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Prolidase is cytosolic manganese dependent exopeptidase responsible for the catabolism of imido di and tripeptides. Prolidase levels have been associated with a number of diseases such as bipolar disorder, erectile dysfunction and varied cancers. Single nucleotide polymorphism present in coding region of proteins (nsSNPs) has the potential to alter the primary structure as well as function of the protein. Hence, it becomes necessary to differentiate the potential harmful nsSNPs from the neutral ones. 19 nsSNPs were predicted as damaging by in-silico analysis of 298 nsSNPs retrieved from dbSNP database. Consurf analysis showed 18 out of 19 substitutions were present in the conserved regions. 4 substitutions (D276N, D287N, E412K, and G448R) that observed to have damaging effect are present in catalytic pocket. Four SNPs listed in splice site region were found to affect splicing of mRNA by altering acceptor site. On 3′UTR scan of 77 SNPs listed in SNP database, 9 SNPs were lead to alter miRNA target sites. These results provide a filtered data to explore the effect of uncharacterized nsSNP and SNP related to UTRs and splice site of prolidase to find their association with the disease susceptibility and to design the target dependent drugs for therapeutics.

The advanced glycation end products (AGEs) participate in the pathogenesis of diabetic nephropathy (DN) by promoting renal inflammation and injury. L-carnosine acts as a quencher of the AGE precursors reactive carbonyl species (RCS), but is rapidly inactivated by carnosinase. In this study, we evaluated the effect of FL-926-16, a carnosinase-resistant and bioavailable carnosine derivative, on the onset and progression of DN in db/db mice.Adult male db/db mice and coeval db/m controls were left untreated or treated with FL-926-16 (30 mg·kgIn the prevention protocol, FL-926-16 significantly attenuated increases in creatinine (-80%), albuminuria (-77%), proteinuria (-75%), mean glomerular area (-34%), fractional (-40%) and mean (-42%) mesangial area in db/db mice. This protective effect was associated with a reduction in glomerular matrix protein expression and cell apoptosis, circulating and tissue oxidative and carbonyl stress, and renal inflammatory markers, including the NLRP3 inflammasome. In the regression protocol, the progression of DN was completely blocked, although not reversed, by FL-926-16. In cultured mesangial cells, FL-926-16 prevented NLRP3 expression induced by RCS but not by the AGE NFL-926-16 is effective at preventing the onset of DN and halting its progression in db/db mice by quenching RCS, thereby reducing the accumulation of their protein adducts and the consequent inflammatory response. In a future perspective, this novel compound may represent a promising AGE-reducing approach for DN therapy.

Dipeptidyl peptidase 9 (DPP9) was recently identified as fusion gene in ovarian high-grade serous carcinoma (HGSC). The aim of this study was to analyze the expression and clinical relevance of DPP8 and DPP9 in ovarian carcinoma, with focus on HGSC. mRNA expression by qRT-PCR of DPP8 and DPP9 was analyzed in 232 carcinomas, including 114 effusions and 118 surgical specimens (89 ovarian, 29 solid metastases). DPP8 and DPP9 protein expression was analyzed in 92 effusions. DPP8 and DPP9 mRNA was overexpressed in effusions compared to solid lesions in analysis of all histotypes (p

Objective: Coronary artery ectasia (CAE) is defined as an angiographic enlargement of a portion of the coronary artery between 1.5 and 2 times the diameter of the adjacent normal coronary artery. It has been demonstrated that increased serum prolidase activity (SPA) is associated with increased collagen turnover. We aimed to analyze the relationship between CAE and serum SPA levels. Methods: This study used a prospective case protocol design. A total of 40 consecutive patients with isolated right CAE and normal coronary arteries (23 men, 17 women; mean age, 62.4±10.8 years) were evaluated. The control group included the same number of consecutive patients with angiographically normal coronary arteries (20 men, 20 women; mean age, 63.8±11.1 years). Clinical characteristics, laboratory results, cardiovascular risk factors, and medication use were recorded. SPA was measured using a spectrophotometer. Student’s t-test, Mann–Whitney U test, chi-square test, Pearson’s and Spearman’s correlations, logistic regression analysis, and ROC curve analysis were used for statistical analysis. Results: SPA was significantly higher in the CAE group compared with the control group (1635.2±492.0 U/L and 986.2±422.3 U/L, respectively; p

A particular allele of the carnosinase gene (CNDP1) is associated with reduced plasma carnosinase activity and reduced risk for nephropathy in diabetic patients. On the one hand, animal and human data suggest that hyperglycemia increases plasma carnosinase activity. On the other hand, we recently reported lower carnosinase activity levels in elite athletes involved in high-intensity exercise compared with untrained controls. Therefore, this study investigates whether exercise training and the consequent reduction in hyperglycemia can suppress carnosinase activity and content in adults with type 2 diabetes. Plasma samples were taken from 243 males and females with type 2 diabetes (mean age = 54.3 yr, SD = 7.1) without major microvascular complications before and after a 6-mo exercise training program [4 groups: sedentary control ( n = 61), aerobic exercise ( n = 59), resistance exercise ( n = 63), and combined exercise training ( n = 60)]. Plasma carnosinase content and activity, hemoglobin (Hb) A1c, lipid profile, and blood pressure were measured. A 6-mo exercise training intervention, irrespective of training modality, did not decrease plasma carnosinase content or activity in type 2 diabetic patients. Plasma carnosinase content and activity showed a high interindividual but very low intraindividual variability over the 6-mo period. Age and sex, but not Hb A1c, were significantly related to the activity or content of this enzyme. It can be concluded that the beneficial effects of exercise training on the incidence of diabetic complications are probably not related to a lowering effect on plasma carnosinase content or activity.

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.

To determine and compare the serum and tear film prolidase activity (PA) between patients with keratoconus and healthy subjects. Also, we aimed to evaluate the serum oxidative stress level and the correlation with serum PA in patients with keratoconus.This prospective, comparative clinical study included 31 patients with keratoconus and 33 age-matched and sex-matched control subjects. All participants underwent a detailed ophthalmologic examination. Serum and tear samples were obtained from all participants. Tears and serum PA and serum oxidative stress markers were measured.No significant differences in demographic characteristics were detected between groups (P0.05). The serum PA was significantly lower in the keratoconus group than in the control group (895.6 ± 198.7 vs. 1145.9 ± 285.4 U/L, P0.001). A tear film comparison showed that PA was lower in the keratoconus group than in the control group; however, this difference was not significant (3075.4 ± 672.2 vs. 3225.8 ± 903.2 U·L⁻¹·g⁻¹ protein, P = 0.45). Oxidative stress markers, such as total oxidant status and oxidative stress index, were found to be significantly higher in the keratoconus group (P0.001).The serum PA was found to be lower in patients with keratoconus than in the controls. Additionally, serum oxidative stress markers were found to be higher than those of the controls. Thus, prolidase and systemic oxidative stress may have a role in the pathogenesis of keratoconus.

