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Antonella Forlino - One of the best experts on this subject based on the ideXlab platform.
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partial rescue of biochemical parameters after hematopoietic stem cell transplantation in a patient with Prolidase Deficiency due to two novel pepd mutations
JIMD reports, 2011Co-Authors: D Caselli, Antonio Rossi, Rolando Cimaz, Roberta Besio, Ersilia De Lorenzi, Raffaella Colombo, Luca Cantarini, S Riva, M Spada, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare recessive disorder resulting from mutations in the Prolidase gene (PEPD); only 17 causative mutant alleles had been so far characterized. Prolidase is a ubiquitous enzyme that hydrolyses dipeptides with C-terminal proline or hydroxyproline residues and indeed, lack of this enzyme activity causes massive urine excretion of undigested iminodipeptides. The clinical manifestations of PD are widely variable, and include intractable skin ulcers, unusual face, different degree of mental retardation, and recurrent infections. No definitive treatment is at present available.
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Identifying the structure of the active sites of human recombinant Prolidase
European Biophysics Journal, 2010Co-Authors: Roberta Besio, Anna Lupi, Antonella Forlino, R. Tenni, Stefania Alleva, Carlo Meneghini, Velia Minicozzi, Antonella Profumo, Francesco Stellato, Silvia MoranteAbstract:In this paper we provide a detailed biochemical and structural characterization of the active site of recombinant human Prolidase, a dimeric metalloenzyme, whose misfunctioning causes a recessive connective tissue disorder (Prolidase Deficiency) characterized by severe skin lesions, mental retardation and respiratory tract infections. It is known that the protein can host two metal ions in the active site of each constituent monomer. We prove that two different kinds of metals (Mn and Zn) can be simultaneously present in the protein active sites with the protein partially maintaining its enzymatic activity. Structural information extracted from X-ray absorption spectroscopy measurements have been used to yield a full reconstruction of the atomic environment around each one of the two monomeric active sites. In particular, as for the metal ion occupation configuration of the recombinant human Prolidase, we have found that one of the two active sites is occupied by two Zn ions and the second one by one Zn and one Mn ion. In both dinuclear units a histidine residue is bound to a Zn ion.
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Human Prolidase and Prolidase Deficiency: an overview on the characterization of the enzyme involved in proline recycling and on the effects of its mutations
Amino Acids, 2008Co-Authors: A. Lupi, Giuseppe Cetta, R. Tenni, A. Rossi, Antonella ForlinoAbstract:Here we summarized what is known at the present about function, structure and effect of mutations in the human Prolidase. Among the peptidases, Prolidase is the only metalloenzyme that cleaves the iminodipeptides containing a proline or hydroxyproline residue at the C-terminal end. It is relevant in the latest stage of protein catabolism, particularly of those molecules rich in imino acids such as collagens, thus being involved in matrix remodelling. Beside its intracellular functions, Prolidase has an antitoxic effect against some organophosphorus molecules, can be used in dietary industry as bitterness reducing agent and recently has been used as target enzyme for specific melanoma prodrug activation. Recombinant human Prolidase was produced in prokaryotic and eukaryotic hosts with biochemical properties similar to the endogenous enzyme and represents a valid tool both to better understand the structure and biological function of the enzyme and to develop an enzyme replacement therapy for the Prolidase Deficiency (PD). Prolidase Deficiency is a rare recessive disorder caused by mutations in the Prolidase gene and characterized by severe skin lesions. Single amino acid substitutions, exon splicing, deletions and a duplication were described as causative for the disease and are mainly located at highly conserved amino acids in the sequence of Prolidase from different species. The pathophysiology of PD is still poorly understood; we offer here a review of the molecular mechanisms so far hypothesized.
