Altered Expression of DAAM1 and PREP Induced by Cadmium Toxicity Is Counteracted by Melatonin in the Rat Testis.

Cadmium (Cd) is one of the most toxic pollutants for health due to its accumulation in several tissues, including testis. This report confirms that Cd increased oxidative stress and apoptosis of germ and somatic cells and provoked testicular injury, as documented by biomolecular and histological alterations, i.e., CAT and SOD activity, the protein level of steroidogenic enzymes (StAR and 3ß-HSD), and morphometric parameters. Additionally, it further documents the melatonin (MLT) coadministration produces affects in mitigating Cd-induced toxicity on adult rat testis, as demonstrated by the reduction of oxidative stress and apoptosis, with reversal of the observed histological changes; moreover, a role of MLT in partially restoring steroidogenic enzymes expression was evidenced. Importantly, the cytoarchitecture of testicular cells was perturbed by Cd exposure, as highlighted by impairment of the expression and localization of two cytoskeleton-associated proteins DAAM1 and PREP, which are involved in the germ cells' differentiation into spermatozoa, altering the normal spermatogenesis. Here, for the first time, we found that the co-treatment with MLT attenuated the Cd-induced toxicity on the testicular DAAM1 and PREP expression. The combined findings provide additional clues about a protective effect of MLT against Cd-induced testicular toxicity by acting on DAAM1 and PREP expression, encouraging further studies to prove its effectiveness in human health.

Prolyl endopeptidase disruption reduces hepatic inflammation and oxidative stress in methionine-choline-deficient diet-induced steatohepatitis.

AIMS: Prolyl endopeptidase (PREP) is a serine endopeptidase widely distributed in the body, and accumulated evidence suggests that PREP participates in inflammation and oxidative stress. Here, we explored the effect of PREP gene disruption on hepatic inflammation and oxidative stress status in a methionine-choline-deficient (MCD)-induced nonalcoholic steatohepatitis (NASH) model. MAIN METHODS: PREP gene disruption (PREPgt) mice and wild-type (WT) littermates were placed on a control or an MCD diet for 4 weeks, respectively. The liver histopathological analysis and the number of inflammatory cells were determined by hematoxylin-eosin (HE) and immunohistochemical staining. Inflammation-associated genes and cytokine levels in liver tissue were evaluated by quantitative PCR and ELISA. The levels of P53, Sesn2, Nrf2, HO-1, and oxidative stress indicators in mice and the palmitic acid (PA)-treated human hepatocellular carcinoma cells (HepG2) were examined by immunoblotting and commercially available kits, respectively. KEY FINDINGS: We found that PREP expression was upregulated in the MCD-induced NASH model. In addition, PREP disruption alleviated MCD-induced hepatic inflammation accompanied by diminished infiltration of inflammatory cells and secretion of inflammatory mediators. More importantly, the results of this study indicate that targeting PREP can improve oxidative stress status in the liver of MCD-diet mice and PA-exposed HepG2 cells. The effect is most likely mediated by the activation of P53 and its downstream signaling pathways (Sesn2/Nrf2/HO-1). SIGNIFICANCE: Our results showed that PREP disruption (or inhibition) could decrease oxidative stress and inflammation and improve liver function, indicating that targeting PREP might be a new potential therapeutic option for NAFLD/NASH.

Characterization and rational design for substrate specificity of a prolyl endopeptidase from Stenotrophomonas maltophilia.

A novel prolyl endopeptidase from Stenotrophomonas maltophilia, SmPEP, was discovered and characterized. The specific activity of the recombinant SmPEP expressed by Escherichia coli BL21 (DE3), was 68.3 U/mg at pH 8.0 and 37 °C. In order to improve the substrate specificity for long-chain peptide, rational design was applied based on the structure constructed by homology modeling. Inter-domain sites within the ß-propeller domain were chosen for the mutation to weaken the inter-domain interaction and form an open conformation for long-chain substrate entering into the active site. The substrate specificity on a designed long-chain substrate, PQPQLPYPQPQLP, of the mutants F263A and E184 G increased 8.77 and 5.75 times respectively versus wild-type. After the saturated mutation of the both sites, the reactive rate of mutant F263 V on 13-mer peptide was 10.2 times higher than that of the wild-type. Then the mutant F263 V was used in the hydrolysis of casein, and the ACE inhibitory activity of the hydrolysate was significantly improved compared with wild type enzyme, which verified the efficiency of the design strategy.

Cadmium-induced toxicity increases prolyl endopeptidase (PREP) expression in the rat testis.

During the differentiation of the male gamete, there is a massive remodeling in the shape and architecture of all the cells of the seminiferous epithelium. The cytoskeleton, as well as many associated proteins with it, plays a pivotal role in this process. The testis is particularly susceptible to environmental pollutant, which can lead to injury and impairment of normal spermatozoa production. Cadmium (Cd) is one of the major chemical environmental toxicants in economically developed countries. Food and cigarettes are the main sources of exposure to this element. Here, the protective role of zinc (Zn) to prevent the testicular toxicity in male adult rats after prenatal and during lactation exposure to Cd has been assessed. Altered testicular histology at the interstitial and germinal levels was found, whereas Zn supply completely corrected Cd toxicity. Moreover, the effects of these metals on the testicular expression and localization of the protease prolyl endopeptidase (PREP) were evaluated. Interestingly, the results showed an increase of PREP messenger RNA and protein. Data were corroborated by immunofluorescence. This study raises the possibility of using PREP as a new fertility marker.

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A.

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson's disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of death-associated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.