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.

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (

Degradation of extracellular matrix components is central to many pathological features of chronic destructive lung disorders. Desmosine and isodesmosine are elastin-derived cross-linked amino acids whose urine levels are considered representative of elastin breakdown.The aim of this study was to apply a novel methodology, based on high-performance capillary electrophoresis, to the quantification of desmosine and isodesmosine in 11 patients with stable chronic obstructive pulmonary disease (COPD), 10 with an exacerbation of COPD, nine with a 1 -antitrypsin deficiency, 13 with bronchiectasis, and 11 adults with cystic fibrosis, in comparison to 24 controls.It was found that, in patients with stable COPD, urinary desmosine levels were higher than in controls (p=0.03), but lower than in COPD subjects with an exacerbation (p#0.05). The highest desmosine levels were found in subjects with a 1 -antitrypsin deficiency, bronchiectasis and cystic fibrosis (p<0.001 versus stable COPD). In a shortterm longitudinal study, five stable COPD patients showed a constant rate of desmosine excretion (mean coefficient of variation <8% over three consecutive days).In conclusion, the present method is simple and suitable for the determination of elastin-derived cross-linked amino acid excretion in urine, giving results similar to those obtained using other separation methods. In addition, evidence is presented that urinary desmosine excretion is increased in conditions characterized by airway inflammation, such as exacerbations of chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. Results obtained in subjects with a 1 -antitrypsin deficiency suggest that this method might be used to evaluate the putative efficacy of replacement therapy. Eur Respir J 2000; 15: 1039±1045. Chronic obstructive pulmonary disease (COPD), with or without inherited deficiency of a 1 -antitrypsin (AAT), disseminated bronchiectasis (Bx) and cystic fibrosis (CF) are destructive lung disorders characterized by chronic and irreversible airflow limitation. Irrespective of the aetiological factors implicated, there is a solid body of evidence suggesting that degradation of the extracellular matrix component, mainly elastin, due to an imbalance between proteinases and their naturally occurring inhibitors, is central to many of the pathological features of these conditions [1±4].Excess lung elastin degradation results in the excretion of elastin-derived peptides containing desmosine (DES) and isodesmosine (IDES) in urine [5]. Since these crosslinked amino acids are unique to mature elastin, their urine levels are considered to be representative of body elastin breakdown. Urinary excretion of DES and IDES has been reported to be increased in patients with COPD, current smokers with normal lung function [6] and adults with CF [7] compared to appropriate controls.There are two validated means of detecting DES and IDES in body fluids: a separation method, based on isotope -dilution/high -performance liquid chromatography (HPLC) [8], and ...

This study used proteomic and transcriptomic techniques to understand the molecular basis of the phenotypic variability in the bone disorder osteogenesis imperfecta (OI). Calvarial bone mRNA expression was evaluated by microarray, real-time, and comparative RT-PCR and the bone proteome profile was analyzed by 2-DE, MS, and immunoblotting in the OI murine model BrtlIV, which has either a moderate or a lethal OI outcome. Differential expression analysis showed significant changes for eight proteins. The expression of the ER stress-related protein Gadd153 was increased in lethal mice, whereas expression of the chaperone alphaB crystallin was increased in nonlethal mice, suggesting that the intracellular machinery is involved in the modulation of the OI phenotype. Furthermore, in lethal BrtlIV, the increased expression of the cartilaginous proteins Prelp, Bmp6, and Bmp7 and the lower expression of the bone matrix proteins matrilin 4, microfibril-associated glycoprotein 2, and thrombospondin 3 revealed that both a delay in skeletal development and an alteration in extracellular matrix composition influence OI outcomes. Differentially expressed proteins identified in this model offer a starting point for elucidating the molecular basis of phenotypic variability, a characteristic common to many genetic disorders. The first reference 2-DE map for murine calvarial tissue is also reported.

Prolidase, a ubiquitously distributed dipeptidase, is involved in the latter stage of degradation of endogenous and dietary proteins and is particularly important in collagen catabolism. It hydrolyzes dipeptides containing proline or hydroxyproline at the C-terminal position. Mutations in the gene encoding for prolidase cause prolidase deficiency (PD), an autosomal recessive disorder mainly characterized by skin lesions, mental retardation and recurrent infectious. In this work we reported the identification of the molecular defect in five PD patients. Direct sequencing of PCR amplified genomic DNA showed a homozygous G>A transversion in two siblings leading to a G448R substitution. A heterozygous IVS11+1G>C transition causing the skipping of exon 11 and a null allele were detected in a third proband. In two unrelated patients, a homozygous IVS7-1G>A transversion was identified and shown to cause multiple alternative spliced transcripts. All the mutations result in loss of prolidase activity. Long-term cultured fibroblasts from these PD patients were used to develop an in vitro model that allowed investigation of the affected cells. Light and electron microscopy revealed that PD cells were more round and branched out than controls with increased cytosolic vacuolization, interruptions of the plasma membrane, mitochondria swelling, mitochondrial matrix and cristae modifications. JC-1 labeling showed decreased mitochondrial membrane potential. A significant intracellular accumulation of the Gly-Pro dipeptide was detected by capillary electrophoresis analysis. Our results provide the first evidence that absence of prolidase activity causes the activation of a necrosis-like cellular death, which could be responsible for the typical skin lesions in PD.

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Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (k(obs)) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (k(a)). A proflavine displacement assay was used to determine the deacylation rate constant (k(d)). Both Er and FE are good HNE inhibitors, showing a high k(a) and a low k(d). Because the number of turnovers per inactivation of Er was congruent with 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a k(d) only, whereas for FE, in addition to the k(d), an alkylation constant (k(2)) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.