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molecular characterisation of six patients with Prolidase Deficiency identification of the first small duplication in the Prolidase gene and of a mutation generating symptomatic and asymptomatic outcomes within the same family
Journal of Medical Genetics, 2006Co-Authors: Anna Lupi, Antonio Rossi, Elena Campari, Giuseppe Cetta, Fabio Pecora, Allan M Lund, N H Elcioglu, M Gultepe, M Di Rocco, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare autosomal recessive connective tissue disorder caused by mutations in the Prolidase gene. The PD patients show a wide range of clinical outcomes characterised mainly by intractable skin ulcers, mental retardation and recurrent respiratory infections. Here we describe five different PEPD mutations in six European patients. We identified two new PEPD mutant alleles: a 13 bp duplication in exon 8, which is the first reported duplication in the Prolidase gene and a point mutation resulting in a change in amino acid E412, a highly conserved residue among different species. The E412K substitution is responsible for the first reported phenotypic variability within a family with severe and asymptomatic outcomes.
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characterization of a new pepd allele causing Prolidase Deficiency in two unrelated patients natural occurrent mutations as a tool to investigate structure function relationship
Journal of Human Genetics, 2004Co-Authors: Anna Lupi, Antonio De Riso, Sara Della Torre, Antonio Rossi, Elena Campari, Laura Vilarinho, Giuseppe Cetta, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare autosomal recessive disorder characterized mainly by skin lesions of the legs and feet, mental retardation, and respiratory infections. Mutations at the PEPD locus, located on chromosome 19, are responsible for this disease. We identified a new PEPD allele in two unrelated Portuguese PD patients by analyses of reverse transcribed PCR-amplified cDNA. We used SSCP analysis of seven overlapping fragments spanning the entire coding region of the gene and detected abnormal SSCP bands in two of them: PD3 (nt 425–743) and PD4 (nt 661–973). Direct sequencing of the mutant cDNA and genomic DNA revealed a new homozygous 3-bp deletion (Y231del) in both cases. Transient expression in PD fibroblasts of wild-type and mutant Prolidase cDNA confirmed reduced activity of the construct carrying the 3-bp deletion. The mutation results in a loss of Prolidase activity in skin fibroblasts. Intracellular accumulation of Gly-Pro dipeptide in long-term cultured fibroblasts was detected by capillary electrophoresis. The mutation falls in the α2 domain of the “pita bread” structure proposed for E. coli and human Prolidase by Bazan et al. on the bases of their sequence homology with E. coli methionine aminopeptidase. Taking into account the effects of the described mutations on stability and activity of the enzyme, we propose the identification of three different functional regions.
Hiroyuki Kodama - One of the best experts on this subject based on the ideXlab platform.
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Prolidase isoenzymes in the rat their organ distribution developmental change and specific inhibitors
Pediatric Research, 2007Co-Authors: Gang Liu, Kazuko Nakayama, Shiro Awata, Shuhan Tang, Noriko Kitaoka, Masanobu Manabe, Hiroyuki KodamaAbstract:Lack of Prolidase I (PD I) leads to Prolidase Deficiency, a disease characterized by intractable skin lesions, recurrent respiratory infections, and mental retardation. The present study was undertaken to characterize and determine the physiologic roles of different Prolidase isoenzymes. Two isoforms of Prolidase were isolated from rat kidney. PD I showed higher activity against seryl-proline and alanyl-proline, whereas PD II was active especially against methionyl-proline. PD I was highly concentrated in the small intestine and kidney, whereas PD II was shown not to vary in the organs examined. Expression of PD I and PD II in the small intestine were maximal within 1 wk of birth, and then rapidly declined. The changes of Prolidase in the kidney and heart were found to differ slightly. N-benzyloxycarbonyl-l-proline and captopril inhibited PD I dose-dependently, but showed no inhibition of PD II at low concentrations. NiCl2 inhibited PD II much more effectively than PD I. Our findings suggest that PD I functions by way of an intestinal peptide carrier, which may also be regulated by the uptake of various iminodipeptides. Similarly, age-related alterations of Prolidase isoenzymes suggest that intestinal PD II also participates in absorption of proline and other amino acids early in life.
