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Objectives The aim of this study was to extend our knowledge about the mechanism involved in the gastroprotective effect of P1G10, a proteolytic fraction rich in cysteine proteinases from Vasconcellea cundinamarcensis (syn. Carica candamarcensis) latex, which demonstrated gastric healing and protection activities in rats. Methods Wistar rats were submitted to gastric lesions by indomethacin and treated with P1G10 (10 mg/kg). Free thiol groups and prostaglandin E2 content were measured in gastric mucosal and gastrin levels in blood samples. To evaluate the participation of nitric oxide ( NO) or proteolytic activity of P1G10 on its gastroprotective effect, animals were treated with an inhibitor of NO production ( L-NAME) or the fraction inhibited by iodoacetamide, respectively. Gastric secretion study (acidity and pepsin activity) was also performed. Key findings P1G10 (10 mg/kg) inhibited the occurrence of gastric lesions by indomethacin, restored the free thiol groups content on gastric mucosa and increased moderately prostaglandin E2 levels (34%). Furthermore, the treatment decreased the gastrin levels (95%), suggesting a possible modulation of secretory activity. This effect was accordant with attenuation of gastric acidity (42%) and pepsin activity (69%) seen in animals subjected to pyloric ligation. The inhibition of NO production or the proteolytic activity of P1G10 does not affect the gastroprotective effect. Conclusions These results can explain the gastroprotective activity of P1G10 and serve a basis for further studies of this active principle. [ABSTRACT FROM AUTHOR]

CMS1MS2 (CC-Ib) from Carica candamarcensis ( Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Crystals of CMS1MS2 complexed with E-64 were obtained by the hanging drop vapor-diffusion method at 291 K using ammonium sulfate and polyethylene glycol 4000/8000 as precipitant. The complex CMS1MS2-E-64 crystallized in the tetragonal space group P422 with unit-cell parameters; a = b = 73.64, c = 118.79 Å. The structure was determined by Molecular Replacement and refined at 1.87 Å resolution to a final R factor of 16.2 % ( R = 19.3 %). Based on the model, the structure of CMS1MS2 (PDB 3IOQ) ranks as one of the least basic cysteine isoforms from C. candamarcensis, is structurally closer to papain, caricain, chymopapain and mexicain than to the other cysteine proteinases, while its activity is twice the activity of papain towards BAPNA substrate. Two differences, one in the S2 subsite and another in the S3 subsite of CMS1MS2 may contribute to the enhanced activity relative to papain. In addition, the model provides a structural basis for the sensitivity of CMS1MS2 to inhibition by cystatin, not shown by other enzymes of the group, e.g., glycyl endopeptidase and CMS2MS2. [ABSTRACT FROM AUTHOR]

The cysteine protease CMS2MS2 from Carica candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In experiments with CMS2MS2 irreversibly inhibited by E-64, the proliferative effect on fibroblasts remains unaffected. ERK phosphorylation mediated by CMS2MS2 was abolished in the presence of PD 98059 or U0126, both MAPK cascade inhibitors. In addition, these inhibitors suppress the mitogenic activity of intact CMS2MS2 or CMS2MS2-E-64. Furthermore, ERK phosphorylation and the mitogenic effect are partially suppressed by a phospholipase C (PLC) inhibitor. These data suggest that the mitogenic effect of CMS2MS2 on fibroblasts is independent of its proteolytic activity, requires ERK phosphorylation, and involves activation of PLC. [ABSTRACT FROM AUTHOR]

Abstract: Cysteine proteinases are involved in virtually every aspect of plant physiology and development. They play a role in development, senescence, programmed cell death, storage and mobilization of germinal proteins, and in response to various types of environmental stress. In this review, we focus on a group of plant defensive enzymes occurring in germinal tissue of Caricaceae. These enzymes elicit a protective response in the unripe fruit after physical stress. We propose that these enzymes follow a strategy similar to mammalian serine proteinases involved in blood clotting and wound healing. We show evidence for the pharmacological role of plant cysteine proteinases in mammalian wound healing, immunomodulation, digestive conditions, and neoplastic alterations. [Copyright &y& Elsevier]

In a recent study we showed that two proteinases (CMS2MS2 and CMS2MS3) from Carica candamarcensis enhance mammalian cell proliferation. The aim of the present study is the determination of the primary structure of CMS2MS2 and prediction of its three-dimensional structure. The protein contains 214 residues, including the catalytic triad composed of Cys25, His159, and Asn175. A phylogenetic tree analysis demonstrated that CMS2MS2 ranks closer to chymopapain than to papain. The overall predicted three-dimensional structure is similar to proteinases from the papain family. These results suggest that minor structural differences within CMS2MS2 must account for its proliferative action. [ABSTRACT FROM AUTHOR]

