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A 4-week-old boy presented to the hospital with symptoms of diarrhoea and vomiting initially thought to be due to cow’s milk allergy. He was discharged with extensively hydrolysed formula. The patient represented with worsening of symptoms with metabolic acidosis and was screened and treated for sepsis. However, his condition deteriorated further and he developed methaemoglobinaemia. He was transferred to the high dependency unit and was given two doses of methylene blue. Further investigations were carried out, including rapid trio exome sequencing, which identified a homozygous pathogenic Peptidase D (PEPD) variant (c.978G>A, p.(Trp326*)). This was consistent with a diagnosis of prolidase deficiency.

The paper presents data on the pathogenesis of systemic lupus erythematosus (SLE), and depicts various molecular mechanisms for the development of SLE and lupus-like syndromes. It describes groups of diseases, such as apoptotic defects; NETosis; interferonopathies; complement deficiency; autotolerance disorders associated with mutations in the RAG1/RAG2 genes; hereditary metabolic diseases (prolidase deficiency, deficiency of adenosine deaminase 2; lysinuric protein intolerance; and α-mannosidase deficiency). The table summarizes clinical data on most of the known lupus-like syndromes and their molecular mechanisms.The pathogenesis of many forms of monogenic lupus-like diseases is being studied. The main sign suggesting in favor of the possible monogenic disease in a patient with SLE is its onset in infancy, especially in males. Attention should be also paid to a compromised family history, including to the marriage between close relatives, the resistance of disease to standard therapy, as well as atypical symptoms.

Prolidase deficiency (PD) is a rare autosomal recessive disorder that has symptoms such as chronic skin ulcers, dysmorphic facies, cognitive retardation, hematological anomalies, splenomegaly, and chronic infections. Bone and joint abnormalities were referred occasionally and included the signs and symptoms of prolidase deficiency, but were not deeply investigated in PD patients. We report a case of two PD Moroccan sisters with osteoarticular deformities, some of them were never described before: toes deformities and equinovalgus with fusion and dislocation of a tarsal bone in radiography x-rays.

Prolidase (peptidase D), encoded by the PEPD gene, is a ubiquitously expressed cytosolic metalloproteinase, the only enzyme capable of cleaving imidodipeptides containing C-terminal proline or hydroxyproline. Prolidase catalyzes the rate-limiting step during collagen recycling and is essential in protein metabolism, collagen turnover, and matrix remodeling. Prolidase, therefore plays a crucial role in several physiological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis. Accordingly, mutations leading to loss of prolidase catalytic activity result in prolidase deficiency a rare autosomal recessive metabolic disorder characterized by defective wound healing. In addition, alterations in prolidase enzyme activity have been documented in numerous pathological conditions, making prolidase a useful biochemical marker to measure disease severity. Furthermore, recent studies underscore the importance of a non-enzymatic role of prolidase in cell regulation and infectious disease. This review aims to provide comprehensive information on prolidase, from its discovery to its role in health and disease, while addressing the current knowledge gaps.

Diffuse alveolar haemorrhage (DAH) is rare in the paediatric population and the biological mechanisms remain poorly understood [1]. We retrospectively studied 12 children at our centre, identified from our pathology database, with idiopathic DAH between 2005–2017 who had undergone lung biopsy (SickKids ethics approval number: 1000029185). Two children in this cohort were offered clinical whole exome sequencing (WES) as an investigational diagnostic procedure based on a family history of lung disease. Both children who underwent WES, including the one presented here and one who we had previously reported [2], had single gene mutations that explained the pathogenesis of their pulmonary haemorrhage., The case of a young boy with pulmonary haemorrhage who was ultimately diagnosed on whole exome sequencing with a rare condition called prolidase deficiency. This case demonstrates the utility of modern genomic testing in paediatric rare lung disease. http://ow.ly/rDGz30o8pcd

