Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

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.

N2-Acetylornithine deacetylase functions as a Cys-Gly dipeptidase in the cytosolic glutathione degradation pathway in Arabidopsis thaliana.

Glutathione (GSH) is required for various physiological processes in plants, including redox regulation and detoxification of harmful compounds. GSH also functions as a repository for assimilated sulfur and is actively catabolized in plants. In Arabidopsis, GSH is mainly degraded initially by cytosolic enzymes, γ-glutamyl cyclotransferase, and γ-glutamyl peptidase, which release cysteinylglycine (Cys-Gly). However, the subsequent enzyme responsible for catabolizing this dipeptide has not been identified to date. In the present study, we identified At4g17830 as a Cys-Gly dipeptidase, namely cysteinylglycine peptidase 1 (CGP1). CGP1 complemented the phenotype of the yeast mutant that cannot degrade Cys-Gly. The Arabidopsis cgp1 mutant had lower Cys-Gly degradation activity than the wild type and showed perturbed concentrations of thiol compounds. Recombinant CGP1 showed reasonable Cys-Gly degradation activity in vitro. Metabolomic analysis revealed that cgp1 exhibited signs of severe sulfur deficiency, such as elevated accumulation of O-acetylserine (OAS) and the decrease in sulfur-containing metabolites. Morphological changes observed in cgp1, including longer primary roots of germinating seeds, were also likely associated with sulfur starvation. Notably, At4g17830 has previously been reported to encode an N2-acetylornithine deacetylase (NAOD) that functions in the ornithine biosynthesis. The cgp1 mutant did not show a decrease in ornithine content, whereas the analysis of CGP1 structure did not rule out the possibility that CGP1 has Cys-Gly dipeptidase and NAOD activities. Therefore, we propose that CGP1 is a Cys-Gly dipeptidase that functions in the cytosolic GSH degradation pathway and may play dual roles in GSH and ornithine metabolism.

PEGylation renders carnosine resistant to hydrolysis by serum carnosinase and increases renal carnosine levels.

Carnosine's protective effect in rodent models of glycoxidative stress have provided a rational for translation of these findings in therapeutic concepts in patient with diabetic kidney disease. In contrast to rodents however, carnosine is rapidly degraded by the carnosinase-1 enzyme. To overcome this hurdle, we sought to protect hydrolysis of carnosine by conjugation to Methoxypolyethylene glycol amine (mPEG-NH2). PEGylated carnosine (PEG-car) was used to study the hydrolysis of carnosine by human serum as well as to compare the pharmacokinetics of PEG-car and L-carnosine in mice after intravenous (IV) injection. While L-carnosine was rapidly hydrolyzed in human serum, PEG-car was highly resistant to hydrolysis. Addition of unconjugated PEG to carnosine or PEG-car did not influence hydrolysis of carnosine in serum. In mice PEG-car and L-carnosine exhibited similar pharmacokinetics in serum but differed in half-life time (t1/2) in kidney, with PEG-car showing a significantly higher t1/2 compared to L-carnosine. Hence, PEGylation of carnosine is an effective approach to prevent carnosine degradations and to achieve higher renal carnosine levels. However, further studies are warranted to test if the protective properties of carnosine are preserved after PEGylation.

Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Aminopeptidases trim Xaa-Pro proteins, initiating their degradation by the Pro/N-degron pathway.

