Unravelling the role of dipeptidyl peptidases-8/9 (DPP-8/9) in inflammatory osteoporosis: a comprehensive study investigating chrysin as a potential anti-osteoporotic agent.

OBJECTIVES: This study aimed to investigate the role of dipeptidyl peptidase-8 and 9 (DPP-8/9) enzymes in inflammatory bone loss using a 4-vinylcyclohexene diepoxide (VCD)-induced model in Wistar rats. Additionally, we evaluated the therapeutic potential of inhibiting these enzymes with the flavonoid chrysin. METHODS: Inflammatory osteoporosis was induced by administering VCD that elevated interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) levels. DPP-8/9 enzyme expression and various bone markers were assayed using serum. Further analysis included bone microarchitecture, histology, and immunohistochemistry. Additionally, chrysin's potential to inhibit DPP-8/9 and mitigate VCD-induced inflammatory bone loss was also evaluated. KEY FINDINGS: VCD administration in rats caused ovotoxicity that increased IL-6 and TNF-α levels, resulting in significant bone loss. Serum analysis revealed elevated bone resorption markers and DPP-8/9 enzyme levels. Inhibiting DPP-8/9 with 1G244 reversed these effects, confirmed by histology, immunohistochemistry, and micro-CT scans. Moreover, chrysin significantly reduced DPP-8/9 levels compared with the untreated group, improved bone markers, and lower inflammatory cytokines, indicating reduced osteoclastogenesis. CONCLUSION: This study highlights the role of DPP-8/9 in inflammation-induced osteoporosis. Following inhibition of DPP-8/9, we observed improved bone markers with preservation of trabecular bone mineral density in rats. Additionally, chrysin demonstrated potential as an anti-DPP-8/9 agent, suggesting its viability for future therapeutic interventions in DPP-8/9-related inflammatory diseases.

Active site-directed probes targeting dipeptidyl peptidases 8 and 9.

Dipeptidyl peptidases (DPP) 8 and 9 are intracellular serine proteases that play key roles in various biological processes and recent findings highlight DPP8 and DPP9 as potential therapeutic targets for hematological and inflammasome-related diseases. Despite the substantial progress, the precise biological functions of these proteases remain elusive, and the lack of selective chemical tools hampers ongoing research. In this paper, we describe the synthesis and biochemical evaluation of the first active site-directed DPP8/9 probes which are derived from DPP8/9 inhibitors developed in-house. Specifically, we synthesized fluorescent inhibitors containing nitrobenzoxadiazole (NBD), dansyl (DNS) and cyanine-3 (Cy3) reporters to visualize intracellular DPP8/9. We demonstrate that the fluorescent inhibitors have high affinity and selectivity towards DPP8/9 over related S9 family members. The NBD-labeled DPP8/9 inhibitors were nominated as the best in class compounds to visualize DPP8/9 in human cells. Furthermore, a method has been developed for selective labeling and visualization of active DPP8/9 in vitro by fluorescence microscopy. A collection of potent and selective biotinylated DPP8/9-targeting probes was also prepared by replacing the fluorescent reporter with a biotin group. The present work provides the first DPP8/9-targeting fluorescent compounds as useful chemical tools for the study of DPP8 and DPP9's biological functions.

Dipeptidyl-peptidase 9 regulates the dynamics of tumorigenesis and metastasis in breast cancer.