Previous studies showed that P1G10, a proteolytic fraction from Vasconcellea cundinamarcensis latex, reduced the tumor mass in animals bearing melanoma, increased in vitro DNA fragmentation and decreased cell adhesion. Here, we present some molecular and cellular events related to the antimetastatic effect induced by the CMS-2 fraction derived from P1G10 in metastatic melanoma B16-F10 and melanocyte Melan-a. Using difference gel electrophoresis and mass spectrometry, we identified four proteins overexpressed in tumor cells, all of them related to proliferation, survival, migration and cell invasion, that had their expression normalized upon treatment with CMS-2: nucleophosmin 1, heat shock protein 65, calcyclin binding protein and eukaryotic translation initiation factor 4H. In addition, some antioxidant and glycolytic enzymes show increased expression after exposure to CMS-2, along with an induction of melanogenesis (differentiation marker). The down regulation of cofilin 1, a protein involved in cell motility, may explain the inhibition of cell migration and dendritic-like outgrowth in B16-F10 and Melan-a, observed after CMS-2 treatment. Taken together, it is argued that CMS-2 modulates the expression of proteins related to metastatic development, driving the cell to a more differentiated-like state. These effects support the CMS-2 antimetastatic activity and place this fraction in the category of anticancer agent. [ABSTRACT FROM AUTHOR]

Abstract: The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg180 located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin. [Copyright &y& Elsevier]

Abstract: Carica candamarcensis is a species from the Caricaceae family whose immature fruit contains latex with large amounts of cysteine proteinases. In prior studies, we isolated two of these enzymes displaying mitogenic activity when incubated with L929 fibroblastic cells. One of the fractions containing these enzymes (P1G10) was shown to enhance wound healing of skin and to accelerate healing of chemically induced gastric ulcer. In this study we evaluate the effect of P1G10 on heat-induced, third-degree burn using a rodent model. The results show that 0.1% P1G10 accelerates epithelisation while the effect of 1% or 0.01% P1G10 is not significantly different to 1% silver sulphadiazine, 2% papain or the hydrosoluble vehicle used as control. In a double-blind randomised experiment comparing the healing response of 0.1%, 1% and the vehicle alone, we confirmed the enhanced healing property of P1G10. Histological analysis of burn-tissue sections following treatment with P1G10 support these observations. These results extend the healing properties of these groups of enzymes to a different type of trauma and open the way to future clinical applications. [Copyright &y& Elsevier]

Abstract: Prior evidence suggests that proteinases in latex from Caricaceae protect against injuries induced by physical wounding. While the proteolytic enzymes from Carica papaya are well characterized, the homologues from Carica candamarcensis were not given similar attention, probably because its distribution is restricted to South American regions. We describe the chromatographic steps to fractionate 14 components from C. candamarcensis, 12 of them displaying amidase activity. The mass of these proteins plus two others isolated by HPLC rank between 23,943 and 22,991Da, and their N-terminal sequences showed similarities or identities with the enzymes described earlier in this species. Following CM-Sephadex chromatography two major peaks containing proteolytic activity were resolved. Each of these peaks was further resolved by Mono S chromatography yielding several purified fractions. The kinetic parameters of two of the Mono S purified enzymes originated from each of the CMS-Sephadex peaks were determined. While the K m with (Pyr-Phe-Leu-pNA), is similar in both enzymes, the k cat for one of them is 10-fold lower than the other. Based on these differences it is proposed that two groups of proteinases exist in latex of C. candamarcensis. [Copyright &y& Elsevier]

Abstract: A group of cysteine-proteolytic enzymes from C. candamarcensis latex, designated as P1G10 displays pharmacological properties in animal models following various types of lesions. This enzyme fraction expresses in vitro fibrinolytic effect without need for plasminogen activation. Based on this evidence, we assessed by intravital microscopy the effect of P1G10 on recanalization of microvessels after thrombus induction in the ear of hairless mice. Video playback of intravital microscopic images allowed measurement of blood flow velocity (mm/s) during the experimental procedure. Groups treated with 5 or 7.5mg/Kg P1G10 showed thrombolysis between 7–15min, without vessel obstruction. Ex vivo experiments demonstrated that platelet activation by ADP is impaired in a dose dependent manner following treatment with P1G10. The P1G10 action on plasma coagulation also showed that prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (aPTT, μg/uL) are increased in a dose dependent manner. In addition, P1G10 displayed fibrinogenolytic and fibrinolytic activities, both in a dose dependent manner. Each of these effects was suppressed by inhibition of the proteolytic activity of the fraction. The antithrombotic action of P1G10 can be explained by proteolytic cleavage of fibrinogen and fibrin, both key factors during formation of a stable thrombus. These results combined with prior evidence suggest that P1G10 has potential as thrombolytic agent. [Copyright &y& Elsevier]