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Measurement of Iminodipeptides in the Serum of Patients with Prolidase Deficiency Using Liquid Chromatography-Mass Spectrometry
1993Co-Authors: Eur J. Clin, Chem Clin Biochem, Kazunori Sugaharal, Zhang Jianying, Yasuo Yamamoto, Kayo Yasuda, Hajime Kodama, Hiroyuki KodamaAbstract:Summary: Iminodipeptides containing C-terminal proline or hydroxyproline were determined in sera from patients with Prolidase Deficiency, in their mother's serum, and in the sera of unfelated controls, using liquid chromatogra-phy-mass Spectrometry with an atmospheric pressure ionization interface System. The Separation was carried out on a reversed phase column using l g/l aqueous trifluoroacetic acid-methanol (75 + 25, by vol.). The quasi-molecular ions ([M-l · H])"1 " of various Iminodipeptides containing C-terminal proline and hydroxyproline were observed in the sera of patients with Prolidase Deficiency, using selected ipn monitoring. The quasi-molecular ions ([M + H])+ of iminodipeptides containing C-terminal proline were not observed in the sera of normal subjects or the patients' mother, but the latter did contain various iminodipeptides with C-terminal hydroxyproline. This method proved usefiil for the determination of iminodipeptides in the sera of patients with Prolidase Deficiency
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The use of liquid chromatography-mass spectrometry for the identification and quantification of urinary iminodipeptides in Prolidase Deficiency
1993Co-Authors: Sugahara Takashi Ohno, Jiro Arata, Hiroyuki KodamaAbstract:Summary: It has been reported that the urine of patients with Prolidase Deficiency contains various iminodi-peptides with a carboxyl-terminal proline (hydroxyproline). These iminodipeptides have hitherto been detected indirectly by acid hydrolysis or enzymatic digestion, followed by amino acid analysis. In the present study, it was shown that X-Pro could be distinguished from Pro-X when the iminodipeptides were analysed directly by liquid chromatography coupled with atmospheric pressure ionization mass Spectrometry (LC/API-MS), with scanning of the protonated molecule ions ([M + H]+). The same procedure also successfully quantified urinary iminodipeptides from patients with Prolidase Deficiency. A quantitative investigation of two siblings with Prolidase Deficiency revealed that the patient with severe clinical symptoms excreted more iminodipeptides than the other who did not have serious symptoms. LC/API-MS also revealed iminodipeptides (Gly-Hyp and Pro^Hyp) in the urine of the mother of the patients and in normal volunteers. Patients excreted much more Pro-Hyp than normal volunteers, whereas no quantitative differences were found between the mother and controls. In patients, the excretion of large quantities of X-Pro is due to their very low Prolidase activity towards this type of substrate. In the erythrocytes of patients, Prolidase activity towards X-Hyp was extremely low; even in the mother and normal volunteers, it was remarkably low in comparison with the activity against X-Pro
Antonio Rossi - One of the best experts on this subject based on the ideXlab platform.
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Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue
2013Co-Authors: Roberta Besio, R. Tenni, Antonella Profumo, Roberta Gioia, Federica Cossu, Enrico Monzani, Stefania Nicolis, Lucia Cucca, Martino Bolognesi, Antonio RossiAbstract:Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase Deficiency (PD) is an intractable loss of function disease, characterized by mutations in the Prolidase gene. The exact causes of activity impairment in mutant Prolidase are still unknown. We generated three recombinant Prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial Prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the Prolidase activity in patients ’ fibroblasts through the induction of Heath Shock Protein
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partial rescue of biochemical parameters after hematopoietic stem cell transplantation in a patient with Prolidase Deficiency due to two novel pepd mutations
JIMD reports, 2011Co-Authors: D Caselli, Antonio Rossi, Rolando Cimaz, Roberta Besio, Ersilia De Lorenzi, Raffaella Colombo, Luca Cantarini, S Riva, M Spada, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare recessive disorder resulting from mutations in the Prolidase gene (PEPD); only 17 causative mutant alleles had been so far characterized. Prolidase is a ubiquitous enzyme that hydrolyses dipeptides with C-terminal proline or hydroxyproline residues and indeed, lack of this enzyme activity causes massive urine excretion of undigested iminodipeptides. The clinical manifestations of PD are widely variable, and include intractable skin ulcers, unusual face, different degree of mental retardation, and recurrent infections. No definitive treatment is at present available.