Background A 4-week-old infant presented to hospital with diarrhea and vomiting. An initial diagnosis of cow’s milk allergy was made and he was discharged with extensively hydrolysed formula. However the infant was readmitted within a week with ongoing symptoms and associated metabolic acidosis. He was suspected to have sepsis and treated with intravenous antibiotics. However, he deteriorated further with worsening metabolic acidosis despite treatment. Methaemoglobinaemia was then identified and he was treated with methylene blue and transferred to HDU for further management. Objectives Consider Methaemoglobinaemia as a differential diagnosis in infants with unexplained metabolic acidosis. Raise awareness on Prolidase deficiency – a rare genetic condition affecting 1 in 1 million of worldwide population. Methods Literature search on Ovid using Medline/Embase was undertaken. Results Conclusions Further investigations results of Imidopeptiduria and rapid exome sequencing confirmed diagnosis of Prolidase deficiency (PD) in this infant. PD is a rare autosomal recessive genetic condition caused by mutations in PEPD gene, which codes for Prolidase, an enzyme involved in the final stage of the degradation of collagen and other proline containing proteins including dietary proteins. PD affects approximately 1 in 1 million worldwide. The symptoms of PD include dysmorphic features, skin lesions, recurrent infections, hepatosplenomegaly and intellectual disability. In this case, the child had symptoms of diarrhoea, likely secondary to PD. As the diarrhoea was persistent, he then developed methaemoglobinaemia as a result. Initial methaemoglobin was 48% which subsequently improved to 3% following 3 doses of methylene blue with good clinical response. Further investigations excluded Systemic lupus erythematosus and Crohn’s disease, which have been reported as associated with PD. The infant’s recovery was supported with parental nutrition and discharged home with ongoing follow up with Paediatric metabolic specialists. Management of PD is mainly supportive with consideration of use of co-factors that can improve collagen stability, suppression of collagenase and topical application of ointments containing L-proline. Enzyme replacement is also being actively researched. This case illustrates the importance of prompt recognition and treatment of methaemoglobinaemia in infants with unexplained metabolic acidosis. It also highlights awareness of PD, which although, is extremely rare, can be diagnosed through metabolic screening tests and genetic testing, thereby leading to earlier supportive management.

Prolidase Deficiency (PD) is an autosomal recessive rare disorder caused by loss or reduction of prolidase enzymatic activity due to variants in the PEPD gene. PD clinical features vary among affected individuals: skin ulcerations, recurrent infections, and developmental delay are common. In this study, we describe a 16-year-old boy with a mild PD phenotype comprising chronic eczema, recurrent infections and elevated IgE. Whole exome sequencing analysis revealed three PEPD variants: c.575T>C p.(Leu192Pro) inherited from the mother, and c.692_694del p.(Tyr231del) and c.1409G>A p.(Arg470His), both inherited from the father. The variant p.(Tyr231del) has been previously characterized by high-resolution X-ray structure analysis as altering protein dynamics/flexibility. In order to study the effects of the other two prolidase variants, we performed site directed mutagenesis purification and crystallization studies. A high-resolution X-ray structure could only be obtained for the p.(Arg470His) variant, which showed no significant structural differences in comparison to WT prolidase. On the other hand, the p.(Leu192Pro) variant led to significant protein destabilization. Hence, we conclude that the maternal p.(Leu192Pro) variant was likely causally associated with the proband´s disease, together with the known pathogenic paternal variant p.(Tyr231del). Our results demonstrated the utility of exome sequencing to perform diagnosis in PD cases with mild phenotype.

Prolidase is a metal-dependent peptidase specialized in the cleavage of dipeptides containing proline or hydroxyproline on their C-termini. Prolidase homologues are found in all kingdoms of life. The importance of prolidase in human health is underlined by a rare hereditary syndrome referred to as Prolidase Deficiency. A growing number of studies highlight the importance of prolidase in various other human conditions, including cancer. Some recent studies link prolidase's activity-independent regulatory role to tumorigenesis. Furthermore, the enzyme or engineered variants have some applications in biotechnology. In this short review, we aim to highlight different aspects of the protein the importance of which is increasingly recognized over the last years.

Prolidase [EC 3.4.13.9], known as PEPD, cleaves di- and tripeptides containing carboxyl-terminal proline or hydroxyproline. For decades, prolidase has been thoroughly investigated, and several mechanisms regulating its activity are known, including the activation of the β1-integrin receptor, insulin-like growth factor 1 receptor (IGF-1) receptor, and transforming growth factor (TGF)-β1 receptor. This process may result in increased availability of proline in the mitochondrial proline cycle, thus making proline serve as a substrate for the resynthesis of collagen, an intracellular signaling molecule. However, as a ligand, PEPD can bind directly to the epidermal growth factor receptor (EGFR, epidermal growth factor receptor 2 (HER2)) and regulate cellular metabolism. Recent reports have indicated that PEPD protects p53 from uncontrolled p53 subcellular activation and its translocation between cellular compartments. PEPD also participates in the maturation of the interferon α/β receptor by regulating its expression. In addition to the biological effects, prolidase demonstrates clinical significance reflected in the disease known as prolidase deficiency. It is also known that prolidase activity is affected in collagen metabolism disorders, metabolic, and oncological conditions. In this article, we review the latest knowledge about prolidase and highlight its biological function, and thus provide an in-depth understanding of prolidase as a dipeptidase and protein regulating the function of key biomolecules in cellular metabolism.

