ser 1The abbreviations used are: NMEN-terminal methionine excisionCDcatalytic domainDAPI4′-6-diamidino-2-phenylindoleFoN-formylGFPgreen fluorescent proteinHEKhuman embryonic kidneyEcE. coliHsH. sapiensMAPmethionine aminopeptidaseORFopen reading framePDFpeptide deformylasePDFIpeptide deformylase inhibitorspNAp-nitroanilideRFPred fluorescent proteinTCEPTris(2-carboxyethyl)-phosphineAtA. thaliana. of EcPDF are numbered relative to position 1 and take a negative sign. N-terminal methionine excision catalytic domain 4′-6-diamidino-2-phenylindole N-formyl green fluorescent protein human embryonic kidney E. coli H. sapiens methionine aminopeptidase open reading frame peptide deformylase peptide deformylase inhibitors p-nitroanilide red fluorescent protein Tris(2-carboxyethyl)-phosphine A. thaliana. General Methods—Strain PAL421Tr (fmsΔ1, galK,rpsL, recA56, srl-300::Tn10) and CAG1284 (λ-, tolC210::Tn10, rph-) have been described elsewhere (32Meinnel T. Blanquet S. J. Bacteriol. 1994; 176: 7387-7390Crossref PubMed Google Scholar, 33Singer M. Baker T.A. Schnitzler G. Deischel S.M. Goel M. Dove W. Jaacks K.J. Grossman A.D. Erickson J.W. Gross C.A. Microbiol. Rev. 1989; 53: 1-24Crossref PubMed Google Scholar). Strain GIF1 was derived from CAG1284 and contains a chloramphenicol-resistant plasmid, pLysS, supplying tRNAs for AGG, AGA, AUA, CUA, CCC, and GGA. The human fetus RACE library was purchased from Clontech. HsMAP1D was amplified in one step with PfuTurbo DNA polymerase (Stratagene). Mutations were introduced into DNA by oligonucleotide site-directed mutagenesis, using the double-stranded plasmid with the QuickChange™ site-directed mutagenesis kit (Stratagene). Nucleotide sequences were determined by the Big-Dye Terminator V3 method with a 16-capillary ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Cloning in GFP Fusion Vectors—Full-length HsPDF and HsMAP1D sequences and sequences with deletions were inserted between the XhoI and XmaI restriction sites of pEGFP-N1 (Clontech). The plasmid pCB6 (34Legros F. Lombes A. Frachon P. Rojo M. Mol. Biol. Cell. 2002; 13: 4343-4354Crossref PubMed Scopus (490) Google Scholar) encodes the N-terminal mitochondrial domain of mitofusin fused to DsRed1-RFP (mt/RFP), which was used as a control for mitochondrial location. We inserted sequences into pSmRSGFP, expressing GFP under the control of the 35S promoter, between the unique XbaI-BamHI sites, as previously described (7Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (181) Google Scholar). Redesign of the HsPDF ORF—The starting material was pQHsdefΔN62ori, a plasmid encoding codons 63-244 of HsPDF inserted between restriction sites NcoI and HindIII of pQE60 (Qiagen) in-frame with an additional N-terminal Met-Ala dipeptide and a C-terminal His6 tag. Block I encodes the first 66 codons of the catalytic domain of HsPDF (i.e. codons 62-127 of HsPDF). We introduced a BamHI site into block I, at the 5′-end (bases 34-39), for subsequent block II insertion. Block II encodes the N-terminal targeting sequence of HsPDF (i.e. codons 1-70; Fig. 1). Both synthesized fragments (each ∼200 bp in length) were built by assembling in vitro six 50-75 bp oligonucleotides, as previously described (35Meinnel T. Mechulam Y. Fayat G. Nucleic Acids Res. 1988; 16: 8095-8096Crossref PubMed Scopus (83) Google Scholar). The initial remodeling step involved replacement of the NcoI-SacI fragment of HsPDFΔN62 with block I to yield pQHsdefΔN62mod. We then inserted block II between the NcoI and BamHI sites of pQHsdefΔN62mod to yield the full-length redesigned wild-type HsPDF ORF (Hsdefmod). The final optimized nucleotide sequence of Hsdefmod encoding full-length HsPDF is available from GenBank™ under accession number AY368205. Strain GIF1 Susceptibility Tests—We designed a susceptibility test to determine the susceptibility to drugs of the tolC strain GIF1 derivative producing a given PDF. Insertion of the PDF sequence into the pBAD vector (Invitrogen) render the synthesis of this protein dependent on arabinose concentration (36Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3876) Google Scholar). The GIF1 strain, which is highly susceptible to several antibiotics including chloramphenicol (37Sulavik M.C. Houseweart C. Cramer C. Jiwani N. Murgolo N. Greene J. DiDomenico B. Shaw K.J. Miller G.H. Hare R. Shimer G. Antimicrob. Agents Chemother. 