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molecular characterisation of six patients with Prolidase Deficiency identification of the first small duplication in the Prolidase gene and of a mutation generating symptomatic and asymptomatic outcomes within the same family
Journal of Medical Genetics, 2006Co-Authors: Anna Lupi, Antonio Rossi, Elena Campari, Giuseppe Cetta, Fabio Pecora, Allan M Lund, N H Elcioglu, M Gultepe, M Di Rocco, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare autosomal recessive connective tissue disorder caused by mutations in the Prolidase gene. The PD patients show a wide range of clinical outcomes characterised mainly by intractable skin ulcers, mental retardation and recurrent respiratory infections. Here we describe five different PEPD mutations in six European patients. We identified two new PEPD mutant alleles: a 13 bp duplication in exon 8, which is the first reported duplication in the Prolidase gene and a point mutation resulting in a change in amino acid E412, a highly conserved residue among different species. The E412K substitution is responsible for the first reported phenotypic variability within a family with severe and asymptomatic outcomes.
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characterization of a new pepd allele causing Prolidase Deficiency in two unrelated patients natural occurrent mutations as a tool to investigate structure function relationship
Journal of Human Genetics, 2004Co-Authors: Anna Lupi, Antonio De Riso, Sara Della Torre, Antonio Rossi, Elena Campari, Laura Vilarinho, Giuseppe Cetta, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare autosomal recessive disorder characterized mainly by skin lesions of the legs and feet, mental retardation, and respiratory infections. Mutations at the PEPD locus, located on chromosome 19, are responsible for this disease. We identified a new PEPD allele in two unrelated Portuguese PD patients by analyses of reverse transcribed PCR-amplified cDNA. We used SSCP analysis of seven overlapping fragments spanning the entire coding region of the gene and detected abnormal SSCP bands in two of them: PD3 (nt 425–743) and PD4 (nt 661–973). Direct sequencing of the mutant cDNA and genomic DNA revealed a new homozygous 3-bp deletion (Y231del) in both cases. Transient expression in PD fibroblasts of wild-type and mutant Prolidase cDNA confirmed reduced activity of the construct carrying the 3-bp deletion. The mutation results in a loss of Prolidase activity in skin fibroblasts. Intracellular accumulation of Gly-Pro dipeptide in long-term cultured fibroblasts was detected by capillary electrophoresis. The mutation falls in the α2 domain of the “pita bread” structure proposed for E. coli and human Prolidase by Bazan et al. on the bases of their sequence homology with E. coli methionine aminopeptidase. Taking into account the effects of the described mutations on stability and activity of the enzyme, we propose the identification of three different functional regions.
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Prolidase Deficiency: biochemical study of erythrocyte and skin fibroblast Prolidase activity in Italian patients
1994Co-Authors: Katharine M. Dyne, Antonio Rossi, Virginia MonafoAbstract:rolidase Deficiency (PD), a rare autoso-mal recessive phenotype in which defi-cient Prolidase activity leads to massive urinary excretion of imidodipeptides (X-Pro and X-Hyp), and it is associated with a range of clinical manifestations.1,2 Prolidase (imino-dipeptidase E.C. 3.4.13.9), a dimer with a mole-cular subunit mass of about 54.3 kD is respon-sible for hydrolyzing dipeptides containing C-terminal prolyl or hydoxyprolyl residues in the final stages of protein catabolism. It is an ubiq-uitous enzyme whose activity has been docu-mented in erythrocytes, leukocytes, plasma, dermal fibroblasts, the kidney, brain, heart, thy-mus and uterus. Given the relatively high quan-tity of iminoacids in collagen and the presence of skin alterations, it is tempting to speculate that this enzyme Deficiency results in altered collagen metabolism even though protein catabolism in general is affected. The enzyme probably plays an important role in the re-uti-lization of proline residues for de novo protein synthesis (Figure 1), although the exact correla-tion between deficient Prolidase activity and the pathogenesis of clinical manifestations is still not fully understood
Roberta Besio - One of the best experts on this subject based on the ideXlab platform.