Prolidase deficiency is a rare disorder inherited through an autosomal recessive gene. The hallmark of the disorder are iminodipeptiduria, chronic skin ulcers, recurring infections, mental retardation and characteristic facial appearance, although prolidase deficiency can occur with no clinical manifestation. The primary biological function of the enzyme involves the metabolism of collagen degradation products and the recycling of proline for collagen resynthesis. We describe two patients with prolidase deficiency and review the different clinical manifestations suggesting the pathogenetic mechanism through few hypotheses.

Prolidase catalyzes the cleavage of dipeptides containing proline on their C terminus. The reduction in prolidase activity is the cause of a rare disease named 'Prolidase Deficiency'. Local structural disorder was indicated as one of the causes for diminished prolidase activity. Previous studies showed that heat shock proteins can partially recover prolidase activity in vivo. To analyze this mechanism of enzymatic activity rescue, we compared the crystal structures of selected prolidase mutants expressed in the absence and in the presence of chaperones. Our results confirm that protein chaperones facilitate the formation of more ordered structures by their substrate protein. These results also suggest that the protein expression system needs to be considered as an important parameter in structural studies. DATABASES: The reported crystal structures and their associated structure factor amplitudes were deposited in the Protein Data Bank under the accession codes 6SRE, 6SRF, and 6SRG, respectively.

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Prolidase is a metallopeptidase that cleaves iminodipeptides containing a proline (Pro) or hydroxyproline (Hyp) residue at their C-terminal end. The disease prolidase deficiency (PD) is a rare recessive human disorder characterized by reduced prolidase activity. PD manifests itself by a wide range of severe clinical symptoms, most commonly as skin ulceration, recurrent infections of the respiratory tract, and mental retardation. Several mutations in the PEPD gene have been identified that are responsible for the loss or the reduction of prolidase activity. In contrast, the structural basis of enzyme inactivation has so far remained elusive. In this study, we present high resolution crystal structures of a number of human prolidase (HsProl) variants, in which single amino acids are either substituted by others or deleted. The observed implications of the mutations on the three-dimensional structure of HsProl are reported and discussed and related to their enzymatic activity. The resulting structures may be divided into four groups depending on the presumed effect of the corresponding mutations on the reaction mechanism. The four possible inactivation mechanisms, which could be elucidated, are disruption of the catalytic Mn2 (OH- )-center, introduction of chain disorder along with the displacement of important active site residues, rigidification of the active site, and flexibilization of the active site. Database All refined structure coordinates as well as the corresponding structure factor amplitudes have been deposited in the PDB under the accession numbers 5MBY, 5MBZ, 5MC0, 5MC1, 5MC2, 5MC3, 5MC4, 5MC5, 6H2P, 6H2Q.

Prolidase deficiency (PD) is a rare inherited autosomal recessive disorder of amino acid metabolism with an incidence of 1 to 2 for every one million.1, 2 First described in 1968,1 PD is caused by many different types of mutations in the peptidase D (PEPD) gene encoding prolidase, an enzyme that breaks down iminodipeptides containing proline and hydroxyproline.3 To date, 29 different mutations have been detected in this gene.4 Here we present a 20-year-old man with PD diagnosed through PEPD gene analysis. To our knowledge, the novel mutation detected in this case has never been reported. Therefore, our case provides important genetic information pertaining to PD.

Background We report the Case of Prolidase - deficiency syndrome in a 3-year-old boy suffering from atraumatic compartment syndrome in both hands. Case description We discuss four areas of controversy. First, what could be the reason for an atraumatic compartment syndrome in both hands? Second, should we operate on a 3-year-old boy or use a conservative approach? Third, would we have chosen an operative approach knowing the underlying condition? Fourth, how should we proceed postoperatively? Conclusion We chose to operate him and performed a compartment release operation. He was treated interdisciplinary and discharged after fifteen days.

Prolidase deficiency and solitary mastocytoma of the eyelid are both exceedingly rare. Prolidase deficiency is an inherited connective tissue disorder that has systemic sequelae, such as intractable skin ulceration, poor wound healing, recurrent infections, and intellectual impairment. Cutaneous mastocytoma is an isolated, aberrant cutaneous aggregation of mast cells. A case of an adult with severe prolidase deficiency who developed cutaneous mastocytoma of the eyelid was presented. To the authors' knowledge, adult-onset solitary mastocytoma of the eyelid has never been reported previously.