N-degron pathways are proteolytic systems that recognize proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Our previous work identified Gid4 as a recognition component (N-recognin) of the Saccharomyces cerevisiae proteolytic system termed the proline (Pro)/N-degron pathway. Gid4 is a subunit of the oligomeric glucose-induced degradation (GID) ubiquitin ligase. Gid4 targets proteins through the binding to their Nt-Pro residue. Gid4 is also required for degradation of Nt-Xaa-Pro (Xaa is any amino acid residue) proteins such as Nt-[Ala-Pro]-Aro10 and Nt-[Ser-Pro]-Pck1, with Pro at position 2. Here, we show that specific aminopeptidases function as components of the Pro/N-degron pathway by removing Nt-Ala or Nt-Ser and yielding Nt-Pro, which can be recognized by Gid4-GID. Nt-Ala is removed by the previously uncharacterized aminopeptidase Fra1. The enzymatic activity of Fra1 is shown to be essential for the GID-dependent degradation of Nt-[Ala-Pro]-Aro10. Fra1 can also trim Nt-[Ala-Pro-Pro-Pro] (stopping immediately before the last Pro) and thereby can target for degradation a protein bearing this Nt sequence. Nt-Ser is removed largely by the mitochondrial/cytosolic/nuclear aminopeptidase Icp55. These advances are relevant to eukaryotes from fungi to animals and plants, as Fra1, Icp55, and the GID ubiquitin ligase are conserved in evolution. In addition to discovering the mechanism of targeting of Xaa-Pro proteins, these insights have also expanded the diversity of substrates of the Pro/N-degron pathway.

State of the Art in the Development of Human Serum Carnosinase Inhibitors.

Human serum carnosinase is an enzyme that operates the preferential hydrolysis of dipeptides with a C-terminus histidine. Only higher primates excrete such an enzyme in serum and cerebrospinal fluid. In humans, the serum hydrolytic rate has high interindividual variability owing to gene polymorphism, although age, gender, diet, and also diseases and surgical interventions can modify serum activity. Human genetic diseases with altered carnosinase activity have been identified and associated with neurological disorders and age-related cognitive decline. On the contrary, low peripheral carnosinase activity has been associated with kidney protection, especially in diabetic nephropathy. Therefore, serum carnosinase is a druggable target for the development of selective inhibitors. However, only one molecule (i.e., carnostatine) has been discovered with the purpose of developing serum carnosinase inhibitors. Bestatin is the only inhibitor reported other than carnostatine, although its activity is not selective towards serum carnosinase. Herein, we present a review of the most critical findings on human serum carnosinase, including enzyme expression, localization and substrate selectivity, along with factors affecting the hydrolytic activity, its implication in human diseases and the properties of known inhibitors of the enzyme.

Prolidase activity in women with polycystic ovarian syndrome undergoing assisted conception.

Background: Prolidase is a manganese (Mn)-dependent cytosolic exopeptidase that degrades imidodipeptides with C-terminal proline or hydroxyproline. Prolidase recycling from imidodipeptides plays a critical role in collagen resynthesis and extracellular matrix (ECM) remodelling. Following an increase in gonadotropins, ovarian and follicular collagen undergo substantial degradation. Abnormal ovarian ECM composition is associated with polycystic ovary syndrome (PCOS). This study aimed to examine prolidase activity in the serum and follicular fluid (FF) of women undergoing in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) treatment, comparing those with PCOS to those with normal ovarian function.Methods: This prospective study enrolled 50 participants, of whom 44 were included. PCOS diagnosis followed the Rotterdam consensus criteria, with 20 patients constituting the study group. The control group comprised 24 individuals with mild-to-moderate male infertility. Prolidase enzyme activity in serum and FF was measured using the Chinard reagent via spectrophotometric analysis and compared between the groups.Results: Serum and FF prolidase levels were significantly lower in patients with PCOS (p < 0.05). A direct correlation was observed between serum and FF prolidase levels (p < 0.05). Although blastocyst quality scoring (BQS) significantly decreased in PCOS patients, no statistical difference was observed in the clinical pregnancy rate between the groups (p < 0.05) (p > 0.05). A negative correlation existed between serum prolidase levels and total antral follicle (AF) count (p < 0.05). Conversely, both serum and FF prolidase levels positively correlated with BQS (r = 0.574)(p < 0.05) (r = 0.650)(p < 0.05).Conclusions: Patients with PCOS showed lower serum and FF prolidase levels, indicating abnormal degradation of ovarian and follicular collagen, potentially causing anovulation.