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Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue
2013Co-Authors: Roberta Besio, R. Tenni, Antonella Profumo, Roberta Gioia, Federica Cossu, Enrico Monzani, Stefania Nicolis, Lucia Cucca, Martino Bolognesi, Antonio RossiAbstract:Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase Deficiency (PD) is an intractable loss of function disease, characterized by mutations in the Prolidase gene. The exact causes of activity impairment in mutant Prolidase are still unknown. We generated three recombinant Prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial Prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the Prolidase activity in patients ’ fibroblasts through the induction of Heath Shock Protein
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partial rescue of biochemical parameters after hematopoietic stem cell transplantation in a patient with Prolidase Deficiency due to two novel pepd mutations
JIMD reports, 2011Co-Authors: D Caselli, Antonio Rossi, Rolando Cimaz, Roberta Besio, Ersilia De Lorenzi, Raffaella Colombo, Luca Cantarini, S Riva, M Spada, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare recessive disorder resulting from mutations in the Prolidase gene (PEPD); only 17 causative mutant alleles had been so far characterized. Prolidase is a ubiquitous enzyme that hydrolyses dipeptides with C-terminal proline or hydroxyproline residues and indeed, lack of this enzyme activity causes massive urine excretion of undigested iminodipeptides. The clinical manifestations of PD are widely variable, and include intractable skin ulcers, unusual face, different degree of mental retardation, and recurrent infections. No definitive treatment is at present available.
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Identifying the structure of the active sites of human recombinant Prolidase
European Biophysics Journal, 2010Co-Authors: Roberta Besio, Anna Lupi, Antonella Forlino, R. Tenni, Stefania Alleva, Carlo Meneghini, Velia Minicozzi, Antonella Profumo, Francesco Stellato, Silvia MoranteAbstract:In this paper we provide a detailed biochemical and structural characterization of the active site of recombinant human Prolidase, a dimeric metalloenzyme, whose misfunctioning causes a recessive connective tissue disorder (Prolidase Deficiency) characterized by severe skin lesions, mental retardation and respiratory tract infections. It is known that the protein can host two metal ions in the active site of each constituent monomer. We prove that two different kinds of metals (Mn and Zn) can be simultaneously present in the protein active sites with the protein partially maintaining its enzymatic activity. Structural information extracted from X-ray absorption spectroscopy measurements have been used to yield a full reconstruction of the atomic environment around each one of the two monomeric active sites. In particular, as for the metal ion occupation configuration of the recombinant human Prolidase, we have found that one of the two active sites is occupied by two Zn ions and the second one by one Zn and one Mn ion. In both dinuclear units a histidine residue is bound to a Zn ion.
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Kinetic and structural evidences on human Prolidase pathological mutants suggest strategies for enzyme functional rescue.
Public Library of Science (PLoS), 2024Co-Authors: Roberta Besio, R. Tenni, Antonella Profumo, Roberta Gioia, Federica Cossu, Enrico Monzani, Stefania Nicolis, Lucia Cucca, Luigi Casella, Martino BolognesiAbstract:Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase Deficiency (PD) is an intractable loss of function disease, characterized by mutations in the Prolidase gene. The exact causes of activity impairment in mutant Prolidase are still unknown. We generated three recombinant Prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial Prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the Prolidase activity in patients' fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment
Anna Lupi - One of the best experts on this subject based on the ideXlab platform.