The extracellular matrix is essential for brain development, lamination, and synaptogenesis. In particular, the basement membrane below the pial meninx (pBM) is required for correct cortical development. The last step in the catabolism of the most abundant protein in pBM, collagen Type IV, requires prolidase, an exopeptidase cleaving the imidodipeptides containing pro or hyp at the C-terminal end. Mutations impairing prolidase activity lead in humans to the rare disease prolidase deficiency characterized by severe skin ulcers and mental impairment. Thus, the dark-like (dal) mouse, in which the prolidase is knocked-out, was used to investigate whether the deficiency of prolidase affects the neuronal maturation during development of a brain cortex area. Focusing on the cerebellar cortex, thinner collagen fibers and disorganized pBM were found. Aberrant cortical granule cell proliferation and migration occurred, associated to defects in brain lamination, and in particular in maturation of Purkinje neurons and formation of synaptic contacts. This study deeply elucidates a link between prolidase activity and neuronal maturation shedding new light on the molecular basis of functional aspects in the prolidase deficiency.

Prolidase deficiency is a rare, autosomal-recessive, inborn error of collagen metabolism. We report a 20-year old girl with prolidase deficiency, who presented with spontaneous, refractory, lower extremity ulceration, elevated immunoglobulin E levels (IgE) and normal intellect. Hyperimmunoglobulin E syndrome was considered as a possible diagnosis but Th1 and Th17 T cell subsets were normal. Thin layer chromatography for examination of amino acids in random urine sample revealed proline containing peptides. Prolidase enzyme activity was undetectable in dried blood spots as well as in plasma using glycylproline as substrate. Sequence analysis of PEPD gene revealed a single nucleotide substitution (G > A) at the splice acceptor consensus sequence of AG resulting in AA at 3′ of intron 11 in both the alleles leading to skipping of entire exon 12, frame shift and premature stop codon at 278.Comparative expression profile of selected inflammatory genes was carried out in the index patient with prolidase deficiency and high immunoglobulin E levels, another patient with hyperimmunoglobulin E syndrome and a normal control. Increased expression of IL-23 and TNF-α in prolidase deficient patient while IL-6 and TNF-a in hyperimmunoglobulinemia E patient was noted. This indicates a pro-inflammatory state in hyperimmunoglobulin E syndrome compared to the stable regulatory immunological state in prolidase deficiency. To the best of our knowledge, 819-1G > A is a novel mutation of PEPD gene in patients with prolidase deficiency and elevated immunoglobulin E levels.

Summary Background Prolidase deficiency is a rare autosomal recessive disease, in which pulmonary manifestations have been sporadically reported. Aims We have encountered two patients who presented with severe pulmonary cystic lesions leading to respiratory failure. This led us to retrospectively evaluate pulmonary involvement in patients with prolidase deficiency treated in our hospital. Results Of 21 patients (including the 2 mentioned above), 12 had a history of recurrent pulmonary infections and 10 were diagnosed as having chronic lung disease. Of seven chest CT scans performed, four patients had subpleural cysts, two patients had bronchiectatic changes, and one had diffused ground glass attenuation and minor linear atelectasis. Three patients died, with all deaths being attributed to respiratory insufficiency. Conclusions Prolidase deficiency is frequently associated with various pulmonary manifestations, including extensive cystic changes that may be life endangering. The differential diagnosis of bilateral cystic changes should include prolidase deficiency, and pulmonary evaluation should be performed in patients with prolidase deficiency. Pediatr Pulmonol. © 2016 Wiley Periodicals, Inc.

The association of moderate to severe eczema and elevated plasma levels of immunoglobulin E is a characteristic not only of atopic dermatitis but also of various genodermatoses: hyperimmunoglobulin E syndromes, Omenn syndrome, Netherton syndrome, peeling skin syndrome type B, severe dermatitis, multiple allergies, and metabolic wasting syndrome, Wiskott-Aldrich syndrome, prolidase deficiency, Loeys-Dietz syndrome, IPEX syndrome, STAT5B deficiency, and pentasomy X. The clinical presentation of these genodermatoses -typically in children- is consistent with severe atopic dermatitis. Immunoglobulin E is elevated from birth and response to conventional treatments is poor. Diagnosis is further complicated by the fact that these genodermatoses often share other clinical manifestations and laboratory findings. We present practical guidelines for differentiating among these various entities, with the aim of helping physicians decide what type of genetic test should be carried out -and when- in order to establish a definitive diagnosis.