The cytosolic dipeptidyl-aminopeptidase 9 (DPP9) cleaves protein N-termini post-proline or -alanine. Our analysis of DPP9 mRNA expression from the TCGA 'breast cancer' data set revealed that low/intermediate DPP9 levels are associated with poor overall survival of breast cancer patients. To unravel the impact of DPP9 on breast cancer development and progression, the transgenic MMTV-PyMT mouse model of metastasizing breast cancer was used. In addition, tissue- and time-controlled genetic deletion of DPP9 by the Cre-loxP recombination system was done. Despite a delay of tumor onset, a higher number of lung metastases were measured in DPP9-deficient mice compared to controls. In human mammary epithelial cells with oncogenic RAS pathway activation, DPP9 deficiency delayed tumorigenic transformation and accelerated TGF-ß1 induced epithelial-to-mesenchymal transition (EMT) of spheroids. For further analysis of the mechanism, primary breast tumor cells were isolated from the MMTV-PyMT model. DPP9 deficiency in these cells caused cancer cell migration and invasion accompanied by EMT. In absence of DPP9, the EMT transcription factor ZEB1 was stabilized due to insufficient degradation by the proteasome. In summary, low expression of DPP9 appears to decelerate mammary tumorigenesis but favors EMT and metastasis, which establishes DPP9 as a novel dynamic regulator of breast cancer initiation and progression.

Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import.

Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.

The effect of CD26-deficiency on dipeptidyl peptidase 8 and 9 expression profiles in a mouse model of Crohn's disease.

The involvement of dipeptidyl peptidase (DP) IV/CD26 (DPP IV/CD26) family members in the pathogenesis of Crohn's disease (CD), an autoimmune inflammatory condition of the gut, is effected mainly through proteolytic cleavage of immunomodulatory substrates and DPP IV/CD26's costimulatory function. DP8 and DP9 are proteases with diverse functions including cell interactions, apoptosis, and immune response but their localization remains to be clarified. We assessed the impact of DPP IV/CD26 deficiency (CD26-/- ) on the expression profiles of DP8 and DP9 by qPCR and immunodetection as well as quantified DP8/9 enzyme activity in distinctive phases of a chemically-induced CD model in mice. CD26-/- did not affect colon DP8 mRNA expression, while the physiological concentration of DP8 protein is decreased in CD26-/- mice but rises in inflammation (P < 0.05). On the other hand, DP9 mRNA level is significantly increased in CD26-/- mice in inflammation as well as healing with the DP9 concentration being almost twofold increased (P < 0.05) in all experimental points in CD26-/- mice compared to wild-type indicating the expected up-regulation in CD26-/- conditions. Surprisingly, dominantly intracellular DP8 and DP9 were found in abundance in serum. DP8/9 activity is decreased in the inflamed colon, whereas its contribution to the overall serum DPP IV/CD26-like activity is negligible, suggesting the importance of their extra-enzymatic functions. To summarize, CD induction generated gene, protein and enzymatic changes of DP8 and DP9 so their involvement in inflammation development and/or healing process is implicated, especially in CD26-/- , and the question of their subcellular localization should be revised.

Crystal structure of Porphyromonas gingivalis dipeptidyl peptidase 4 and structure-activity relationships based on inhibitor profiling.

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 Å resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.

The expression of proline-specific enzymes in the human lung.

The pathophysiology of lung diseases is very complex and proteolytic enzymes may play a role or could be used as biomarkers. In this review, the literature was searched to make an overview of what is known on the expression of the proline-specific peptidases dipeptidyl peptidase (DPP) 4, 8, 9, prolyl oligopeptidase (PREP) and fibroblast activation protein α (FAP) in the healthy and diseased lung. Search terms included asthma, chronic obstructive pulmonary disease (COPD), lung cancer, fibrosis, ischemia reperfusion injury and pneumonia. Knowledge on the loss or gain of protein expression and activity during disease might tie these enzymes to certain cell types, substrates or interaction partners that are involved in the pathophysiology of the disease, ultimately leading to the elucidation of their functional roles and a potential therapeutic target. Most data could be found on DPP4, while the other enzymes are less explored. Published data however often appear to be conflicting, the applied methods divers and the specificity of the assays used questionable. In conclusion, information on the expression of the proline-specific peptidases in the healthy and diseased lung is lacking, begging for further well-designed research.

Expression, subcellular localisation, and possible roles of dipeptidyl peptidase 9 (DPP9) in murine macrophages.