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Identifying the structure of the active sites of human recombinant Prolidase
European Biophysics Journal, 2010Co-Authors: Roberta Besio, Anna Lupi, Antonella Forlino, R. Tenni, Stefania Alleva, Carlo Meneghini, Velia Minicozzi, Antonella Profumo, Francesco Stellato, Silvia MoranteAbstract:In this paper we provide a detailed biochemical and structural characterization of the active site of recombinant human Prolidase, a dimeric metalloenzyme, whose misfunctioning causes a recessive connective tissue disorder (Prolidase Deficiency) characterized by severe skin lesions, mental retardation and respiratory tract infections. It is known that the protein can host two metal ions in the active site of each constituent monomer. We prove that two different kinds of metals (Mn and Zn) can be simultaneously present in the protein active sites with the protein partially maintaining its enzymatic activity. Structural information extracted from X-ray absorption spectroscopy measurements have been used to yield a full reconstruction of the atomic environment around each one of the two monomeric active sites. In particular, as for the metal ion occupation configuration of the recombinant human Prolidase, we have found that one of the two active sites is occupied by two Zn ions and the second one by one Zn and one Mn ion. In both dinuclear units a histidine residue is bound to a Zn ion.
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molecular characterisation of six patients with Prolidase Deficiency identification of the first small duplication in the Prolidase gene and of a mutation generating symptomatic and asymptomatic outcomes within the same family
Journal of Medical Genetics, 2006Co-Authors: Anna Lupi, Antonio Rossi, Elena Campari, Giuseppe Cetta, Fabio Pecora, Allan M Lund, N H Elcioglu, M Gultepe, M Di Rocco, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare autosomal recessive connective tissue disorder caused by mutations in the Prolidase gene. The PD patients show a wide range of clinical outcomes characterised mainly by intractable skin ulcers, mental retardation and recurrent respiratory infections. Here we describe five different PEPD mutations in six European patients. We identified two new PEPD mutant alleles: a 13 bp duplication in exon 8, which is the first reported duplication in the Prolidase gene and a point mutation resulting in a change in amino acid E412, a highly conserved residue among different species. The E412K substitution is responsible for the first reported phenotypic variability within a family with severe and asymptomatic outcomes.
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characterization of a new pepd allele causing Prolidase Deficiency in two unrelated patients natural occurrent mutations as a tool to investigate structure function relationship
Journal of Human Genetics, 2004Co-Authors: Anna Lupi, Antonio De Riso, Sara Della Torre, Antonio Rossi, Elena Campari, Laura Vilarinho, Giuseppe Cetta, Antonella ForlinoAbstract:Prolidase Deficiency (PD) is a rare autosomal recessive disorder characterized mainly by skin lesions of the legs and feet, mental retardation, and respiratory infections. Mutations at the PEPD locus, located on chromosome 19, are responsible for this disease. We identified a new PEPD allele in two unrelated Portuguese PD patients by analyses of reverse transcribed PCR-amplified cDNA. We used SSCP analysis of seven overlapping fragments spanning the entire coding region of the gene and detected abnormal SSCP bands in two of them: PD3 (nt 425–743) and PD4 (nt 661–973). Direct sequencing of the mutant cDNA and genomic DNA revealed a new homozygous 3-bp deletion (Y231del) in both cases. Transient expression in PD fibroblasts of wild-type and mutant Prolidase cDNA confirmed reduced activity of the construct carrying the 3-bp deletion. The mutation results in a loss of Prolidase activity in skin fibroblasts. Intracellular accumulation of Gly-Pro dipeptide in long-term cultured fibroblasts was detected by capillary electrophoresis. The mutation falls in the α2 domain of the “pita bread” structure proposed for E. coli and human Prolidase by Bazan et al. on the bases of their sequence homology with E. coli methionine aminopeptidase. Taking into account the effects of the described mutations on stability and activity of the enzyme, we propose the identification of three different functional regions.