2001; 45: 1126-1136Crossref PubMed Scopus (353) Google Scholar), was first transformed with the pBAD construct and selected on Luria-Bertani (LB) medium supplemented with 50 μg/ml ampicillin, 3.4 μg/ml chloramphenicol, and 0.5% glucose. Bacteria were cultured overnight in this medium at 37 °C and the culture was then diluted 1:100 and used to inoculate 3 ml of medium. When the OD600 reached 0.9, we diluted the suspension appropriately and layered 2 × 104 of bacteria in 100 μl on 30 ml of solid LB supplemented with 50 μg/ml ampicillin, actinonin (0.1-3 μm), and either glucose (0.5%) or arabinose (0.0002-0.2%) in Petri dishes. The minimum inhibitory concentration was defined as the lowest concentration of actinonin causing no growth after 18 h of incubation at 37 °C. Homologous Studies—Mammalian cell lines were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) with Glutamax-I (Invitrogen) at 37 °C, 5% CO2. HEK293 cells were plated on poly-l-lysine (Sigma)-coated cover slips 12 h before transfection and cultured in 6-well plates in fresh medium at 37 °C, 5% CO2. Cells were transfected in the 6-well plates according to the calcium phosphate transfection protocol (CalPhos mammalian transfection kit; Clontech) with 2 μg of plasmid DNA/well. Cells were washed with phosphate-buffered saline after 7 h to remove plasmid DNA and fixed in 4% formaldehyde/4% sucrose 48 h post-transfection for localization studies. Fixed cells were permeabilized with 0.05% Triton X-100 for 5 min and stained with 4′-6-diamidino-2-phenylindole (DAPI, 15 mg/ml; 1:10000 dilution; Molecular Probes) for nuclear DNA. Coverslips were mounted in Vectashield (Vector Laboratories, Inc. CA) for further examination at the confocal microscope. Cells were imaged by a Leica SP confocal microscope through a ×100 1.4 NA Planapochromat oil immersion objective. eGFP was excited by a 488 line of an Argon laser and RFP by a 543 nm line of Green HeNe laser. In order to avoid bleed-through, the fluorophores were excited sequentially. The emitted fluorescence was collected separately through a triple dichroic mirror 488/543/633. The emission filter bands for GFP and RFP were restricted to 500-568 nm and 570-705 nm, respectively. DAPI staining of nuclear DNA was excited by a 351 nm line of an UV laser and emission fluorescence collected by a 396-508 bandpass filter. Stacks of confocal sections separated by 0.2 μm increments were taken and images analyzed by Metamorph 5.0 software (Universal Imaging Corporation). Heterologous Studies—We bombarded onion epidermal cells with DNA constructs using the PDS-1000/He instrument (Bio-Rad) as previously described (7Giglione C. Serero A. Pierre M. Boisson B. Meinnel T. EMBO J. 2000; 19: 5916-5929Crossref PubMed Scopus (181) Google Scholar). Transient GFP production was examined with an up-right Axioplan 2 imaging fluorescence microscope (Zeiss) with interferential contrast and CCD camera (Sony ICX285). GFP was excited at 460-480 nm and collected at 505-530 nm (Chroma Technology filters). Images were analyzed by Metamorph 5.0 software. Purification of Peptide Deformylase Variants—BL21-pRares (Rosetta) cells (Novagen) expressing a given plasmid construct of the pET series were cultured to an OD600 of ∼0.9 at 22 °C for 8-12 h in 2× TY medium supplemented with 50 μg/ml ampicillin and 34 μg/ml chloramphenicol. Cells were induced by incubation with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside for another 5-12 h, with shaking. The cells were harvested by centrifugation, and resuspended in 10-20 ml of buffer A, consisting of 20 mm sodium phosphate buffer pH 7.3 plus 10 mm 2-mercaptoethanol. The samples were subjected to sonication, and cell debris removed by centrifugation. The supernatant (5-15 ml) was applied to a Hi-Trap chelating HP (0.7 by 2.5 cm; Amersham Biosciences) nickel affinity column equilibrated in buffer B (buffer A plus 0.5 m NaCl). The sample was eluted at a flow rate of 0.5 ml/min, in two steps, with buffer C (buffer B plus 0.5 m imidazole) at concentrations of 0.17 and 0.5 m imidazole, respectively. The purity of the