The crystal structure of recombinant prolidase fromThermococcus sibiricuswas determined by X-ray diffraction at a resolution of 2.6 Å and was found to contain a tetramer in the asymmetric unit. A protein crystal grown in microgravity using the counter-diffusion method was used for X-ray studies. The crystal belonged to space groupP21221, with unit-cell parametersa= 97.60,b= 123.72,c= 136.52 Å, α = β = γ = 90°. The structure was refined to anRcrystof 22.1% and anRfreeof 29.6%. The structure revealed flexible folding of the N-terminal domain of the protein as well as high variability in the positions of the bound metal ions. The coordinates of the resulting model were deposited in the Protein Data Bank as entry 4rgz.

AIM: To determine the predictive value of increased prolidase activity that reflects increased collagen turnover in patients with hepatocellular carcinoma (HCC). METHODS: Sixty-eight patients with HCC (mean age of 69.1 ± 10.1), 31 cirrhosis patients (mean age of 59.3 ± 6.3) and 33 healthy volunteers (mean age of 51.4 ± 12.6) were enrolled in this study. Univariate and multivariate analysis were used to evaluate the association of serum ?-fetoprotein (AFP) values with HCC clinicopathological features, such as tumor size, number and presence of vascular and macrovascular invasion. The patients with HCC were divided into groups according to tumor size, number and presence of vascular invasion (diameters; ? 3 cm, 3-5 cm and ? 5 cm, number; 1, 2 and ? 3, macrovascular invasion; yes/no). Barcelona-clinic liver cancer (BCLC) criteria were used to stage HCC patients. Serum samples for measurement of prolidase and alphafetoprotein levels were kept at-80 °C until use. Prolidase levels were measured spectrophotometrically and AFP concentrations were determined by a chemiluminescence immunometric commercial diagnostic assay. RESULTS: In patients with HCC, prolidase and AFP values were evaluated according to tumor size, number, presence of macrovascular invasion and BCLC staging classification. Prolidase values were significantly higher in patients with HCC compared with controls (P < 0.001). Prolidase levels were significantly associated with tumor size and number (P < 0.001, P = 0.002, respectively). Prolidase levels also differed in patients in terms of BCLC staging classification (P < 0.001). Furthermore the prolidase levels in HCC patients showed a significant difference compared with patients with cirrhosis (P < 0.001). In HCC patients grouped according to tumor size, number and BCLC staging classification, AFP values differed separately (P = 0.032, P = 0.038, P = 0.015, respectively). In patients with HCC, there was a significant correlation (r = 0.616; P < 0.001) between prolidase and AFP values in terms of tumor size, number and BCLC staging classification, whereas the presence of macrovascular invasion did not show a positive association with serum prolidase and AFP levels. CONCLUSION: Considering the levels of both serum prolidase and AFP could contribute to the early diagnosing of hepatocellular carcinoma. © 2015 Baishideng Publishing Group Inc. All rights reserved.

Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.

Glutathione homoeostasis is critical to plant life and its adaptation to stress. The γ-glutamyl cycle of glutathione biosynthesis and degradation plays a pre-eminent role in glutathione homoeostasis. The genes encoding two enzymatic steps of glutathione degradation, the γ-glutamyl cyclotransferase (GGCT; acting on γ-glutamyl amino acids) and the Cys-Gly dipeptidase, have, however, lacked identification. We have investigated the family of GGCTs in Arabidopsis thaliana . We show through in vivo functional assays in yeast that all three members of the ChaC/GCG subfamily show significant activity towards glutathione but no detectable activity towards γ-glutamyl methionine. Biochemical characterization of the purified recombinant enzymes GGCT2;2 and GGCT2;3 further confirmed that they act specifically to degrade glutathione to yield 5-oxoproline and Cys-Gly peptide and show no significant activity towards γ-glutamyl cysteine. The K m for glutathione was 1.7 and 4.96 mM for GGCT2;2 and GGCT2;3 respectively and was physiologically relevant. Evaluation of representative members of other subfamilies indicates the absence of GGCTs from plants showing significant activity towards γ-glutamyl-amino acids as envisaged in the classical γ-glutamyl cycle. To identify the Cys-Gly peptidase, we evaluated leucine aminopeptidases (LAPs) as candidate enzymes. The cytosolic AtLAP1 ( A. thaliana leucine aminopeptidase 1) and the putative chloroplastic AtLAP3 displayed activity towards Cys-Gly peptide through in vivo functional assays in yeast. Biochemical characterization of the in vitro purified hexameric AtLAP1 enzyme revealed a K m for Cys-Gly of 1.3 mM that was physiologically relevant and indicated that AtLAP1 represents a cytosolic Cys-Gly peptidase activity of A. thaliana . The studies provide new insights into the functioning of the γ-glutamyl cycle in plants.

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

The aim of this study was to evaluate serum prolidase levels and its association with oxidative stress in autoimmune thyroid disease (AITD). 25 with Hashimoto thyroiditis (HT) and 25 patients Graves’ disease (GD), and 27 healthy controls were enrolled in the study. The patients with signs of Graves’ ophthalmopathy were excluded from the study. Serum samples were obtained in euthyroid period at the third month of treatment. Serum prolidase, total antioxidant status (TAS), total oxidative stress (TOS), and total free sulfhydryl (–SH) levels were measured. Serum prolidase levels were significantly higher in the patients with GD compared to the HT and the healthy control group. Oxidative stress index (OSI) and TOS levels of the patients with both GD and HT were significantly higher compared to those of the control group (p