Prolidase deficiency is a new genetic cause of combined pulmonary fibrosis and emphysema syndrome in the adulthttp://bit.ly/2QRgqMH

Prolidase (PEPD) catalyses the cleavage of dipeptides with high affinity for proline at the C-terminal end. This function is required in almost all living organisms and orthologues of PEPD were thus detected across a broad taxonomic range. In order to detect strongly conserved residues in PEPD, we analysed PEPD orthologous sequences identified in data sets of animals, plants, fungi, archaea, and bacteria. Due to conservation over very long evolutionary time, conserved residues are likely to be of functional relevance. Single amino acid mutations in PEPD cause an autosomal disorder called prolidase deficiency and were associated with various cancer types. We provide new insights into 15 additional residues with putative roles in prolidase deficiency and cancer. Moreover, our results confirm previous reports identifying five residues involved in the binding of metal cofactors as highly conserved and enable the classification of several non-synonymous single nucleotide polymorphisms as likely pathogenic and seven as putative polymorphisms. Moreover, more than 50 conserved residues across species, which were not previously described, were identified. Conservation degree per residue across the animal kingdom were mapped to the human PEPD 3D structure revealing the strongest conservation close to the active site accompanied with a higher functional implication and pathogenic potential, validating the importance of a characteristic active site fold for prolidase identity.

BACKGROUND The aim of this study was to determine prolidase activity in conjunctival tissue and its relationship with pterygium. MATERIAL AND METHODS Prolidase activity was measured in 23 pterygium and 25 healthy conjunctival tissues and the 2 groups were compared. RESULTS Prolidase enzyme activity could not be measured in either the healthy conjunctival or in pterygium tissues. The mean serum prolidase levels of the control and pterygium groups were 967.46±353.64 and 858.29±301.83, respectively. Statistically, there was no significant difference between the groups with regard to serum prolidase levels (p>0.05). CONCLUSIONS In conclusion, absence of prolidase activity in pterygium tissue indicates that there is no collagen turnover in this tissue. We may explain this finding with the elastin-rich structure of the conjunctiva.

The degradation of the main fibrillar collagens, collagens I and II, is a crucial process for skeletal development. The most abundant dipeptides generated from the catabolism of collagens contain proline and hydroxyproline. In humans, prolidase is the only enzyme able to hydrolyze dipeptides containing these amino acids at their C-terminal end, thus being a key player in collagen synthesis and turnover. Mutations in the prolidase gene cause prolidase deficiency (PD), a rare recessive disorder. Here we describe 12 PD patients, 9 of whom were molecularly characterized in this study. Following a retrospective analysis of all of them a skeletal phenotype associated with short stature, hypertelorism, nose abnormalities, microcephaly, osteopenia and genu valgum, independent of both the type of mutation and the presence of the mutant protein was identified. In order to understand the molecular basis of the bone phenotype associated with PD, we analyzed a recently identified mouse model for the disease, the dark-like (dal) mutant. The dal/dal mice showed a short snout, they were smaller than controls, their femurs were significantly shorter and pQCT and mu CT analyses of long bones revealed compromised bone properties at the cortical and at the trabecular level in both male and female animals. The differences were more pronounce at 1 month being the most parameters normalized by 2 months of age. A delay in the formation of the second ossification center was evident at postnatal day 10. Our work reveals that reduced bone growth was due to impaired chondrocyte proliferation and increased apoptosis rate in the proliferative zone associated with reduced hyperthrophic zone height. These data suggest that lack of prolidase, a cytosolic enzyme involved in the final stage of protein catabolism, is required for normal skeletogenesis especially at early age when the requirement for collagen synthesis and degradation is the highest. (C) 2014 Elsevier Inc. All rights reserved.

