Construction of a Collagen-like Protein Based on Elastin-like Polypeptide Fusion and Evaluation of Its Performance in Promoting Wound Healing.

In the healing of wounds, human-like collagen (hCol) is essential. However, collagen-based composite dressings have poor stability in vivo, which severely limits their current therapeutic potential. Based on the above, we have developed a recombinant fusion protein named hCol-ELP, which consists of hCol and an elastin-like peptide (ELP). Then, we examined the physicochemical and biological properties of hCol-ELP. The results indicated that the stability of the hCol-ELP fusion protein exhibited a more compact and homogeneous lamellar microstructure along with collagen properties, it was found to be significantly superior to the stability of free hCol. The compound hCol-ELP demonstrated a remarkable capacity to induce the proliferation and migration of mouse embryo fibroblast cells (NIH/3T3), as well as enhance collagen synthesis in human skin fibroblasts (HSF) when tested in vitro. In vivo, hCol-ELP demonstrated significant enhancements in healing rate and a reduction in the time required for scab removal, thereby exhibiting a scar-free healing effect. The findings provide a crucial theoretical foundation for the implementation of an hCol-ELP protein dressing in fields associated with the healing of traumatic injuries.

Discovery of novel reversible monoacylglycerol lipase inhibitors via docking-based virtual screening.

Monoacylglycerol lipase (MAGL) is the major enzyme that catalyzes the hydrolysis of monoacylglycerols (MAGs). MAGL is responsible for degrading 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain and specific tissues. The inhibition of MAGL could attenuate the inflammatory response. Here, we report a series of reversible non-covalent MAGL inhibitors via virtual screening combined with biochemical analysis. The hit, DC630-8 showed low-micromolar activity against MAGL in vitro, and exhibited significant anti-inflammatory effects.

Fusion protein of FGF21 and elastin-like peptide improves wound healing in diabetic mice via inflammation modulation, collagen synthesis, and vascular network formation.

Chronic-healing skin wounds are a common complication in diabetic individuals. To alleviate patient suffering, there is a pressing demand for more effective strategies to expedite the repair of diabetic wounds. Fibroblast growth factor 21(FGF21) has been proven to accelerate wound healing, but its stability and ability to assist in the healing of diabetic ulcers have not met expectations. Therefore, we have fused FGF21 with an elastin-like peptide (ELP) to create a recombinant fusion protein (abbreviated as "ELF") to increase the bioactivity and stability in vitro or in vivo. Our results demonstrated that ELF significantly improved the efficiency of FGF21 purification due to the inverse temperature responsive phase transition property of ELP. Meanwhile, the fusion strategy did not impair the structure of FGF21 or diminish its activity, as demonstrated by the highly similar secondary structure of ELF and FGF21, and their considerable inhibitory activity in the glucose consumption experiment of Huh-7 cells. An in vitro migration assay revealed that ELF promoted healing more effectively than either free FGF21 or ELP. Further in vivo study revealed the ability of ELF to improve skin wound healing quality, manifested by lower levels of inflammatory factors, more collagen formation and deposition, and the formation of robust vascular networks, though there was no significant difference in healing rate among the ELF, FGF21, and ELP groups. In conclusion, our study indicated that FGF21 and ELP fusion molecules could be developed as more efficient and cost-effective therapeutic strategies for diabetic wound healing.

17­AAG synergizes with Belinostat to exhibit a negative effect on the proliferation and invasion of MDA­MB­231 breast cancer cells.

Breast cancer is one of the most common malignancies that threaten the health of women. Although there are a few chemotherapies for the clinical treatment of breast cancer, these therapies are faced with the problems of drug­resistance and metastasis. Drug combination can help to reduce the adverse side effects of chemotherapies using single drugs, and also help to overcome common drug­resistance during clinical treatment of breast cancer. The present study reported the synergistic effect of the heat shock protein 90 inhibitor 17­AAG and the histone deacetylase 6 inhibitor Belinostat in triple­negative breast cancer (TNBC) MDA­MB­231 cells, by detection of proliferation, apoptosis and cell cycle arrest following treatment with this combination. Subsequently, RNA sequencing (RNA­seq) data was collected and analyzed to investigate the synergistic mechanism of this combination. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways revealed by RNA­seq data analysis, a wound­healing assay was used to investigate the effect of this combination on the migration of MDA­MB­231 cells. Compared with treatment with 17­AAG or Belinostat alone, both the viability inhibition and apoptosis rate of MDA­MB­231 cells were significantly enhanced in the combination group. The combination index values were <1 in three concentration groups. Revealed by the RNA­seq data analysis, the most significantly enriched KEGG pathways in the combination group were closely associated with cell migration. Based on these findings, the anti­migration effect of this combination was investigated. It was revealed that the migration of MDA­MB­231 cells was significantly suppressed in the combination group compared with in the groups treated with 17­AAG or Belinostat alone. In terms of specific genes, the mRNA expression levels of TEA domain family proteins were significantly decreased in the combination group, whereas the phosphorylation of YY1 associated protein 1 and modulator of VRAC current 1 was significantly enhanced in the combination group. These alterations may help to explain the anti­migration effect of this combination. Belinostat has already been approved as a treatment for T­cell lymphoma and 17­AAG is undergoing clinical trials. These findings could provide a beneficial reference for the clinical treatment of patients with TNBC.

Molecular dynamics simulation revealed the intrinsic conformational change of cellular inhibitor of apoptosis protein-1.

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis, and protein targets for the development of anti-cancer drugs. Cellular inhibitor of apoptosis protein-1 (cIAP1) is an important member of IAPs. Peptides or small-molecular antagonists can induce the dimerization, auto-ubiquitination, and proteasomal degradation of the cellular inhibitor of apoptosis protein-1 (cIAP1). While in the absence of antagonists, several mutations of the cIAP1 protein also lead to its dimerization and auto-ubiquitination. Even though the crystal structure of cIAP1 protein has been determined, the intrinsic mechanism of its dimerization remains unexplored. Accumulating evidence indicated that intrinsic conformational change existed during the binding of antagonists with cIAP1 protein, or introduction of mutations. To reveal this intrinsic conformational change, molecular dynamics simulations at microsecond scale were applied for the wild-type and mutant-type cIAP1 proteins. Compared to the crystal structure, significant conformational change was observed during the simulations, which could explain the importance of previously identified key mutations. To validate these findings revealed by our simulations, a new mutation D303A was constructed and the following native polyacrylamide gel electrophoresis (native-PAGE) assay observed a proportion of spontaneous dimerization, in comparison with the wild-type control. Taken together, these computational and experimental results revealed the intrinsic conformational change of cIAP1, which could not only explain previously identified key mutations, but also be exploited for further design and development of anti-tumor compounds that target the cIAP1 protein.Communicated by Ramaswamy H. Sarma.