To characterize the hydrolysis of the peptide prodrug pomaglumetad methionil (LY2140023; (1R,4S,5S,6S)-4-(L-methionylamino)-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide), to the active drug LY404039 [(1R,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide], a series of in vitro studies were performed in various matrices, including human intestinal, liver, kidney homogenate, and human plasma. The studies were performed to determine the tissue(s) and enzyme(s) responsible for the conversion of the prodrug to the active molecule. This could enable an assessment of the risk for drug interactions, an evaluation of pharmacogenomic implications, as well as the development of a Physiologically Based Pharmacokinetic (PBPK) model for formation of the active drug. Of the matrices examined, hydrolysis of pomaglumetad methionil was observed in intestinal and kidney homogenate preparations and plasma, but not in liver homogenate. Clearance values calculated after applying standard scaling factors suggest the intestine and kidney as primary sites of hydrolysis. Studies with peptidase inhibitors were performed in an attempt to identify the enzyme(s) catalyzing the conversion. Near complete inhibition of LY404039 formation was observed in intestinal and kidney homogenate and human plasma with the selective dehydropeptidase1 (DPEP1) inhibitor cilastatin. Human recombinant DPEP1 was expressed and shown to catalyze the hydrolysis, which was completely inhibited by cilastatin. These studies demonstrate pomaglumetad methionil can be converted to LY404039 via one or multiple enzymes completely inhibited by cilastatin, likely DPEP1, in plasma, the intestine, and the kidney, with the plasma and kidney involved in the clearance of the circulating prodrug. These experiments define a strategy for the characterization of enzymes responsible for the metabolism of other peptide-like compounds.

Histoplasmosis, one of the most important mycoses, needs to be diagnosed rapidly and accurately. The main method used to diagnose histoplasmosis is serological detection of antibodies to the Histoplasma capsulatum H and M antigens. Several other protein antigens have been reported in H. capsulatum; however, they have not been used for diagnosis. In this study, we explored novel antigens that were detected during H. capsulatum infection. We obtained a protein mixture from H. capsulatum yeast cells after vigorous mixing in a 0.1% Triton X-100 solution. From the resultant pool, we detected nine spots that reacted with sera from patients with histoplasmosis and identified eight seroactive proteins with mass spectrometry. The seroactive proteins were purified, and their antigenicities were tested with an enzyme-linked immunosorbent assay (ELISA). ELISA revealed that the titer of the patients' sera to N-acetylated α-linked acidic dipeptidase was significantly higher than those of healthy volunteers (P0.01). These data indicate that N-acetylated α-linked acidic dipeptidase of H. capsulatum is recognized as a major antigen during histoplasmosis.

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.

The aim of this study was to investigate whether anticoagulant and antiaggregant agents have protective effects against oxidative damage induced by peripheral ischemia–reperfusion (I/R). Groups were created as follows: control group, I/R group (sham group), I/R plus acetylsalicylic acid (Group I), I/R+clopidogrel (Group II), I/R+rivaroxaban (Group III), I/R+bemiparin sodium (Group IV), and I/R+enoxaparin sodium (Group V). In Groups I, II, III, IV, and V, drugs were administered daily for 1 week before I/R creation. Peripheral I/R was induced in the I/R groups by clamping the right femoral artery. The rats were sacrificed 1 hour after reperfusion. Nitrogen oxide levels, malondialdehyde (MDA) levels, paraoxonase-1 (PON1) activity, and prolidase activity were evaluated in both cardiac and renal tissues. There was no significant difference in nitrogen oxide levels between the groups. However, cardiac and renal MDA were significantly higher and PON1 activity was markedly lower in the I/R groups compared with the control group (p

The oxythiamine (OXY) is antivitamin of thiamine. The finding that OXY increases the cytoplasmic concentration of pyruvate, known to enhance collagen biosynthesis, led us to investigate the mechanism of this antivitamin action on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (30-1,000 µM) of OXY for 24 and 48 h. It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells. Since phosphoenolpyruvate (PEP) is known as an inhibitor of prolidase-the enzyme that plays important role in collagen biosynthesis, the mechanism of pyruvate interconversion was considered as a regulatory switch in collagen biosynthesis. In fact, 3-MPA, specific inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), contributed to up-regulation of prolidase activity, suggesting that down-regulation of PEP formation is an underlying mechanism. Since collagen biosynthesis and prolidase activity are regulated by signal induced by activated α2β1 integrin receptor as well as insulin-like growth factor-I receptor (IGF-IR), the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to OXY contributed to decrease in IGF-IR, α2β1 integrin receptor, pERK1/2, and NF-κB p65 expressions. It was accompanied by increase in total ERK1/2 expression and induction of phosphorylation of Akt protein. The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

Introduction Dipeptidyl peptidases (DPPs) 8 and 9 are homologous, cytoplasmic postproline-cutting enzymes, which have similar enzymatic activity and preferred substrates as DPP4. DPP4 is a well-known target for treating diabetes mellitus. With the increased concern of non-selectivity and toxicities caused by DPP4 inhibitors, it is essential to establish new ex vivo system to investigate DPP4 inhibitors' effect on DPP8 and DPP9. Method Here we reported a newly established cell model system by cloning and transfecting human DPP8/9 genes into HEK 293 cells. We then used this model to evaluate the clinically applied DPP4 inhibitors' effect on DPP8/9, by direct enzymatic activity assay. Given the difference of cellular locations between DPP4 and DPP8/9, we also evaluated the influence of these drugs on intracellular DPP8/9 activity and cell viability by extracellular treatment with different inhibitors. Results Direct enzymatic activity assay revealed significant and concentration-dependent inhibition effect of vildagliptin, saxagliptin on DPP8/9. Extracellular incubation of DPP8/9 over expressed cells with sitagliptin, vildagliptin, saxagliptin, alogliptin and linagliptin, showed only mild inhibition on DPP8/9. Moreover, all of these drugs showed no significant influence on cell viability. Discussion Our results demonstrated that the DPP8/9 over-expressing cell model system is a very useful and promising system for investigating the selectivity and associated toxicity of DPP4 inhibitors on DPP8/9.

Carnosine dipeptidase II (CN2), a metallopeptidase present in the cytosol of various vertebrate tissues, catalyzes the hydrolysis of carnosine and several other dipeptides in the presence of Mn2+. Although the metal-binding center of mouse CN2 is also able to associate with Zn2+ in vitro, it was not known whether the zinc form of CN2 has any enzymatic activity. In the present study, we show that Zn2+ has a higher affinity for binding to CN2 than Mn2+, as evidenced by native mass spectrometry. The issue of whether the zinc form of CN2 has enzymatic activity was also examined using various dipeptides as substrates. The findings indicate that the zinc form of CN2 catalyzes the hydrolysis of several different dipeptides including Leu-His, Met-His and Ala-His at a reaction rate comparable to that for its manganese form. On the other hand, the zinc form of CN2 did not catalyze the hydrolysis of carnosine and several other dipeptides that are hydrolyzed by the manganese form of CN2. Substrate specificity was also examined in HEK293T cells expressing CN2, and the findings indicate that Leu-His, Met-His, but not carnosine, were hydrolyzed in the cell culture. These results suggest that the zinc form of CN2 is an active enzyme, but with a different substrate specificity from that of the manganese form.