Prolidase is a ubiquitously distributed dipeptidase and the only dipeptidase in humans capable of cleaving the peptide bond preceding the amino acids proline (Pro) or hydroxyproline (Hyp). It is mainly implicated in the degradation of dietary and endogenous proteins. It is also involved in the terminal steps of collagen catabolism by hydrolyzing Pro and Hyp-containing dipeptides. Finally, it is believed to play a role in the regulation of peptidic hormones. Diminished or absent prolidase activity is related to a rare autosomal disease, referred to as prolidase deficiency (PD). This disease manifests itself by a variety of clinical symptoms. To date, there is no definitive cure to PD. This may in part be due to an incomplete understanding of the wild-type (wt) enzyme with respect to substrate-binding mode and consequently the mechanism of the catalyzed reaction. In this work, we describe the high-resolution crystal structures of the wt human prolidase in the ligand-free form as well as in substrate-bound states and in complex with the cleavage product Pro. This series of structures provides much relevant information for the definition of substrate-binding and the reaction mechanism. A recent study on Escherichia coli prolidase revealed how substrates of different length are discriminated. Here, based on our own structural results, we evaluate and extend this analysis. Moreover, we describe and analyze substrate and product binding in the active site and we propose that the crucial catalytic moiety is actually a hydroxide ion. This information significantly advances our understanding of prolidase-based pathologies. Database: The refined structure coordinates as well as the corresponding structure factor amplitudes have been deposited in the PDB under the accession numbers 5M4G, 5M4J, 5M4L, and 5M4Q. (Less)

Aim Prolidase deficiency is a rare autosomal recessive disease in which one of the last steps of collagen metabolism, cleavage of proline-containing dipeptides, is impaired. Only about 93 patients have been reported with about 10% also having systemic lupus erythematosus (SLE). Methods We studied a large extended Amish pedigree with four prolidase deficiency patients and three heterozygous individuals for lupus-associated autoimmunity. Eight unaffected Amish children served as normal controls. Prolidase genetics and enzyme activity were confirmed. Antinuclear antibodies (ANA) were determined using indirect immunofluorescence and antibodies against extractable nuclear antigens were determined by various methods, including double immunodiffusion, immunoprecipitation and multiplex bead assay. Serum C1q levels were determined by enzyme-linked immunosorbent assay. Results Two of the four homozygous prolidase deficiency subjects had a positive ANA. One had anti-double-stranded DNA, while another had precipitating anti-Ro. By the simultaneous microbead assay, three of the four had anti-Sm and anti-chromatin. One of the three heterozygous subjects had a positive ANA and immunoprecipitation of a 75 000 molecular weight protein. The unaffected controls had normal prolidase activity and were negative for autoantibodies. Conclusions Prolidase deficiency may be associated with the loss of immune tolerance to lupus-associated autoantigens even without clinical SLE.

Prolidase deficiency (PD) is a rare inborn disorder of collagen metabolism characterized by chronic recurrent cutaneous ulceration. We report a novel 3 bp insertion in the 12th exon of the PEPD gene in two Kashmiri siblings with prolidase deficiency phenotype. This mutation results in addition of an extra alanine residue at the amino-acid position number 304 of prolidase peptide. The structural analysis showed that this Ala insertion is located at the helix (a.a. 300–320), which contains several important hydrogen bonds between residues essential for structural folding for the enzyme activity. In silico analysis suggests that this insertion mutation might distort or bend the helical feature to affect the hydrogen-bond network between residues of neighboring secondary structures and deform the metal-binding geometry of the enzyme. Although approximately 70 PEPD gene mutations and polymorphisms have been reported in various ethnic groups, we however report, for the first time, the identification of insertion mutation in human the PEPD gene.

Prolidase deficiency (PD) is a rare genetic disorder caused by mutations in the peptidase D (PEPD) gene, affecting collagen degradation. Features include lower extremity ulcers, facial dysmorphism, frequent respiratory infections, and intellectual disability, though there is significant intra- and interfamilial variability. Twenty-eight mutations have been previously reported, all either small deletions/duplications or point mutations discovered by enzyme or DNA assays. PD has been reported in patients of various ethnic backgrounds, but never in the Mexican-American population. We describe the first Mexican-American patient with PD, who presented with typical facial features, developmental delay, microcephaly, and xerosis. Chromosome microarray analysis (CMA) revealed a homozygous deletion in the region of 19q13.11, estimated to be between 124.79 and 195.72 kb in size, representing the largest PEPD gene deletion reported to date and the first discovered by CMA.