Polycystic ovary syndrome (PCOS), the most prevalent endocrinopathy among reproductive-aged women, affects approximately 3­15% of this demographic. Long-term disorders such as cardiovascular disease, type 2 diabetes mellitus, obesity, and infertility are commonly associated with PCOS, with approximately 70% of affected women experiencing infertility. Although the aetiology of PCOS remains unclear, complex multigenic disorders and environmental factors such as abnormal ovarian extracellular matrix composition, disruption of the inflammatory pathway, and lifestyle factors have been found to be related.This study addresses the aetiology of PCOS, focusing on the close association between abnormal ovarian extracellular matrix composition and the syndrome, as seen in previous reports. Prolidase is a manganese-dependent cytosolic exopeptidase that degrades imidodipeptides using the C-terminal proline or hydroxyproline. Proline recycling from imidodipeptides by prolidase plays a critical role in the resynthesis of collagen and remodelling of the extracellular matrix. Our aim was to evaluate prolidase activity in the serum and follicular fluid of women diagnosed with PCOS. Our findings revealed a direct correlation between serum and follicular fluid prolidase levels, both of which were diminished in women with PCOS. Furthermore, a negative correlation was observed between serum prolidase levels and total antral follicle count indicating a potential link between prolidase activity and ovarian follicle development. In contrast, both serum and follicular fluid prolidase levels were positively correlated with blastocyst quality. In conclusion, PCOS patients showed lower serum and follicular fluid prolidase levels, indicating abnormal degradation of ovarian and follicular collagen, and potentially causing anovulation. Future studies measuring manganese levels in larger numbers of participants are required.

Multiplex gene knockout raises Ala-Gln production by Escherichia coli expressing amino acid ester acyltransferase.

L-Alanyl-L-Glutamine (Ala-Gln) is a common parenteral nutritional supplement. In our previous study, the recombinant whole-cell catalyst Escherichia coli BL21(DE3) overexpressing α-amino acid ester acyltransferase (BPA) to produce Ala-Gln has high activity and has been applied to large-scale production experiments. However, the degradation of Ala-Gln is detected under prolonged incubation, and endogenous broad-spectrum dipeptidase may be the primary cause. In this study, a CRISPR-Cas9 method was used to target pepA, pepB, pepD, pepN, dpp, and dtp to knock out one or more target genes. The deletion combination was optimized, and a triple knockout strain BL21(DE3)-ΔpepADN was constructed. The degradation performance of the knockout chassis was measured, and the results showed that the degradation rate of Ala-Gln was alleviated by 48% compared with the control. On this basis, BpADNPA (BPA-ΔpepADN) was built, and the production of Ala-Gln was 129% of the BPA's accumulation, proving that the ΔpepADN knockout conducive to the accumulation of dipeptide. This study will push forward the industrialization process of Ala-Gln production by whole-cell catalyst Escherichia coli expressing α-amino acid ester acyltransferase. KEY POINTS: • Endogenous dipeptidase knockout alleviates the degradation of Ala-Gln by the chassis • The balanced gene knockout combination is pepA, pepD, and pepN • The accumulation of Ala-Gln with BpADNPA was 129% of the control.

Crystal structure of aspartyl dipeptidase from Xenopus laevis revealed ligand binding induced loop ordering and catalytic triad assembly.

Gene encoding aspartyl dipeptidase from Xenopus levies (PepExl) is upregulated by thyroid hormone and is proposed to play a significant role in resorption of tadpole tail during metamorphosis. However, the importance of peptidase activity for the resorption of the tail remain elusive. Here we report the crystal structures of first eukaryotic S51 peptidase, PepExl, in its ligand-free and Asp-bound states at 1.4 and 1.8 Å resolutions, respectively. The active site is located at dimeric interface and the catalytic triad is found to be dissembled in ligand-free and assembled in Asp-bound state. Structural comparison and molecular dynamic simulations of ligand-free and Asp-bound states shows that distinct loop (loop-A) plays an important role in active site shielding, substrate binding and enzyme activation. This study illuminates the Asp-X dipeptide binding in PepExl is associated with ordering of the loop-A and assembly of residues of catalytic triad in active conformation for enzymatic activity.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

Fruit ripening-associated leucylaminopeptidase with cysteinylglycine dipeptidase activity from durian suggests its involvement in glutathione recycling.