protein was monitored by SDS-PAGE (13%) and the final yield was 3 mg for a 400 ml culture. The pooled purified PDF preparation (5 ml) was first dialyzed against buffer A for 12 h and then against buffer A plus 55% glycerol for 24 h before storage at -20 °C. Protein concentration was measured with the Bio-Rad protein assay kit. Bovine serum albumin was used as the protein standard. Preparation of Cell Extracts—Exponentially growing human cells were collected and frozen at -80 °C. Cells were then resuspended in 200 μl of 50 mm Hepes (pH 7.5), 150 mm NaCl, 15 mm MgCl2, 1% Triton, 5% glycerol, 10-4m phenylmethylsulfonyl fluoride, and anti-protease mixture (Roche Applied Science) and disrupted in a MM 300 mixer mill (Qiagen). The sample was centrifuged and the supernatant collected for further analysis. Arabidopsis thaliana leaf and root tissues were obtained from 10-day-old seedlings. Samples were frozen in liquid nitrogen and prepared as previously described (10Giglione C. Vallon O. Meinnel T. EMBO J. 2003; 22: 13-23Crossref PubMed Scopus (117) Google Scholar). Protein concentrations were measured with the BC assay protein quantification kit (Uptima). Bovine serum albumin was used as the protein standard. Immunological Methods—The rabbit antiserum against A. thaliana AtPDF1A used has been described elsewhere (10Giglione C. Vallon O. Meinnel T. EMBO J. 2003; 22: 13-23Crossref PubMed Scopus (117) Google Scholar). Rabbit antisera against HsPDF were raised at Eurogentech (Herstal, Belgium) and further purified before use. Polyacrylamide gel electrophoresis (PAGE) in SDS-denaturing gels (1.5-mm thick, 20-cm long, 10% polyacrylamide gels) was performed with the PROTEAN II system (BioRad). Proteins were electrotransferred onto nitrocellulose BA85 membranes (Schleicher & Schüll) with a wet transfer unit (Bio-Rad) in the cold room. Western blots were probed with mouse anti-His tag antibodies (dilution 1/2000) and peroxidase-conjugated anti-mouse antibodies from sheep (dilution 1:5000) or with anti-PDF antibodies (1:3000) and peroxidase-conjugated anti-rabbit antibodies from donkey (1:5000), and developed with ECL detection reagents (Amersham Biosciences). Membranes were placed against x-ray films for signal detection (Kodak). Enzyme Assays—We used an assay coupling PDF activity and formate dehydrogenase activity to assess PDF activity. We monitored, at 37 °C, the absorbance at 340 nm of NADH (ϵM = 6,300 m-1·cm-1), essentially as previously described (38Lazennec C. Meinnel T. Anal. Biochem. 1997; 244: 180-182Crossref PubMed Scopus (73) Google Scholar). The reaction was started by adding 5-15 μl of purified enzyme. In each case, the kinetic parameters were derived from iterative non-linear least square fits of the Michaelis-Menten equation, using the experimental data (39Dardel F. Comput. Appl. Biosci. 1994; 10: 273-275PubMed Google Scholar) as previously described (11Serero A. Giglione C. Meinnel T. J. Mol. Biol. 2001; 314: 695-708Crossref PubMed Scopus (72) Google Scholar). Determination of Metal Ions by Atomic Absorption Spectroscopy— Protein samples (200-400 μl) were dialyzed overnight against a buffer (1 liter) consisting of 20 mm Hepes (pH 7.5) and 0.1 m KCl. We used a Varian AA220 spectrophotometer equipped with an air-acetylene burner in “peak height” mode and measured atomic absorption at 213.9 nm for 5 s after the injection of 0.1 ml samples. The concentrations of metal ions in serial dilutions of the enzyme samples were calculated by comparison with serial dilutions of standard ZnCl2 solutions (Merck). Identification in Animal cDNA Libraries and Cloning of Homologs of Plant Mitochondrial PDF1A and MAP1D—We previously reported the identification in human and mouse of cDNAs encod" @default.
• W1972693783 created "2016-06-24" @default.
• W1972693783 creator A5030367209 @default.
• W1972693783 creator A5058977760 @default.
• W1972693783 creator A5072337283 @default.
• W1972693783 creator A5083236007 @default.
• W1972693783 creator A5089355412 @default.
• W1972693783 date "2003-12-01" @default.
• W1972693783 modified "2023-10-17" @default.
• W1972693783 title "An Unusual Peptide Deformylase Features in the Human Mitochondrial N-terminal Methionine Excision Pathway" @default.