Metabolic engineering of Saccharomyces cerevisiae often requires a restriction on the ethanol biosynthesis pathway. The non-ethanol-producing strains, however, are slow growers. In this study, a cDNA library constructed from S. cerevisiae was used to improve the slow growth of non-ethanol-producing S. cerevisiae strains lacking all pyruvate decarboxylase enzymes (Pdc−, YSM021). Among the obtained 120 constructs expressing cDNAs, 34 transformants showed a stable phenotype with quicker growth. Sequence analysis showed that the open reading frames of PDC1, DUG1 (Cys–Gly metallo-di-peptidase in the glutathione degradation pathway), and TEF1 (translational elongation factor EF-1 alpha) genes were inserted into the plasmids of 32, 1, and 1 engineered strains, respectively. DUG1 function was confirmed by the construction of YSM021 pGK416-DUG1 strain because the specific growth rate of YSM021 pGK416-DUG1 (0.032 ± 0.0005 h−1) was significantly higher than that of the control strains (0.029 ± 0.0008 h−1). This suggested that cysteine supplied from glutathione was probably used for cell growth and for construction of Fe-S clusters. The results showed that the overexpression of cDNAs is a promising approach to engineer S. cerevisiae metabolism.

Carnosine (β-alanyl-L-histidine), abundantly found in skeletal muscle, plays an important role during exercise, especially for high-intensity contractions. Variability in muscle carnosine content between individuals exists and may also be explained by different genetic bases, although no study has addressed the association of polymorphisms in genes related to carnosine metabolism in athletes. This study aimed to investigate the frequency of single nucleotide polymorphisms (SNPs) in the carnosinase genes (CNDP1 and CNDP2) in a large Brazilian cohort of athletes and nonathletes. Eight SNPs were compared between a representative cohort of elite athletes from Brazil (n = 908) and a paired group of nonathletes (n = 967). The athletes were stratified into three groups: endurance (n = 328), power (n = 415), and combat (n = 165). The CNDP2 rs6566810 (A/A genotype) is overrepresented in endurance athletes, but only in international-level endurance athletes. Three SNPs (CNDP2 rs3764509, CNDP2-CNDP1 rs2346061, and CNDP1 rs2887) were overrepresented in power athletes compared with nonathletes. Carriers of the minor allele had an increased odds ratio of being a power athlete. For the rs2346061, no significant difference was observed in genotype frequencies between power and combat sports athletes, but for rs2887 the power and combat groups showed an inverse genotype distribution. In conclusion, we found that minor alleles carriers for CNDP2 rs3764509 (G-allele), CNDP2-CNDP1 rs2346061 (C-allele), and CNDP1 rs2887 (A-allele) are more likely to be a power athlete. These polymorphisms may be novel genetic markers for power athletes. Furthermore, these results are suggestive of a distinct CNDP genotype for sporting development.

Radiographic contrast agents cause acute kidney injury (AKI), yet the underlying pathogenesis is poorly understood. Nod-like receptor pyrin containing 3-deficient (Nlrp3-deficient) mice displayed reduced epithelial cell injury and inflammation in the kidney in a model of contrast-induced AKI (CI-AKI). Unexpectedly, contrast agents directly induced tubular epithelial cell death in vitro that was not dependent on Nlrp3. Rather, contrast agents activated the canonical Nlrp3 inflammasome in macrophages. Intravital microscopy revealed diatrizoate (DTA) uptake within minutes in perivascular CX3CR1+ resident phagocytes in the kidney. Following rapid filtration into the tubular luminal space, DTA was reabsorbed and concentrated in tubular epithelial cells via the brush border enzyme dipeptidase-1 in volume-depleted but not euvolemic mice. LysM-GFP+ macrophages recruited to the kidney interstitial space ingested contrast material transported from the urine via direct interactions with tubules. CI-AKI was dependent on resident renal phagocytes, IL-1, leukocyte recruitment, and dipeptidase-1. Levels of the inflammasome-related urinary biomarkers IL-18 and caspase-1 were increased immediately following contrast administration in patients undergoing coronary angiography, consistent with the acute renal effects observed in mice. Taken together, these data show that CI-AKI is a multistep process that involves immune surveillance by resident and infiltrating renal phagocytes, Nlrp3-dependent inflammation, and the tubular reabsorption of contrast via dipeptidase-1.

Tomasz Guszczyn,1 Arkadiusz Surażyński,2 Ilona Zaręba,2 Edyta Rysiak,2 Janusz Popko,1 Jerzy Pałka2 1Department of Pediatric Orthopedics and Traumatology, 2Department of Medicinal Chemistry, Medical University of Białystok, Białystok, Poland Abstract: The study was conducted to evaluate the effects of platelet-rich plasma (PRP), supernatant of PRP (SPRP) obtained by centrifugation, and supernatant of activated PRP (SActi-PRP) obtained by Ca2+ solution-treated PRP on collagen biosynthesis, prolidase activity, and β1-integrin signaling in cultured human skin fibroblasts. Incubation of fibroblasts with 5% PRP for 24 h contributed to ~5-fold increase in collagen biosynthesis compared to the control. In the cells treated with 5% of SPRP or SActi-PRP, collagen biosynthesis showed a 3-fold increase of the control. PRP, SPRP, and SActi-PRP stimulated prolidase activity similar to collagen biosynthesis. Collagen biosynthesis and prolidase activity are regulated by β1-integrin receptor signaling. Incubation of fibroblasts with PRP for 24 h contributed to a dose-dependent increase in the expression of β1-integrin receptor, while SActi-PRP increased the process to a much lower extent. SPRP had no effect on the β1-integrin receptor expression. All the studied fractions of blood increased the expression of FAK as well as the expression of phosphorylated MAP-kinases. However, PRP was found to be the most effective stimulator of expression of these particular kinases. These studies suggest that a complex of factors, including growth factors, adhesion molecules, and prolidase contained in PRP, all evoke growth and collagen-promoting activities in human dermal fibroblasts. Keywords: fibroblasts, FAK, MAPK

