Potential of mesenchymal stem cell-derived conditioned medium/secretome as a therapeutic option for ocular diseases.

Research has shown that the therapeutic effect of mesenchymal stem cells (MSCs) is partially due to its secreted factors as opposed to the implantation of the cells into the treated tissue or tissue replacement. MSC secretome, especially in the form of conditioned medium (MSC-CM) is now being explored as an alternative to MSCs transplantation. Despite the observed benefits of MSC-CM, only a few clinical trials have evaluated it and other secretome components in the treatment of eye diseases. This review provides insight into the potential therapeutic use of MSC-CM in eye conditions, such as corneal diseases, dry eye, glaucoma, retinal diseases and uveitis. We discuss the current evidence, some limitations, and the progress that remains to be achieved before clinical translation becomes possible.

Biochemical and functional characterization of the N-terminal ubiquitin-like domain of human SHARPIN.

SHARPIN, an accessory subunit of the E3 ligase complex LUBAC, participates in the formation of LUBAC through the ubiquitin-like (UBL) domain located in the central region of SHARPIN and interacts with the ubiquitin-associated domain (UBA) of the catalytic subunit HOIP. However, the role of the N-terminal UBL domain of SHARPIN in stable LUBAC formation has not been clarified. In this study, the 1-127 domain, 128-309 domain, and UBL domain of SHARPIN expression vectors were constructed using the molecular biology method. Then the co-expression of SUMO fusion protein combined with SUMO protease (ULP enzyme) in Escherichia coli was successfully applied to improve the soluble expression of target protein. The results of circular dichroism proved that they all belong to the α+ß class of proteins. The results of size exclusion chromatography showed that 128-309 domain could combine with HOIP and HOIL-1L to participate in the stability of LUBAC. Both thermal-induced and urea-induced unfolding experiment results demonstrated that the existence of the N-terminal UBL domain could make the overall structure more stable than the alone UBL domain. Biosensor experiments indicated that the existence of the N-terminal UBL domain strengthened the binding ability of the UBL domain and the UBA domain. These results were conducive to further study the structure and function of SHARPIN.

Effects of removing a highly conserved disulfide bond in ubiquitin-associated domain of human HOIP on biochemical characteristics.

Disulfide bond formed between the cysteine pairs plays a key role in maintaining the integrity of the protein structure and function. The ubiquitin-associated (UBA) domain of human HOIP contains three cysteine residues, Cys504, Cys551, and Cys572. Disulfide bonds formed by Cys504 and Cys551 residues are highly conserved, but the effect of disulfide bonds on the biochemical characteristics of UBA has not been elucidated. In addition, due to the presence of isolated Cys572, inactive inclusion bodies may be formed during protein expression or trigger protein aggregation during protein purification. In this study, the co-expression of SUMO fusion protein combined with SUMO protease (ULP enzyme) in Escherichia coli was successfully applied to improve the soluble expression of UBA domain. Introduced three mutants (UBAC551A, UBAC572A and UBAC551,572A) determined the effects of disulfide bonds on the biochemical characteristics of UBA. Circular dichroism and analytical size exclusion chromatography results showed that the target proteins obtained by co-expression could be folded correctly and had biological activity. Both thermal-induced and urea-induced results demonstrated that the elimination of disulfide bonds would significantly reduce the stability of UBA. Fluorescence spectroscopy result showed that the elimination of disulfide bonds slightly increases the binding affinity of UBA to ligands. In summary, soluble, stable and active UBA domain and its mutants were prepared by co-expression system, which will further contribute to the structural and functional research of UBA.