Dipeptidyl peptidase 9 (DPP9) is a peptidase of the DPPIV gene family, and its role in immune responses has been reported. In this study, we compared the messenger RNA expression profile of DPP9 to that of the related DPP8 and DPPIV in murine haematopoietic and lymphatic tissues. A similar order of expression levels was observed for all 3 peptidases: peritoneal macrophages < bone marrow < spleen ≤ lymph nodes. Also, we examined the subcellular localisation of DPP9 and its possible role(s) in J774 cell line of macrophage origin. DPP9 was dominantly expressed intracellularly. DPPIV-like enzymatic activity was mostly present in cytoplasm, but also in cell membranes and organelles/vesicles. Decreased expression of DPP9 was observed upon activation of J774 cells by combined treatment with interferon gamma and lipopolysaccharide. Changes induced by DPP9 gene silencing in J774 cells suggest possible role of DPP9 in regulation of proliferation and activation status. The colocalisation of DPP9 with endocytosed DQ-OVA demonstrated in endosomes of J774 cells might suggest the role of DPP9 in peptide processing within endosomal/vesicular compartment.

Dipeptidyl peptidase 9 (DPP9) in human skin cells.

BACKGROUND: Dipeptidyl peptidase 9 (DPP9) is a relatively new member of the DPPIV family of prolyl dipeptidases which is ubiquitously expressed. Its role in regulation of immune responses and proliferation of epithelial carcinoma cells was reported. There is no data on possible role of DPP9 expressed in skin epithelial cells (keratinocytes) and in dermal fibroblasts. MATERIALS AND METHODS: Transcriptional and protein expression of DPP9 and DPPIV was examined in fibroblasts and keratinocytes isolated from normal human skin. Localization of DPP9 and its sub-localization in Golgi were determined by immunocytochemistry staining. DPPIV-like enzyme activity was determined in cell lysates and in isolated cell fractions containing membranes (M), cytosol (C) and content of organelles/endosomes/vesicles (V). Relative contribution of DPPIV and DPP8/9 enzyme activity in these fractions was determined by using selective inhibitors: sitagliptin (selective for DPPIV) and 1G244 (selective for DPP9 and a highly homologous DPP8). Possible roles of DPP8/9 via its enzyme activity were analysed by assessment of survival and proliferative capacity of fibroblasts and HaCaT cells of keratinocyte origin in the presence of the inhibitors. Possible role of DPP9 in cell migration and/or adhesion was analysed in fibroblasts and HaCaT cells after DPP9 gene silencing. RESULTS: Fibroblasts and keratinocytes exerted comparable level of DPP9 both at transcriptional and protein level. Fibroblasts strongly expressed DPPIV, whereas in keratinocytes DPPIV expression was low. DPP9 expression was found in cytosol and in perinuclear area of some fibroblasts, or in scattered pattern of keratinocytes, as well as in nuclei of some cells. Only low level of DPP9 sub-localization within Golgi was observed in fibroblasts and keratinocytes. DPPIV-like enzyme activity was about 5 times higher in lysates of fibroblasts than of HaCaT cells. In fibroblasts DPPIV-like enzyme activity was mainly (65%) found in the fraction containing cell membranes (M) and was predominantly (86.9%) due to DPPIV. In contrast, in HaCaT cells the DPPIV-like enzyme activity was mainly (84.2%) found in cytosol (C) and was predominantly (95.6%) due to DPP8/9. Survival and the proliferative capacity were significantly diminished in the presence of 10µM 1G244, both in fibroblasts and in HaCaT cells, suggesting possible role of DPP8/9 enzyme activity in regulation of survival and proliferation of these cells. DPP9 gene silencing resulted in decreased adhesion of fibroblasts, as well as in decreased migration of fibroblasts and HaCaT cells. Accumulation of DPP9 on the edges of plasma membranes of fibroblasts and keratinocytes adhering to surface supports the idea of possible role of DPP9 in cell adhesion. CONCLUSIONS: This is the first study showing protein expression, sub-localization and possible biological roles of DPP9 expressed in isolated human skin cells. The data may be relevant for development of new drugs against skin diseases by targeting DPP9 expressed in the skin cells.