The 7S dodecamer is recognized as an important structural cross-linking domain of collagen IV networks that provide mechanical stability to basement membranes, a specialized form of extracellular matrix essential for the development and maintenance of tissue architecture. Although the 7S dodecamer is stabilized by covalent cross-linking, the molecular mechanism by which such cross-links are formed has not been revealed. Here, we aimed to identify the enzyme(s) that cross-links the 7S dodecamer and characterize its expression in the kidney glomerulus. Pharmacological inhibition of candidate extracellular matrix enzymes revealed that lysyl oxidase activity is required for cross-linking of 7S polypeptides. Among all lysyl oxidase family members, lysyl oxidase-like-2 (LOXL2) was identified as the isoform cross-linking collagen IV in mouse embryonal PFHR-9 cells. Biochemical analyses revealed that LOXL2 readily promoted the formation of lysyl-derived cross-links in the 7S dodecamer but not in the NC1 domain. We also established that LOXL2 is the main lysyl oxidase family member present in the glomerular extracellular matrix. Altogether, we demonstrate that LOXL2 is a novel component of the molecular machinery that forms cross-linked collagen IV networks, which are essential for glomerular basement membrane stability and molecular ultrafiltration function.

Lysyl oxidase–like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV.

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.

Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V_max/K_m ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in V_max values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.

Biogenic-silver nanoparticles emerge as new nanosilver platforms that allow us to obtain silver nanoparticles via “green chemistry”. In our study, biogenic-silver nanoparticles were obtained from Iris tuberosa leaf extract. Nanoparticles were characterized by a UV-vis spectroscopy, dynamical light scattering technique. The transmission electron microscope revealed spheric and irregular nanoparticles with 5 to 50 nm in diameter. Antimicrobial properties were evaluated against typical microbial contaminants found in cosmetic products, showing high antimicrobial properties. Furthermore, natural moisturizing cream was formulated with biogenic-silver nanoparticles to evaluate the preservative efficiency through a challenge test, indicating its promising use as preservative in cosmetics.

The current status of controversy regarding the use of certain preservatives in cosmetic products makes it necessary to seek new ecological alternatives that are free of adverse effects on users. In our study, the natural terpene thymoquinone was encapsulated in chitosan nanoparticles. The nanoparticles were characterized by DLS and TEM, showing a particle size of 20 nm. The chemical structure, thermal properties, and release profile of thymoquinone were evaluated and showed a successful stabilization and sustained release of terpenes. The antimicrobial properties of the nanoparticles were evaluated against typical microbial contaminants found in cosmetic products, showing high antimicrobial properties. Furthermore, natural moisturizing cream inoculated with the aforementioned microorganisms was formulated with thymoquinone-chitosan nanoparticles to evaluate the preservative efficiency, indicating its promising use as a preservative in cosmetics.