Ghrelin proteolysis increases in plasma of men, but not women, with obesity.

AIMS: Since plasma ghrelin can undergo des-acylation and proteolysis, the aim of this study was to investigate the extent to which an enhancement of these reactions is associated to the decrease of ghrelin in plasma after food intake or in individuals with obesity. MAIN METHODS: we performed an intervention cross-sectional study, in which levels of ghrelin, desacyl-ghrelin (DAG), glucose, insulin, ghrelin des-acylation and ghrelin proteolysis were assessed in plasma before and after a test meal in 40 people (n = 21 males) with normal weight (NW, n = 20) or overweight/obesity (OW/OB, n = 20). KEY FINDINGS: Preprandial ghrelin and DAG levels were lower, whereas preprandial ghrelin proteolysis was ∼4.6-fold higher in plasma of males with OW/OB. In males, ghrelin proteolysis positively correlated with glycemia. Ghrelin and DAG levels were also lower in females with OW/OB, but preprandial ghrelin proteolysis was not different between females with NW or OW/OB. Ghrelin and DAG levels decreased postprandially in males and females, independently of BMI, and ghrelin proteolysis increased postprandially ∼2 folds only in individuals with NW. Ghrelin des-acylation remained unaffected by BMI or feeding status in both sexes. SIGNIFICANCE: Current study shows that ghrelin proteolysis increases in males with obesity as well as after meal in lean individuals. Therefore, ghrelin proteolysis may be an important checkpoint and, consequently, a putative pharmacological target to control circulating ghrelin levels in humans.

Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review.

Protease inhibitors (PIs) are regulatory proteins found in numerous animal tissues and fluids, plants, and microorganisms that reduce and inhibit the exacerbated and uncontrolled activity of the target proteases. Specific PIs are also effective tools for inactivating proteases involved in human diseases like arthritis, pancreatitis, hepatitis, cancer, AIDS, thrombosis, emphysema, hypertension, and muscular dystrophy among others. Plant PIs-small peptides with a high content of cystine residues in disulfide bridges-possess a remarkable resistance to heat treatment and a high stability against shifts in pH, denaturing agents, ionic strength, and proteolysis. In recent years, novel biologic activities have been reported for plant PIs, including antimicrobial, anticoagulant, antioxidant action plus inhibition of tumor-cell growth; thus pointing to possible applications in medicine, agriculture, and biotechnology. In this review, we provide a comparative overview of plant-PIs classifying them in four groups according of their thermal and pH stability (high stability and hyperstable -to temperature and to pHs-, respectively), then emphasizing the relevance of the physicochemical characteristics of these proteins for potential biotechnological and industrial applications. Finally, we analyze the biologic activities of the stable protease inhibitors previously characterized that are the most relevant to potential applications in biomedicine, the food industry, and agriculture.

Plasma levels of ghrelin, des-acyl ghrelin and LEAP2 in children with obesity: correlation with age and insulin resistance.

OBJECTIVE: The octanoylated peptide hormone ghrelin regulates appetite and glycaemic control. Des-acyl ghrelin abolishes some effects of ghrelin, but does not bind to ghrelin receptor. LEAP2 is a novel ligand for ghrelin receptor that blocks the effects of ghrelin. Some evidences show that plasma levels of these peptides are altered in adults with obesity, but their levels in childhood obesity remain poorly studied. Therefore, the objective of this study was to assess fasting plasma levels of ghrelin, des-acyl ghrelin and LEAP2 in children with normoweight, overweight/obesity and their association with different anthropometric and metabolic variables. DESIGN: A total of 42 females and 40 males, ages 3-12 years old were enrolled as a cross-sectional cohort. RESULTS: Plasma levels of des-acyl ghrelin and LEAP2 (but not ghrelin) were lower and ghrelin/des-acyl ghrelin ratio was higher in children with overweight/obesity. Des-acyl ghrelin negatively correlated with age, BMI z-score, insulin and HOMA index, and the correlations were stronger in children with overweight/obesity. LEAP2 levels negatively correlated with BMI z-score. No gender differences were found. CONCLUSIONS: Our findings suggest that ghrelin tone is increased in childhood obesity, due to a decrease on plasma levels of des-acyl ghrelin and LEAP2, and that des-acyl ghrelin is associated to insulin resistance, particularly in children with overweight/obesity.

Ghrelin binding to serum albumin and its biological impact.

Ghrelin is an octanoylated peptide hormone that plays a key role in the regulation of the body weight and glucose homeostasis. In plasma, ghrelin circulates bound to larger proteins whose identities are partially established. Here, we used size exclusion chromatography, mass spectrometry and isothermal titration microcalorimetry to show that ghrelin interacts with serum albumin. Furthermore, we found that such interaction displays an estimated dissociation constant (KD) in the micromolar range and involves albumin fatty-acid binding sites as well as the octanoyl moiety of ghrelin. Notably, albumin-ghrelin interaction reduces the spontaneous deacylation of the hormone. Both in vitro experiments-assessing ghrelin ability to inhibit calcium channels-and in vivo studies-evaluating ghrelin orexigenic effects-indicate that the binding to albumin affects the bioactivity of the hormone. In conclusion, our results suggest that ghrelin binds to serum albumin and that this interaction impacts on the biological activity of the hormone.

Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes.

Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.

Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin.

Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ∼4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.

Properties and applications of phytepsins from thistle flowers.

Aqueous extracts of thistle flowers from the genus Cynara-Cardueae tribe Cass. (Cynareae Less.), Asteraceae Dumortier-are traditionally used in the Mediterranean region for production of artisanal cheeses. This is because of the presence of aspartic proteases (APs) with the ability to coagulate milk. Plant APs, collectively known as phytepsins (EC 3.4.23.40), are bilobed endopeptidases present in an ample variety of plant species with activity mainly at acidic pHs, and have two aspartic residues located on each side of a catalytic cleft that are responsible for catalysis. The cleavage of the scissile peptide-bond occurs primarily between residues with large hydrophobic side-chains. Even when aspartylendopeptidase activity in plants is normally present at relatively low levels overall, the flowers of several species of the Cardueae tribe possess APs with extremely high specific activities in certain tissues. For this reason, in the last two decades, APs present in thistle flowers have been the subject of intensive study. Present here is a compilation of work that summarizes the known chemical and biological properties of these proteases, as well as their biomedical and biotechnological applications.

Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae).

Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.

Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex.

Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-L: -leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (K (m) = 0.18 ± 0.03 mM, k (cat) = 1.078 ± 0.055 s(-1), k (cat)/K (m) = 5.99 ± 0.57 s(-1) mM(-l)). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman's degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was suggested by molecular modeling.