BACKGROUND: Durian (Durio zibethinus L.) is a highly popular fruit in Thailand and several other Southeast Asian countries. It is abundant in essential nutrients and sulphur-containing compounds such as glutathione (GSH) and γ-glutamylcysteine (γ-EC). Cysteinylglycine (Cys-Gly) is produced by GSH catabolism and occurs in durian fruit pulp. Cysteine (Cys) is a precursor of sulphur-containing volatiles generated during fruit ripening. The aforementioned substances contribute to the strong odour and flavour of the ripe fruit. However, the genes encoding plant Cys-Gly dipeptidases are unknown. The aim of this study was to measure leucylaminopeptidase (LAP) activity in durian fruit pulp. RESULTS: We identified DzLAP1 and DzLAP2, which the former was highly expressed in the fruit pulp. DzLAP1 was expressed at various ripening stages and in response to ethephon/1-MCP treatment. Hence, DzLAP1 is active at the early stages of fruit ripening. DzLAP1 is a metalloenzyme ~ 63 kDa in size. It is activated by Mg2+ or Mn2+ and, like other LAPs, its optimal alkaline pH is 9.5. Kinetic studies revealed that DzLAP1 has Km = 1.62 mM for its preferred substrate Cys-Gly. DzLAP1-GFP was localised to the cytosol and targeted the plastids. In planta Cys-Gly hydrolysis was confirmed for Nicotiana benthamiana leaves co-infiltrated with Cys-Gly and expressing DzLAP1. CONCLUSIONS: DzLAP1 has Cys-Gly dipeptidase activity in the γ-glutamyl cycle. The present study revealed that the LAPs account for the high sulphur-containing compound levels identified in fully ripened durian fruit pulp.

Dipeptidase PEPDA Is Required for the Conidiation Pattern Shift in Metarhizium acridum.

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involves a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum, which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in ΔpepdA mutant. The defect of MC in ΔpepdA can be rescued when nonpolar amino acids, α-alanine, ß-alanine, or proline, were added into sucrose yeast extract agar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum. IMPORTANCE Conidia, as the asexual propagules in many fungi, are the start and end of the fungal life cycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the ΔpepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.

Profibrotic mechanisms of DPP8 and DPP9 highly expressed in the proximal renal tubule epithelial cells.

BACKGROUND: DPP8 and DPP9 have been demonstrated to play important roles in multiple diseases. Evidence for increased gene expression of DPP8 and DPP9 in tubulointerstitium was found to be associated with the decline of kidney function in chronic kidney disease (CKD) patients, which was observed in the Nephroseq human database. To examine the role of DPP8 and DPP9 in the tubulointerstitial injury, we determined the efficacy of DPP8 and DPP9 on epithelial-to-mesenchymal transition (EMT) and tubulointerstitial fibrosis (TIF) as well as the underlying mechanisms. METHODS: We conducted the immunofluorescence of DPP8 and DPP9 in kidney biopsy specimens of CKD patients, established unilateral ureteral obstruction (UUO) animal model, treated with TC-E5007 (a specific inhibitor of both DPP8 and DPP9) or Saxagliptin (positive control) or saline, and HK-2 cells model. RESULTS: We observed the significantly increased expression of DPP8 and DPP9 in the renal proximal tubule epithelial cells of CKD patients compared to the healthy control subjects. DPP8/DPP9 inhibitor TC-E5007 could significantly attenuate the EMT and extracellular matrix (ECM) synthesis in UUO mice, all these effects were mediated via interfering with the TGF-ß1/Smad signaling. TC-E5007 treatment also presented reduced renal inflammation and improved renal function in the UUO mice compared to the placebo-treated UUO group. Furthermore, the siRNA for DPP8 and DPP9, and TC-E5007 treatment decreased EMT- and ECM-related proteins in TGF-ß1-treated HK-2 cells respectively, which could be reversed significantly by transduction with lentivirus-DPP8 and lentivirus-DPP9. CONCLUSION: These data obtained provide evidence that the DPP8 and DPP9 could be potential therapeutic targets against TIF.

Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer.

Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one ß-propeller and α/ß hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an α-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.

Erythrocytes Prevent Degradation of Carnosine by Human Serum Carnosinase.

The naturally occurring dipeptide carnosine (ß-alanyl-l-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography-mass spectrometry. In addition, we studied carnosine's effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.