Carnosinase (CN1) is a dipeptidase, encoded by the CNDP1 gene, that degrades histidine-containing dipeptides, such as carnosine, anserine and homocarnosine. Loss of CN1 function (also called carnosinase deficiency or aminoacyl-histidine dipeptidase deficiency) has been reported in a small number of patients with highly elevated blood carnosine concentrations, denoted carnosinaemia; it is unclear whether the variety of clinical symptoms in these individuals is causally related to carnosinase deficiency. Reduced CN1 function should increase serum carnosine concentrations but the genetic basis of carnosinaemia has not been formally confirmed to be due to CNDP1 mutations. A CNDP1 polymorphism associated with low CN1 activity correlates with significantly reduced risk for diabetic nephropathy, especially in women with type 2 diabetes, and may slow progression of chronic kidney disease in children with glomerulonephritis. Studies in rodents demonstrate antiproteinuric and vasculoprotective effects of carnosine, the precise molecular mechanisms, however, are still incompletely understood. Thus, carnosinemia due to CN1 deficiency may be a non-disease; in contrast, carnosine may potentially protect against long-term sequelae of reactive metabolites accumulating, e.g. in diabetes and chronic renal failure.

Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects.

Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role., In the prevailing model for assisted protein folding, chaperonins act passively by preventing protein aggregation. Here, the authors use single-molecule fluorescence measurements and cryo-electron microscopy and show that the E. coli GroELS chaperonin system also has an active role in folding the endogenous bacterial protein PepQ.

Background/Aims: It is possible that carnosinase (CNDP1) and cellular nonspecific dipeptidase (CNDP2) have important roles in protecting cells and tissues against the damage of oxidative stress. Oxidative stress and subsequent inflammation are key factors in the development of common chronic metabolic diseases, such as obesity. We aimed to investigate the combined effects of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate and carotene intake on obesity risk. Methods: A total of 1,059 Japanese men were randomly selected from participants who visited a medical center for routine medical checkups. We analyzed the relationships between the genotypes of 4 single-nucleotide polymorphisms (SNPs) (rs12605520, rs7244647, rs4891558, and rs17089368) in the CNDP1/CNDP2 locus and body mass index or prevalence of obesity/overweight taking into account dietary carbohydrate and carotene intake. Results: We found that 2 SNPs (rs7244647 in CNDP1 and rs4891558 in CNDP2) were associated with obesity risk. In addition, these associations were observed only in the group with high carbohydrate and low carotene intake but not in the group with low carbohydrate and high carotene intake. Conclusions: Our findings indicate that the combination of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate/carotene intake modulate obesity risk.

Human prolidase is a proline-specific metallopeptidase that catalyzes the hydrolysis of dipeptides containing a proline residue at the C-terminus. Human prolidase also has weak hydrolytic activity for toxic organophosphorus compounds, including diisopropyl fluorophosphate (DFP), sarin and soman. In this study, we performed in silico analysis to improve the catalytic activity of human prolidase towards DFP hydrolysis. We predicted that single amino acid substitution mutations of either A252R or P365R could form a salt bridge between the A252R mutant residue and the E249 residue or the P365R mutant residue and the E424 residue. The predicted values for ΔGstability and ΔGaffinity due to salt bridge formation were examined: for A252R, ΔGstability = -12.8 kcal/mol and ΔGaffinity = -0.02 kcal/mol; for P365R, ΔGstability = -1.44 kcal/mol and ΔGaffinity = -2.98 kcal/mol. Next, we produced wild-type (WT), A252R, P365R and A252R/P365R recombinant human prolidases from a bacterial expression system with a purity of over 99 %. Finally, we measured the DFPase and prolidase activities of the purified recombinant human prolidases. The catalytic efficiencies of A252R and P365R towards DFP hydrolysis were 1.23- and 1.36-fold increases, respectively, compared to that of the WT, while the prolidase activities of A252R and P365R towards Leu-Pro hydrolysis were 0.88- and 0.78-fold that of the WT, respectively. However, we could not detect any enzymatic activity in the recombinant A252R/P365R double substitution mutant. Thus, our results indicate that substitution mutations of A252R and P365R in human prolidase show improved DFPase activity.

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)

INTRODUCTION Acute pancreatitis is basically considered as activation of inactive proenzymes in the pancreas and digestion of the gland itself. This study was performed to determine if prolidase enzyme, which plays a role in collagen metabolism, could be used as a parameter to assess the severity of pancreatitis in experimentally induced mild and severe pancreatitis. MATERIAL AND METHOD To create experimentally induced acute pancreatitis 0.1 ml of normal saline solution (NSS) was given five times with an interval of one hour to rats in the first group; 50 µg/kg of cerulein five times with an interval of one hour in the second group; 80 µg/kg of cerulein five times with an interval of one hour in the third group, in the form of intraperitoneal injection. RESULTS When the serum prolidase values at beginning, 1st, 5th and 24th hours in group II and III were compared among themselves, there was a statistically significant increase(p < 0.05). The evaluation between groups revealed a statistically significant increase in the value of serum prolidase in group II and group III compared with the control group (p < 0.05). In comparisons performed with tissue values, a statistically significant increase determined in the value of serum prolidase in group II and group III compared with the control group was observed (p < 0.05). CONCLUSION The findings obtained in our study showed that prolidase activity increases directly proportionaly with the severity of pancreatitis. This allows us to postulate that prolidase enzyme activities provide guidance about the metabolism of collagen in patients with acute pancreatitis, serious damage occurring in collagen protein and metabolic control is further distorted depending on the duration and intensity of damage but to be able to speak more precisely, there is a need for further, more detailed and extensive researchs (Tab. 8, Fig. 2, Ref. 30).

Considering that the homozygous CNDP1 (CTG)5 genotype affords protection against diabetic nephropathy (DN) in female patients with type 2 diabetes, this study assessed if this association remains gender-specific when applying clinical inclusion criteria (CIC-DN) or biopsy proof (BP-DN). Additionally, it assessed if the prevalence of the protective genotype changes with diabetes duration and time on hemodialysis and if this occurs in association with serum carnosinase (CN-1) activity. Whereas the distribution of the (CTG)5 homozygous genotype in the no-DN and CIC-DN patients was comparable, a lower frequency was found in the BP-DN patients, particularly in females. We observed a significant trend towards high frequencies of the (CTG)5 homozygous genotype with increased time on dialysis. This was also observed for diabetes duration but only reached significance when both (CTG)5 homo- and heterozygous patients were included. CN-1 activity negatively correlated with time on hemodialysis and was lower in (CTG)5 homozygous patients. The latter remained significant in female subjects after gender stratification. We confirm the association between the CNDP1 genotype and DN to be likely gender-specific. Although our data also suggest that (CTG)5 homozygous patients may have a survival advantage on dialysis and in diabetes, this hypothesis needs to be confirmed in a prospective cohort study.

