Carlos E. Salas

Plant cysteine proteinases: Evaluation of the pharmacological activity

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.

The proteolytic activities in latex from Carica candamarcensis.

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 Km with (Pyr-Phe-Leu-pNA), is similar in both enzymes, the kcat 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.

A Supramolecular Complex between Proteinases and β-Cyclodextrin that Preserves Enzymatic Activity

BackgroundCyclodextrins are suitable drug delivery systems because of their ability to subtly modify the physical, chemical, and biological properties of guest molecules through labile interactions by formation of inclusion and/or association complexes. Plant cysteine proteinases from Caricaceae and Bromeliaceae are the subject of therapeutic interest, because of their anti-inflammatory, antitumoral, immunogenic, and woundhealing properties.MethodsIn this study, we analyzed the association between β-cyclodextrin (βCD) and fraction P1G10 containing the bioactive proteinases from Carica candamarcensis, and described the physicochemical nature of the solid-state self-assembled complexes by Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and nuclear magnetic resonance (NMR), as well as in solution by circular dichroism (CD), isothermal titration calorimetry (ITC), and amidase activity.Results and discussionThe physicochemical analyses suggest the formation of a complex between P1G10 and βCD. Higher secondary interactions, namely hydrophobic interactions, hydrogen bonding and van der Waals forces were observed at higher P1G10: βCD mass ratios. These results provide evidence of the occurrence of strong solid-state supramolecular non-covalent interactions between P1G10 and βCD. Microcalorimetric analysis demonstrates that complexation results in a favorable enthalpic contribution, as has already been described during formation of similar βCD inclusion compounds. The amidase activity of the complex shows that the enzyme activity is not readily available at 24 hours after dissolution of the complex in aqueous buffer; the proteinase becomes biologically active by the second day and remains stable until day 16, when a gradual decrease occurs, with basal activity attained by day 29.ConclusionThe reported results underscore the potential for βCDs as candidates for complexing cysteine proteinases, resulting in supramolecular arrays with sustained proteolytic activity.

Wound-healing activity of a proteolytic fraction from Carica candamarcensis on experimentally induced burn.

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.

Plant Proteinases and Inhibitors: An Overview of Biological Function and Pharmacological Activity

Proteinases play a fundamental metabolic role during the life cycle in the plant kingdom. By interacting with endogenous or exogenous inhibitors, the proteolytic activity is modulated to meet metabolic requirements. By probing proteolytic enzymes with their inhibitors, it is possible to identify novel functions unrelated to their proteolytic activity. A group of plant proteolytic enzymes stands as a line of defence against environmental changes as their activation is triggered following various types of stress. On the other hand, plants also contain proteinase inhibitors as countermeasures for their protection against insects and pests. Both proteinases and inhibitors emerge as useful tools to combat human diseases. This review focuses on the biochemical characterization of plant proteinases, their inhibitors, the pharmacological potential of proteinases and inhibitors, and new putative emerging functions of proteolytically inhibited proteinases.

Skin-healing activity and toxicological evaluation of a proteinase fraction from Carica candamarcensis

Previous studies demonstrated that proteinases from latex of C. candamarcensis act as mitogens on fibroblast and epithelial cells and a subsequent report showed their protective, angiogenic and wound healing effects on gastric ulcers. In this study, we present evidence of skin healing activity by the group of proteinases known as P1G10. By using a hairless mouse model, we compared the healing effect following topical application of various concentrations of P1G10. The data confirm that healing actions take place between 0.1 and 1%, without adverse local irritation or systemic toxicological action after a prolonged period of use. The wound healing effect is unaltered when P1G10 is previously inhibited with iodoacetamide. The low permeation of the hydrosoluble formulation Polawax(®) supports the maintenance of the drug at the site of application. These results extend the healing properties of these groups of enzymes in situations of dermatological trauma and open the way to future clinical applications.

The thrombolytic action of a proteolytic fraction (P1G10) from Carica candamarcensis

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.

Molecular cloning of a mitogenic proteinase from Carica candamarcensis: Its potential use in wound healing

Cysteine proteinases from the Caricaceae belong to the C1 family of the CA clan and display papain-like structured, the archetype enzyme for this group of proteins. Carica candamarcensis, also named Vasconcellea cundinamarcensis, a member of Caricaceae family common to many areas in South America, contains cysteine proteinases with proteolytic activity five to eight-fold higher than those from latex of Carica papaya. The cysteine protease CMS2MS2 from C. candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In this study, the cDNA cloning, expression and evaluation of biological activity of a CMS2MS2-like protein from C. candamarcensis is reported. The 650 bp fragment was cloned in bacteria and the DNA sequence confirmed a cysteine-proteinase similar to CMS2MS2. The recombinant protein is 30 kDa, induces a mitogenic response, and enhances ERK1/2 phosphorylation, like the non-recombinant enzyme, but lacks either amidase or caseinolytic activity. The mitogenic activity of this protein and its lack of proteolytic activity underscore a potential for use in wound healing treatment.

Stimulation of fibroblast proliferation by the plant cysteine protease CMS2MS2 is independent of its proteolytic activity and requires ERK activation.

Abstract 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.

Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex

Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P2(1)), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 A, beta = 96.81 degrees , and diffract X-rays to 1.8 A resolution.