Abstract
The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/trans-isomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1- and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes.
Highlights
The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, important in many biological processes in both prokaryotes and eukaryotes
Another parvulintype peptidyl-prolyl cis/transisomerases (PPIases), hPar14, which has been characterized in human, shows 34% identity to hPin1 and, in contrast to hPin1, prefers positively charged amino acids preceding Pro (Uchida et al, 1999). hPar14 lacks the N-terminal WW domain of hPin1 and carries an unstructured N-terminal extension, which is important for localization of hPar14 to the nucleus and its binding to DNA (Surmacz et al, 2002; Reimer et al, 2003)
In order to identify cDNA clones coding for parvulintype PPIases in L. japonicus, a bioinformatics approach was undertaken
Summary
The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, important in many biological processes in both prokaryotes and eukaryotes. The WW domain consists of 35 to 40 amino acid residues and acts as a protein-protein interaction module found in many different proteins (Sudol, 1996) Both Ess and hPin are characterized as PIN1 (Protein Interacting with NIMA)-type parvulin PPIases, as they preferentially recognize substrates with a phosphorylated Ser or Thr N terminal to Pro residue In addition to its role in mitosis, hPin is involved in p53-mediated control of DNA damage, as hPin was shown to be important in the activation of the p53-mediated apoptotic pathway (Zheng et al, 2002) Another parvulintype PPIase, hPar, which has been characterized in human, shows 34% identity to hPin and, in contrast to hPin, prefers positively charged amino acids preceding Pro (Uchida et al, 1999). Another parvulintype PPIase, hPar, which has been characterized in human, shows 34% identity to hPin and, in contrast to hPin, prefers positively charged amino acids preceding Pro (Uchida et al, 1999). hPar lacks the N-terminal WW domain of hPin and carries an unstructured N-terminal extension, which is important for localization of hPar to the nucleus and its binding to DNA (Surmacz et al, 2002; Reimer et al, 2003)
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