The enzyme prolidase cleaves dipeptides where the C-terminal amino acid corresponds to proline or hydroxyproline. As a consequence, a deficiency of this enzyme leads to accumulation of these dipeptides, which correspondingly are found to be elevated in urine. In fact, the absence of dipeptiduria is sufficient to rule out a diagnosis of prolidase deficiency. However, given the fact that these dipeptides elute at the same position as more common amino acids, the analyzer’s software will instead call an elevation of these corresponding amino acids. Thus, an elevation of glycylproline, aspartylproline, glutamylproline, threonylproline and serylproline, valylproline, leucylproline, isoleucylproline, alanylproline, phenylalanylproline, and lysylproline will instead be interpreted as an elevation of leucine, citrulline, methionine, isoleucine, beta-aminoisobutyric acid, gamma-aminobutyric acid, ethanolamine, tyrosine, histidine, and anserine/carnosine, respectively. This particular profile of elevated amino acids, however, can easily be overlooked. We hope that the recognition of this characteristic pattern of falsely elevated urinary amino acids will aid in the recognition of prolidase deficiency.

Summary Prolidase is a multifunctional enzyme that possesses the unique ability to degrade imidodipeptides in which a proline or hydroxyproline residue is located at the C-terminal end. Prolidases have been isolated from archaea and bacteria, where they are thought to participate in proline recycling. In mammalian species, prolidases are found in the cytoplasm and function primarily to liberate proline in the final stage of protein catabolism, particularly during the biosynthesis and degradation of collagen. Collagen comprises nearly one-third of the total protein in the body, and it is essential in maintaining tissue structure and integrity. Prolidase deficiency (PD), a rare autosomal recessive disorder in which mutations in the PEPD gene affect prolidase functionality, tends to have serious and sometimes life-threatening clinical symptoms. Recombinant prolidases have many applications and have been investigated not only as a possible treatment for PD, but also as a part of anti-cancer strategies, a component of biodecontamination cocktails and in the dairy industry. This review will serve to discuss the many in vivo functions of procaryotic and eucaryotic prolidases, as well as the most recent advances in therapeutic and biotechnological application of prolidases.

BACKGROUND Hypertrophic cardiomyopathy, characterized by thickened ventricular walls and reduced ventricular chamber volume, is a common cause of sudden cardiac death in young people. Most inherited forms result from mutations in genes encoding sarcomeric proteins. METHODS Histologic analysis identified embryonic cardiac hypertrophy in dark-like mutant mice. BrdU analysis was performed to measure proliferation and cardiomyocytes were isolated to measure cell size. The dark-like mutation was identified by positional cloning. RESULTS The dark-like mutation causes cardiomyocyte hypertrophy due to loss-of-function of peptidase d (Pepd), which encodes prolidase, a cytosolic enzyme that recycles proline for collagen re-synthesis. Prolidase deficiency is a rare autosomal recessive disease in humans with a broad phenotypic spectrum not reported to include heart defects, but a conserved role for prolidase in heart development was confirmed by morpholino knockdown in zebrafish. We tested the hypothesis that loss of prolidase function disrupts collagen-mediated integrin signaling and determined that the levels of several key integrin transducers were reduced in the hearts of dark-like mutant embryos. CONCLUSIONS This work identifies dark-like mice as a model of prolidase deficiency that will be valuable for studying the role of proline metabolism in normal physiology and disease processes, and suggests that integrin signaling may regulate the onset of hypertrophic cardiac growth. Birth Defects Research (Part A) 2011. © 2011 Wiley-Liss, Inc.

Three siblings with recalcitrant leg ulceration, splenomegaly, photosensitive rash, and autoantibodies were suspected of having prolidase deficiency. Urine was checked for iminodipeptiduria, fibroblasts were cultured and analyzed for prolidase activity, and DNA was extracted for identifying the causative mutation. Glycyl proline was found as the dominant dipeptide in the urine. The activity of proline dipeptidase in fibroblasts was 2.5% of control fibroblasts. Sequence analysis of the PEPD gene revealed a homozygous nonsense C--G transition at nucleotide 768. In conclusion, prolidase deficiency was diagnosed in siblings with skin ulceration autoantibodies and a lupus-like disease. A novel nonsense mutation was found, associated with the severe outcome of our patients.