Osteoarthritis is characterized by the presence of proinflammatory cytokines and reactive oxygen species. We aimed to clarify the role of prooxidant enzyme content at the synovial membrane level and how it correlates with the inflammatory process in patients with knee osteoarthritis (KOA). In synovial membranes from KOA patients and control group, we analyzed the protein content of prooxidant enzymes such as Nox2, xanthine oxidase (XO), and prolidase as well as the proinflammatory NALP3. Results show that protein content of prolidase and Nox2 increased 4.8- and 8.4-fold, respectively, and XO showed an increasing trend, while the NALP3 inflammasome increased 5.4-fold with respect to control group. Levels of prolidase and XO had a positive correlation between the levels of NALP3 and Nox2. By principal component analysis the protein expression pattern by study groups was evaluated. Three clusters were identified; protein expression patterns were higher for clusters two (prolidase) and three (XO and Nox2) between KOA patients and controls. Data suggest that prooxidant enzymes increase in synovial membrane of KOA patients and may contribute to the inflammatory state and degradation of the articular cartilage.

We previously demonstrated that polymorphisms in the carnosinase-1 gene (CNDP1) determine the risk of nephropathy in type 2 diabetic patients. Carnosine, the substrate of the enzyme encoded by this gene, is considered renoprotective and could possibly be used to treat diabetic nephropathy (DN). In this study, we examined the effect of carnosine treatment in vivo in BTBR (Black and Tan, BRachyuric) ob/ob mice, a type 2 diabetes model which develops a phenotype that closely resembles advanced human DN. Treatment of BTBR ob/ob mice with 4 mM carnosine for 18 weeks reduced plasma glucose and HbA1c, concomitant with elevated insulin and C-peptide levels. Also, albuminuria and kidney weights were reduced in carnosine-treated mice, which showed less glomerular hypertrophy due to a decrease in the surface area of Bowman’s capsule and space. Carnosine treatment restored the glomerular ultrastructure without affecting podocyte number, resulted in a modified molecular composition of the expanded mesangial matrix and led to the formation of carnosine-acrolein adducts. Our results demonstrate that treatment with carnosine improves glucose metabolism, albuminuria and pathology in BTBR ob/ob mice. Hence, carnosine could be a novel therapeutic strategy to treat patients with DN and/or be used to prevent DN in patients with diabetes.

Background Gene-diet interactions have been reported to contribute to the development of type 2 diabetes (T2D). However, to our knowledge, few examples have been consistently replicated to date. Objective We aimed to identify existing evidence for genemacronutrient interactions and T2D and to examine the reported interactions in a large-scale study. Design We systematically reviewed studies reporting genemacronutrient interactions and T2D. We searched the MEDLINE, Human Genome Epidemiology Network, and WHO International Clinical Trials Registry Platform electronic databases to identify studies published up to October 2015. Eligibility criteria included assessment of macronutrient quantity (e.g., total carbohydrate) or indicators of quality (e.g., dietary fiber) by use of self-report or objective biomarkers of intake. Interactions identified in the review were subsequently examined in the EPIC (European Prospective Investigation into Cancer)-InterAct case-cohort study (n = 21,148, with 9403 T2D cases; 8 European countries). Prentice-weighted Cox regression was used to estimate countryspecific HRs, 95% CIs, and P-interaction values, which were then pooled by random-effects meta-analysis. A primary model was fitted by using the same covariates as reported in the published studies, and a second model adjusted for additional covariates and estimated the effects of isocaloric macronutrient substitution. Results Thirteen observational studies met the eligibility criteria (n , 1700 cases). Eight unique interactions were reported to be significant between macronutrients [carbohydrate, fat, saturated fat, dietary fiber, and glycemic load derived from self-report of dietary intake and circulating n–3 (v-3) polyunsaturated fatty acids] and genetic variants in or near transcription factor 7–like 2 (TCF7L2), gastric inhibitory polypeptide receptor (GIPR), caveolin 2 (CAV2), and peptidase D (PEPD) (P-interaction , 0.05). We found no evidence of interaction when we tried to replicate previously reported interactions. In addition, no interactions were detected in models with additional covariates. Conclusions Eight gene-macronutrient interactions were identified for the risk of T2D from the literature. These interactions were not replicated in the EPIC-InterAct study, which mirrored the analyses undertaken in the original reports. Our findings highlight the importance of independent replication of reported interactions.

Isoaspartyl dipeptidase (IadA) is an enzyme that catalyzes the hydrolysis of an isoaspartyl dipeptide-like moiety, which can be inappropriately formed in proteins, between the β-carboxyl group side chain of Asp and the amino group of the following amino acid. Here, we have determined the structures of an isoaspartyl dipeptidase (CpsIadA) from Colwellia psychrerythraea, both ligand-free and that complexed with β-isoaspartyl lysine, at 1.85-Å and 2.33-Å resolution, respectively. In both structures, CpsIadA formed an octamer with two Zn ions in the active site. A structural comparison with Escherichia coli isoaspartyl dipeptidase (EcoIadA) revealed a major difference in the structure of the active site. For metal ion coordination, CpsIadA has a Glu166 residue in the active site, whereas EcoIadA has a post-translationally carbamylated-lysine 162 residue. Site-directed mutagenesis studies confirmed that the Glu166 residue is critical for CpsIadA enzymatic activity. This residue substitution from lysine to glutamate induces the protrusion of the β12-α8 loop into the active site to compensate for the loss of length of the side chain. In addition, the α3-β9 loop of CpsIadA adopts a different conformation compared to EcoIadA, which induces a change in the structure of the substrate-binding pocket. Despite CpsIadA having a different active-site residue composition and substrate-binding pocket, there is only a slight difference in CpsIadA substrate specificity compared with EcoIadA. Comparative sequence analysis classified IadA-containing bacteria and archaea into two groups based on the active-site residue composition, with Type I IadAs having a glutamate residue and Type II IadAs having a carbamylated-lysine residue. CpsIadA has maximal activity at pH 8-8.5 and 45°C, and was completely inactivated at 60°C. Despite being isolated from a psychrophilic bacteria, CpsIadA is thermostable probably owing to its octameric structure. This is the first conclusive description of the structure and properties of a Type I IadA.

