The effects of free Cys residues on the structure, activity, and tetrameric stability of mammalian uricase.

Mammalian uricases contain four conserved cysteine (Cys) residues, but little is known about their structures and functions. In this study, we first confirmed that all four Cys residues are free and not involved in disulfide bond formation, using canine uricase as a model protein. Cys residues had a greater effect on stability than on activity based on single Cys-to-Ser (serine) substitutions. Circular dichroism (CD) and homology modeling indicated that C188S reduces ß-sheet contents and inter- and intra-subunit hydrophobic interaction, potentially impairing the core tetrameric ß-barrel structure of the tunneling-fold protein, and ultimately decreased the tetrameric stability. Additionally, the inactivation of C188S during the stability tests may be a complex process involving depolymerization followed by irregular aggregation. Double mutations or thiol blockage of Cys188 and Cys195 significantly disrupted the formation and stability of tetrameric uricase, which may be mediated by the free thiols in Cys residues. The present results demonstrated that the free Cys residues are essential for tetrameric formation and stability in mammalian uricase. This implies that free cysteine residues, although not involved in disulfide bonding, may play important structural roles in certain proteins, underscoring the significance of the hydrophobic characteristics of the free thiols in Cys residues. KEY POINTS: • Four Cys residues are not involved in disulfide bonding in mammalian uricase. • The hydrophobicity of free thiols is critical for tetrameric stability in uricase. • Free Cys residues can serve structural roles without involving in disulfide bonds.

Preparation of Conotoxin-Encapsulated Chitosan Nanoparticles and Evaluation of Their Skin Permeability.

µ-Conotoxin CnIIIC (conotoxin, CTX)-loaded chitosan nanoparticles (CTX-NPs) were prepared using the ionic cross-linking method. The CTX-NPs were spherical and well with a polydispersity index of 0.292 ± 0.039, drug loading efficiency of 25.9 ± 1.2%, and encapsulation efficiency of 95.6 ± 1.3%. In vitro release studies showed that the release behavior of CTX-NPs in a pH 5.0 acetate buffer followed zero-order kinetics. In vitro transdermal experiments using Franz diffusion cells mounted with mouse abdominal skin demonstrated that the cumulative intradermal deposition amount of CTX per unit area in 8 h (D8) and permeability coefficient (Pf) of CTX loaded on CTX-NPs were 2.30- and 7.71-times that of the CTX solution. In vivo transdermal experiments in mice showed that the amount of CTX deposited in the skin after 8 h of CTX saline administration was significantly lower than that of CTX deposited in the skin after administration of CTX-NPs. In vitro fluorescence labeling transdermal studies through Franz diffusion cells mounted with mouse abdominal skin indicated that CTX-NPs aggregated at hair follicles. Skin irritation tests in mice indicated that the irritation due to CTX-NPs was negligible. The cytotoxicity experiment showed that the viability of Balb/c 3T3 cells with CTX-NPs containing 230 µg/mL (0.08 µM) CTX was greater than 75%. CTX-NPs increase intradermal deposition of CTX by accumulating in hair follicles, which has positive implications for transdermal penetration of CTX.

A ratiometric and two-photon fluorescent probe for imaging hydrogen peroxide in living cells.

As an important reactive oxygen species (ROS), hydrogen peroxide plays a significant role in the life activity system, and its abnormal levels are closely related to many diseases. Developing effective fluorescent probes for detecting hydrogen peroxide is very urgent. Therefore, we constructed a probe Z that can detect hydrogen peroxide in ratio. It has naphthimide as the fluorophore and phenylboronic acid pinacol esters as the recognition group. It shows higher sensitivity, lower detection limit, higher selectivity, and broad pH applicability. Moreover, probe Z has low cytotoxicity that can be used to detect exogenous hydrogen peroxide in HeLa cells and might be a potential tool for studying hydrogen peroxide in physiological activities.