l-Proline concentration is primarily related to the balance of enzymatic activities of proline dehydrogenase [proline oxidase (POX)] and Delta-1-pyrroline-5-carboxylate (P5C) reductase. As a result, P5C plays a pivotal role in maintaining the concentration of proline in body fluids and inborn errors of P5C metabolism lead to disturbance of proline metabolism. Several inborn errors of proline metabolism have been described. Hyperprolinemia type I (HPI) is a result of a deficiency in POX. The POX gene (PRODH) is located on chromosome 22 (22q11.2) and this region is deleted in velo-cardio-facial syndrome, a congenital malformation syndrome. In addition, this gene locus is related to susceptibility to schizophrenia. The other type of hyperprolinemia is HPII. It is caused by a deficiency in P5C dehydrogenase activity. Hypoprolinemia, on the other hand, is found in the recently described deficiency of P5C synthetase. This enzyme defect leads to hyperammonemia associated with hypoornithinemia, hypocitrullinemia, and hypoargininemia other than hypoprolinemia. Hyperhydroxyprolinemia is an autosomal recessive inheritance disorder caused by the deficiency of hydroxyproline oxidase. There are no symptoms and it is believed to be a benign metabolic disorder. The deficiency of ornithine aminotransferase causes transient hyperammonemia during early infancy due to deficiency of ornithine in the urea cycle. In later life, gyrate atrophy of the retina occurs due to hyperornithinemia, a paradoxical phenomenon. Finally, prolidase deficiency is a rare autosomal recessive hereditary disease. Prolidase catalyzes hydrolysis of dipeptide or oligopeptide with a C-terminal proline or hydroxyproline and its deficiency can cause mental retardation and severe skin ulcers.

Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6+/-2.1%) as well as the suitable physical characteristics (mean diameter of 365.5+/-35.1 nm and a positive zeta-potential of 17.94+/-0.12 mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5 microg of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).

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.

The hyper-immunoglobulin E syndrome (HIES) is a rare primary immunodeficiency characterized by recurrent infections, elevated serum IgE-levels, and involvement of the soft- and bony tissues. We speculated that this complex disease may be caused by a microdeletion syndrome. We therefore analyzed 30 sporadic HIES patients for the presence of chromosomal imbalances using Affymetrix 50k XbaI and 23 of the 30 patients with the higher-resolution 250k StyI SNP mapping arrays. We detected only eight different copy number alterations in six patients with the 50k approach, and seven of these presented known polymorphic regions not associated with disease. However, one patient showed a unique gain on chromosome 20p. 250k array analysis identified this gain as a rare polymorphism segregating in the patient's family, but not associated with the HIES phenotype. In addition, 265 known and novel copy number variants (CNVs) were identified with the 250k arrays, but no recurrent imbalances reminescent of a microdeletion syndrome were found. We aligned the identified CNVs with loci that have been associated with HIES or phenotypically overlapping syndromes. Doing so, a 2-Mb deletion spanning the PEPD gene on 19q13.11 was identified on one allele of one patient. Homozygous mutations in PEPD are responsible for the autosomal-recessive prolidase deficiency which resembles HIES in some aspects. Sequencing of the healthy allele, however, revealed a wild-type sequence. In summary, our results suggest that HIES is not likely to be a microdeletion syndrome.

Hypoxia-inducible factor-1 (HIF-1) plays an important role in stress-responsive gene expression. Although primarily sensitive to hypoxia, HIF-1 signaling can be regulated by a number of stress factors including metabolic stress, growth factors and molecules present in the extracellular matrix (ECM). Degradation of ECM by metalloproteinases (MMP) is important for tumor progression, invasion and metastasis. ECM is predominantly collagen, and the imino acids (Pro and HyPro) comprise 25% of collagen residues. The final step in collagen degradation is catalyzed by prolidase, the obligate peptidase for imidodipeptides with Pro and HyPro in the carboxyl terminus. Defective wound healing in patients with inherited prolidase deficiency is associated with histologic features of angiopathy suggesting that prolidase may play a role in angiogenesis. Because HIF-1α is central to angiogenesis, we considered that prolidase may modulate this pathway. To test this hypothesis, we made expression constructs of human prolidase and obtained stable transfectants in colorectal cancer cells (RKO). Overexpression of prolidase resulted in increased nuclear hypoxia inducible factor (HIF-1α) levels and elevated expression of HIF-1−dependent gene products, vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). The activation of HIF-1-dependent transcription was shown by prolidase-dependent activation of hypoxia response element (HRE)-luciferase expression. We used an oxygen-dependent degradation domain (ODD)-luciferase reporter construct as a surrogate for HIF-1α as an in situ prolyl-hydroxylase assay. Since this reporter is degraded by VHL-dependent mechanisms, the increased levels of luciferase observed with prolidase expression reflected the decreased HIF-1α prolyl hydroxylase activity. Additionally, the differential expression of prolidase in 2 breast cancer cell lines showed prolidase-dependent differences in HIF-1α levels. These findings show that metabolism of imidodipeptides by prolidase plays a previously unrecognized role in angiogenic signaling. © 2007 Wiley-Liss, Inc.