Dug1p, a M20 family metallopeptidase and human orthologue of carnosinase, hydrolyzes Cys-Gly dipeptide, the last step of glutathione (GSH) degradation in Saccharomyces cerevisiae. Molecular bases of peptide recognition by Dug1p and other M20 family peptidases remain unclear in the absence of structural information about enzyme-peptide complexes. We report the crystal structure of Dug1p at 2.55 Å resolution in complex with a Gly-Cys dipeptide and two Zn(2+) ions. The dipeptide is trapped in the tunnel-like active site; its C-terminus is held by residues at the S1' binding pocket, whereas the S1 pocket coordinates Zn(2+) ions and the N-terminus of the peptide. Superposition with the carnosinase structure shows that peptide mimics the inhibitor bestatin, but active site features are altered upon peptide binding. The space occupied by the N-terminus of bestatin is left unoccupied in the Dug1p structure, suggesting that tripeptides could bind. Modeling of tripeptides into the Dug1p active site showed tripeptides fit well. Guided by the structure and modeling, we examined the ability of Dug1p to hydrolyze tripeptides, and results show that Dug1p hydrolyzes tripeptides selectively. Point mutations of catalytic residues do not abolish the peptide binding but abolish the hydrolytic activity, suggesting a noncooperative mode in peptide recognition. In summary, results reveal that peptides are recognized primarily through their amino and carboxyl termini, but hydrolysis depends on the properties of peptide substrates, dictated by their respective sequences. Structural similarity between the Dug1p-peptide complex and the bestatin-bound complex of CN2 suggests that the Dug1p-peptide structure can be used as a template for designing natural peptide inhibitors.

Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.

Xaa-Pro dipeptidase (XPD; prolidase; EC 3.4.13.9) specifically hydrolyzes dipeptides with a prolyl residue at the carboxy-terminus.Xanthomonasspp. possess two different isoforms of XPD (48 and 43 kDa) which share ∼24% sequence identity. The XPD of 43 kDa in size (XPD43) fromXanthomonasspp. is unusual as it lacks the strictly conserved tyrosine residue (equivalent to Tyr387 inEscherichia coliaminopeptidase P) that is suggested to be important in the proton-shuttle transfer required for catalysis in the M24B (MEROPS) family. Here, the crystallization and preliminary X-ray analysis of XPD43 fromX. campestris(GenBank accession No. NP_637763) are reported. Recombinant XPD43 was crystallized using the microbatch-under-oil technique. Diffraction data were collected on the recently commissioned protein crystallography beamline (PX-BL21) at the Indian synchrotron (Indus-2, 2.5 GeV) to 1.83 Å resolution with 100% completeness. The crystal belonged to space groupP212121, with unit-cell parametersa= 84.32,b= 105.51,c= 111.35 Å. Two monomers are expected to be present in the asymmetric unit of the crystal, corresponding to a solvent content of 58%. Structural analysis of XPD43 will provide new insights into the role of the conserved residues in catalysis in the M24B family.

Most previous reports have shown that the basic mechanism of inguinal hernia involves insufficient collagen strength and metabolism. The aim of this study was to evaluate whether joint hypermobility is involved in the development of inguinal hernia in children and to investigate oxidative stress parameters and prolidase activity in tissue samples from children with inguinal hernia.This cross-sectional study involving 41 patients (age, 6.36 ± 2.96 years) with inguinal hernia treated in the pediatric surgery department of our institution and 40 age- and sex-matched controls (age, 6.02 ± 3.13 years) was performed from May to December 2011. Joint hypermobility was assessed using the Beighton criteria in all patients. Hernia sacs were analyzed with respect to the total antioxidative/oxidative status and prolidase activity. The patients were divided into two groups (inguinal hernia with and without hypermobility) according to a Beighton score cut-off of ≥6.A total of 81 subjects aged 3-10 years participated. The ratio of joint hypermobility was significantly higher in patients than in controls (p = 0.01). The prolidase activity, total oxidant status, and oxidative stress index were higher in tissue samples from patients with joint hypermobility (p 0.001).Our results show that joint hypermobility syndrome is associated with inguinal hernia in children and that increased prolidase activity and oxidative stress in tissue samples from patients with joint hypermobility syndrome are related to collagen tissue damage and turnover.

Carnosinases are Xaa-His dipeptidases that play diverse functions throughout all kingdoms of life. Human isoforms of carnosinase (CN1 and CN2) under appropriate conditions catalyze the hydrolysis of the dipeptides carnosine (?-alanyl-L-histidine) and homocarnosine (?-aminobutyryl-L- histidine). Alterations of serum carnosinase (CN1) activity has been associated with several pathological conditions, such as neurological disorders, chronic diseases and cancer. For this reason the use of carnosinase levels as a biomarker in cerebrospinal fluid (CSF) has been questioned. The hydrolysis of imidazolerelated dipeptides in prokaryotes and eukaryotes is also catalyzed by aminoacyl-histidine dipeptidases like PepD (EC 3.4.13.3), PepV (EC 3.4.13.19) and anserinase (EC 3.4.13.5). The review deals with the structure and function of this class of enzymes in physiological and pathological conditions. The main substrates of these enzymes, i.e., carnosine, homocarnosine and anserine (?-alanyl-3-methyl-L-histidine) will also be described. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

The purpose of this article is to characterize skin lesions in cynomolgus monkeys following vildagliptin (dipeptidyl peptidase-4 inhibitor) treatment. Oral vildagliptin administration caused dose-dependent and reversible blister formation, peeling and flaking skin, erosions, ulcerations, scabs, and sores involving the extremities at ≥5 mg/kg/day and necrosis of the tail and the pinnae at ≥80 mg/kg/day after 3 weeks of treatment. At the affected sites, the media and the endothelium of dermal arterioles showed hypertrophy/hyperplasia. Skin lesion formation was prevented by elevating ambient temperature. Vildagliptin treatment also produced an increase in blood pressure and heart rate likely via increased sympathetic tone. Following treatment with vildagliptin at 80 mg/kg/day, the recovery time after lowering the temperature in the feet of monkeys and inducing cold stress was prolonged. Ex vivo investigations showed that small digital arteries from skin biopsies of vildagliptin-treated monkeys exhibited an increase in neuropeptide Y–induced vasoconstriction. This finding correlated with a specific increase in NPY and in NPY1 receptors observed in the skin of vildagliptin-treated monkeys. Present data provide evidence that skin effects in monkeys are of vascular origin and that the effects on the NPY system in combination with increased peripheral sympathetic tone play an important pathomechanistic role in the pathogenesis of cutaneous toxicity.

