Maquiberry Cystatins: Recombinant Expression, Characterization, and Use to Protect Tooth Dentin and Enamel.

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products.

Review: Unraveling the origin of the structural and functional diversity of plant cystatins.

The regulation of protease activity is a critical factor for the physiological balance during plant growth and development. Among the proteins involved in controlling protease activity are the cystatins, well-described inhibitors of cysteine proteases present in viruses, bacteria and most Eukaryotes. Plant cystatins, commonly called phytocystatins, display unique structural and functional diversity and are classified according to their molecular weight as type-I, -II, and -III. Their gene structure is highly conserved across Viridiplantae and provides insights into their evolutionary relationships. Many type-I phytocystatins with introns share sequence similarities with type-II phytocystatins. New data shows that they could have originated from recent losses of the carboxy-terminal extension present in type-II phytocystatins. Intronless type-I phytocystatins originated from a single event shared by flowering plants. Pieces of evidence show multiple events of gene duplications, intron losses, and gains throughout the expansion and diversity of the phytocystatin family. Gene duplication events in Gymnosperms and Eudicots resulted in inhibitors with amino acid substitutions that may modify their interaction with target proteases and other proteins. This review brings a phylogenomic analysis of plant cystatin evolution and contributes to a broader understanding of their origins. A complete functional genomic analysis among phytocystatins and their roles in plant development and responses to abiotic and biotic stresses remains a question to be fully solved.

Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S.

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

Leishmanicidal candidate LASSBio-1736, a cysteine protease inhibitor with favorable pharmacokinetics: low clearance and good distribution.

1. LASSBio-1736 ((E)-1-4(trifluoromethyl) benzylidene)-5-(2-4-dichlorozoyl) carbonylhydrazine) is proposed to be an oral cysteine protease leishmanicidal inhibitor. 2. This work aimed to investigate plasma pharmacokinetics, protein binding and tissue distribution of LASSBio-1736 in male Wistar rats. 3. LASSBio-1736 was administered to male Wistar rats at doses of 3.2 mg/kg intravenously and 12.6 mg/kg oral and intraperitoneal. The individual plasma-concentration profiles were determined by HPLC-UV and evaluated by non-compartmental and population pharmacokinetic analysis (Monolix 2016R1, Lixoft). Tissue distribution was evaluated after iv injection of 3.2 mg/kg drug by non-compartmental approach. 4. After intravenous administration, Vdss (1.79 L/kg), t ½ (23.1 h) and CLtot (56.1 mL/h/kg) were determined, and they were statistically similar (α =0.05) to oral and intraperitoneal pharmacokinetic parameters. The plasma profiles obtained after intravenous, oral and intraperitoneal administration of the compound were best fitted to a three-compartment and one-compartment open model with first-order absorption. 5. The intraperitoneal and oral bioavailability were around 40 and 15%, respectively. 6. Liver, spleen and skin tissues showed penetration of 340, 130 and 40%, respectively, with t ½ like plasma values. 7. LASSBio-1736 protein binding was 95 ± 2%. 8. The t ½, CLtot and tissue distribution of the compound agreed with the desired drug characteristics for leishmanicidal activity.

Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases.

Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.