Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the current coronavirus 2019 (COVID-19) pandemic, uses its spike (S1) protein for host cell attachment and entry. Apart from angiotensin-converting enzyme 2, neuropilin-1 (NRP1) has been recently found to serve as another host factor for SARS-CoV-2 infection; thus, blocking S1–NRP1 interaction can be a potential treatment for COVID-19. Herein, molecular recognition between SARS-CoV-2 S1 C-end rule (CendR) heptapeptide including small-molecule antagonists (EG00229 and EG01377) and the NRP1 was investigated using molecular dynamics simulations and binding free energy calculations based on MM/PBSA method. The binding affinity and the number of hot-spot residues of EG01377/NRP1 complex were higher than those of CendR and EG00229 systems, in line with the reported experimental data as well as with the lower water accessibility at the ligand-binding site. The (i) T316, P317, and D320 and (ii) S346, T349, and Y353 residues were confirmed to respectively form H-bonds with the positively charged guanidinium group and the negatively charged carboxyl moiety of all studied ligands. Moreover, Rosetta protein design was employed to improve CendR peptide binding affinity to NRP1. The newly designed peptides, especially R683G and A684M, exhibited higher binding efficiency than the native CendR heptapeptide as well as the small-molecule EG00229 by forming more H-bonds and hydrophobic interactions with NPR1, suggesting that these designed peptides could be promising NRP1 inhibitors to combat SARS-CoV-2 infection.
Inulosucrase is an enzyme that synthesizes inulin-type β-2,1-linked
fructooligosaccharides (IFOS) from sucrose. Previous studies have
shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here,
mutational analyses of four conserved calcium-binding site I (Ca-I)
residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for
these residues not only reduced the stability and activity of LrInu,
but also modulated the pattern of the IFOS produced. Circular dichroism
spectroscopy and molecular dynamics simulation indicated that these
mutations had limited impact on the overall conformation of the enzyme.
One of Ca-I residues most critical for controlling LrInu-mediated
polymerization of IFOS, Asp418, was also subjected to mutagenesis,
generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity
of these mutants demonstrated that the IFOS chain length could be
controlled by a single mutation at the Ca-I site.
Levan-typed fructooligosaccharide (LFOS), a β-2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.
Mesenchymal stem cells (MSCs) are self-renewal and capable of differentiating to various functional cell types, including osteocytes, adipocytes, myoblasts, and chondrocytes. They are, therefore, regarded as a potential source for stem cell therapy. Fisetin is a bioactive flavonoid known as an active antioxidant molecule that has been reported to inhibit cell growth in various cell types. Fisetin was shown to play a role in regulating osteogenic differentiation in animal-derived MSCs; however, its molecular mechanism is not well understood. We, therefore, studied the effect of fisetin on the biological properties of human MSCs derived from chorion tissue and its role in human osteogenesis using MSCs and osteoblast-like cells (SaOs-2) as a model. We found that fisetin inhibited proliferation, migration, and osteogenic differentiation of MSCs as well as human SaOs-2 cells. Fisetin could reduce Yes-associated protein (YAP) activity, which results in downregulation of osteogenic genes and upregulation of fibroblast genes. Further analysis using molecular docking and molecular dynamics simulations suggests that fisetin occupied the hydrophobic TEAD pocket preventing YAP from associating with TEA domain (TEAD). This finding supports the potential application of flavonoids like fisetin as a protein–protein interaction disruptor and also suggesting an implication of fisetin in regulating human osteogenesis.
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