DPEP1 expression promotes proliferation and survival of leukaemia cells and correlates with relapse in adults with common B cell acute lymphoblastic leukaemia.

Dehydropeptidase-1 (DPEP1) is a zinc-dependent metalloproteinase abnormally expressed in many cancers. However, its potential role in adults with B cell acute lymphoblastic leukaemia (ALL) is unknown. We found that in adults with common B cell ALL high DPEP1, transcript levels at diagnosis were independently associated with an increased cumulative incidence of relapse (CIR) and worse relapse-free survival (RFS) compared with subjects with low transcript levels. We show an increased proliferation and prosurvival role of DPEP1 in B cell ALL cells via regulation of phosphCREB and p53, which may be the biological basis of the clinical correlation we report. Our data implicate DPEP1 expression in the biology of common B cell ALL in adults. We report clinical correlates and provide a potential biological basis for these correlations. If confirmed, analysing DPEP1 transcript levels at diagnosis could help predict therapy outcomes. Moreover, regulation of DPEP1 expression could be a therapy target in B cell ALL.

Overexpression of Prolidase Induces Autophagic Death in MCF-7 Breast Cancer Cells.

BACKGROUND/AIMS: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. METHODS: The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. CONCLUSION: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death.

DPEP1 promotes the proliferation of colon cancer cells via the DPEP1/MYC feedback loop regulation.

DPEP1 is highly expressed in the colorectal carcinoma tissues and colon cancer cells. However, the function and underlying mechanism of DPEP1 in the colon cancer cells are still poorly understood. Here, we found that transcription factor MYC could occupy on the DPEP1 promoter and activate its activities, and DPEP1 was up-regulated by MYC proteins in mRNA and protein levels in a dose-dependent manner in colon cancer cells. The expression levels of DPEP1 were positively correlated with that of MYC in colorectal tumor tissues. Moreover, Laser confocal images and Co-immunoprecipitation (Co-IP) revealed that DPEP1 and MYC proteins could bind to each other in the colon cancer cells. In turn, DPEP1 could enhance the stability of MYC proteins by extending the half-life of MYC proteins in colon cancer cells. Thus, DPEP1 and MYC proteins might form a positive feedback loop to maintain their high expression levels in colon cancer cells. In function, the MTT, EdU, Clone Formation assays and xenograft tumors assays demonstrated that DPEP1 could boost the proliferation of colon cancer cells through the DPEP1/MYC positive feedback loop in vitro and in vivo. Theoretically, DPEP1 may serve as a colon cancer biomarker and a novel target of colorectal carcinogenesis therapy.

Characterization of substrate specificity and novel autoprocessing mechanism of dipeptidase A from Prevotella intermedia.

Prevotella intermedia, a Gram-negative anaerobic rod, is frequently observed in subgingival polymicrobial biofilms from adults with chronic periodontitis. Peptidases in periodontopathic bacteria are considered to function as etiological reagents. Prevotella intermedia OMA14 cells abundantly express an unidentified cysteine peptidase specific for Arg-4-methycoumaryl-7-amide (MCA). BAU17746 (locus tag, PIOMA14_I_1238) and BAU18827 (locus tag, PIOMA14_II_0322) emerged as candidates of this peptidase from the substrate specificity and sequence similarity with C69-family Streptococcus gordonii Arg-aminopeptidase. The recombinant form of the former solely exhibited hydrolyzing activity toward Arg-MCA, and BAU17746 possesses a 26.6% amino acid identity with the C69-family Lactobacillus helveticus dipeptidase A. It was found that BAU17746 as well as L. helveticus dipeptidase A was a P1-position Arg-specific dipeptidase A, although the L. helveticus entity, a representative of the C69 family, had been reported to be specific for Leu and Phe. The full-length form of BAU17746 was intramolecularly processed to a mature form carrying the N-terminus of Cys15. In conclusion, the marked Arg-MCA-hydrolyzing activity in Pre. intermedia was mediated by BAU17746 belonging to the C69-family dipeptidase A, in which the mature form carries an essential cysteine at the N-terminus.