Influenza A virus (IAV) entry is a multistep process that requires the interaction of the virus with numerous host factors. In this study, we demonstrate that prolidase (PEPD) is a cellular factor required by IAV for successful entry into target cells. PEPD was selected as a candidate during an entry screen performed on nonvalidated primary hits from previously published genome-wide small interfering RNA (siRNA) screens. siRNA-mediated depletion of PEPD resulted in the decreased growth of IAV during mono- and multicycle growth. This growth defect was independent of cell type or virus strain. Furthermore, IAV restriction was apparent as early as 3 h postinfection, and experiments in the absence of protein biosynthesis revealed that the nuclear import of viral ribonucleoprotein complexes (vRNPs) was already blocked in the absence of PEPD. These results led us to investigate which step during entry was affected. Receptor expression, IAV attachment, or IAV internalization was not dependent on the presence of PEPD. However, when looking at the distribution of incoming IAV particles in PEPD-knockdown cells, we found a localization pattern that differed from that in control cells: IAV mostly localized to the cell periphery, and consequently, viral particles displayed reduced colocalization with early and late endosome markers and fusion between viral and endosomal membranes was strongly reduced. Finally, experiments using a competitive inhibitor of PEPD catalytic activity suggested that the enzymatic function of the dipeptidase is required for its proviral effect on IAV entry. In sum, this study establishes PEPD as a novel entry factor required for early endosomal trafficking of IAV. IMPORTANCE Influenza A virus (IAV) continues to be a constant threat to public health. As IAV relies on its host cell for replication, the identification of host factors required by the virus is of importance. First, such studies often reveal novel functions of cellular factors and can extend our knowledge of cellular processes. Second, we can further our understanding of processes that are required for the entry of IAV into target cells. Third, the identification of host factors that contribute to IAV entry will increase the number of potential targets for the development of novel antiviral drugs that are of urgent need. Our study identifies prolidase (PEPD) to be a novel entry factor required by IAV for correct routing within the endosomal compartment following virus internalization. Thereby, we link PEPD, which has been shown to play a role during collagen recycling and growth factor signaling, to early events of viral infection.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

In this study, silica-coated magnetic nanoparticles (SiMNPs) with isocyanatopropyltriethoxysilane as a metal-chelating ligand were prepared for the immobilization of His6-tagged Escherichia coli prolidase (His6-EcPepQ). Under one-hour coupling, the enzyme-loading capacity for the Ni2+-functionalized SiMNPs (NiNTASiMNPs) was 1.5 mg/mg support, corresponding to about 58.6% recovery of the initial activity. Native and enzyme-bound NiNTASiMNPs were subsequently characterized by transmission electron microscopy (TEM), superparamagnetic analysis, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. As compared to free enzyme, His6-EcPepQ@NiNTASiMNPs had significantly higher activity at 70 &deg, C and pH ranges of 5.5 to 10, and exhibited a greater stability during a storage period of 60 days and could be recycled 20 times with approximately 80% retention of the initial activity. The immobilized enzyme was further applied in the hydrolysis of two different organophosphorus compounds, dimethyl p-nitrophenyl phosphate (methyl paraoxon) and diethyl p-nitrophenyl phosphate (ethyl paraoxon). The experimental results showed that methyl paraoxon was a preferred substrate for His6-EcPepQ and the kinetic behavior of free and immobilized enzymes towards this substance was obviously different. Taken together, the immobilization strategy surely provides an efficient means to deposit active enzymes onto NiNTASiMNPs for His6-EcPepQ-mediated biocatalysis.

We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum β-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC ( T >MIC ) below clinically achievable levels in humans. These findings provide evidence supporting the potential of β-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

Previous studies have suggested that prolidase and nitric oxide (NO) regulate many processes, such as collagen synthesis and matrix remodeling. Oxidative stress plays an important role in the development of microvascular complications in diabetic patients. Data on serum prolidase activity in patients with diabetes mellitus or diabetic neuropathy (DN) are limited and conflicting. The aim of this study was to measure serum prolidase activity, NO, total antioxidant status (TAS), and malondialdehyde (MDA) levels in patients with DN. Forty-five patients with DN and 40 healthy controls were enrolled. Serum prolidase activity, TAS, MDA, and NO levels were determined. Serum MDA and NO levels were significantly higher in DN patients than controls (p = 0.002, p = 0.001, respectively), while prolidase activity and TAS levels were lower (p = 0.003, p = 0.001, respectively). Prolidase activity was negatively correlated with NO and MDA (r = -0.911, p < 0.001; r = -0.905, p < 0.001, respectively), while positively correlated with TAS (r = 0.981, p < 0.001) in DN patients. The current study is the first showing the decreased serum prolidase enzyme activity. Our results suggest that decreased collagen turnover may occur in DN patients, who have increased oxidative stress and increased NO levels. Decreased prolidase activity seems to be associated with increased NO levels and oxidative stress along with decreased antioxidant levels in DN. Therefore, decreased prolidase activity may play a role in pathogenesis of DN. Prospective clinical studies are necessary to confirm these findings.

DPP8 and DPP9 are recently identified members of the dipeptidyl peptidase IV (DPPIV) enzyme family, which is characterized by the rare ability to cleave a post-proline bond two residues from the N-terminus of a substrate. DPP8 and DPP9 have unique cellular localization patterns, are ubiquitously expressed in tissues and cell lines, and evidence suggests important contributions to various biological processes including: cell behavior, cancer biology, disease pathogenesis, and immune responses. Importantly, functional differences between these two proteins have emerged, such as DPP8 may be more associated with gut inflammation whereas DPP9 is involved in antigen presentation and intracellular signaling. Similarly, the DPP9 connections with H-Ras and SUMO1, and its role in AKT1 pathway downregulation provide essential insights into the molecular mechanisms of DPP9 action. The recent discovery of novel natural substrates of DPP8 and DPP9 highlights the potential role of these proteases in energy metabolism and homeostasis. This review focuses on the recent progress made with these post-proline dipeptidyl peptidases and underscores their emerging importance. Mol Cancer Res; 11(12); 1487–96. ©2013 AACR.