• W1972693783 cites W1509733399 @default.
• W1972693783 cites W1526754730 @default.
• W1972693783 cites W1540958392 @default.
• W1972693783 cites W1915895531 @default.
• W1972693783 cites W1932542519 @default.
• W1972693783 cites W1938326461 @default.
• W1972693783 cites W1964697533 @default.
• W1972693783 cites W1969001974 @default.
• W1972693783 cites W1971579210 @default.
• W1972693783 cites W1971707621 @default.
• W1972693783 cites W1973733047 @default.
• W1972693783 cites W1983185780 @default.
• W1972693783 cites W1993654759 @default.
• W1972693783 cites W1996175903 @default.
• W1972693783 cites W2002260362 @default.
• W1972693783 cites W2002373450 @default.
• W1972693783 cites W2005392593 @default.
• W1972693783 cites W2007301486 @default.
• W1972693783 cites W2010601153 @default.
• W1972693783 cites W2011271534 @default.
• W1972693783 cites W2018590530 @default.
• W1972693783 cites W2020420692 @default.
• W1972693783 cites W2022569902 @default.
• W1972693783 cites W2028358008 @default.
• W1972693783 cites W2031545189 @default.
• W1972693783 cites W2034390577 @default.
• W1972693783 cites W2039254203 @default.
• W1972693783 cites W2042397530 @default.
• W1972693783 cites W2042661769 @default.
• W1972693783 cites W2043337626 @default.
• W1972693783 cites W2043821463 @default.
• W1972693783 cites W2050134681 @default.
• W1972693783 cites W2050928252 @default.
• W1972693783 cites W2052582622 @default.
• W1972693783 cites W2055749408 @default.
• W1972693783 cites W2057183165 @default.
• W1972693783 cites W2065892366 @default.
• W1972693783 cites W2066665513 @default.
• W1972693783 cites W2071958540 @default.
• W1972693783 cites W2073695712 @default.
• W1972693783 cites W2077817524 @default.
• W1972693783 cites W2091418093 @default.
• W1972693783 cites W2116817481 @default.
• W1972693783 cites W2118285751 @default.
• W1972693783 cites W2126824920 @default.
• W1972693783 cites W2130091680 @default.
• W1972693783 cites W2132897827 @default.
• W1972693783 cites W2146611542 @default.
• W1972693783 cites W2148301225 @default.
• W1972693783 cites W2152173135 @default.
• W1972693783 cites W2158714788 @default.
• W1972693783 cites W2160084442 @default.
• W1972693783 cites W2164174765 @default.
• W1972693783 cites W2167701793 @default.
• W1972693783 cites W2169110988 @default.
• W1972693783 cites W2791126481 @default.
• W1972693783 doi "https://doi.org/10.1074/jbc.m309770200" @default.
• W1972693783 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14532271" @default.
• W1972693783 hasPublicationYear "2003" @default.
• W1972693783 type Work @default.
• W1972693783 sameAs 1972693783 @default.
• W1972693783 citedByCount "120" @default.
• W1972693783 countsByYear W19726937832012 @default.
• W1972693783 countsByYear W19726937832013 @default.
• W1972693783 countsByYear W19726937832014 @default.
• W1972693783 countsByYear W19726937832015 @default.
• W1972693783 countsByYear W19726937832016 @default.
• W1972693783 countsByYear W19726937832017 @default.
• W1972693783 countsByYear W19726937832018 @default.
• W1972693783 countsByYear W19726937832019 @default.
• W1972693783 countsByYear W19726937832020 @default.
• W1972693783 countsByYear W19726937832021 @default.
• W1972693783 countsByYear W19726937832022 @default.
• W1972693783 countsByYear W19726937832023 @default.
• W1972693783 crossrefType "journal-article" @default.
• W1972693783 hasAuthorship W1972693783A5030367209 @default.
• W1972693783 hasAuthorship W1972693783A5058977760 @default.
• W1972693783 hasAuthorship W1972693783A5072337283 @default.
• W1972693783 hasAuthorship W1972693783A5083236007 @default.
• W1972693783 hasAuthorship W1972693783A5089355412 @default.
• W1972693783 hasBestOaLocation W19726937831 @default.
• W1972693783 hasConcept C153911025 @default.
• W1972693783 hasConcept C185592680 @default.
• W1972693783 hasConcept C2779281246 @default.
• W1972693783 hasConcept C2779664074 @default.
• W1972693783 hasConcept C2780912031 @default.
• W1972693783 hasConcept C41008148 @default.
• W1972693783 hasConcept C515207424 @default.

• next