A mounting evidence exists for the despicable role of the aberrant immune response in the pathogenesis of rheumatoid arthritis (RA), where toll-like receptor 4 (TLR4) can activate synovial fibroblasts that lead to the chronic inflammation and joint destruction, thus making TLR4 a potent drug target in RA. We report that novel TLR4-antagonizing peptide, PIP2, inhibits the induction of inflammatory biomarkers in vitro as well as in vivo. Systemically, PIP2 inhibits the lipopolysaccharide (LPS)-elicited TNF-α, IL-6, and IL-12p40 in a mouse model. The rationally designed cyclic derivative, cPIP2, is capable of inhibiting LPS-induced proinflammatory cytokines at significantly lower concentration as compared to PIP2 (PIP2 IC50 = 20 μM, cPIP2 IC50 = 5 μM). Finally, cPIP2 was able to relieve the inflammatory symptoms and synovial tissue destruction in the RA rat model. Cumulatively, these data suggest that PIP2 and cPIP2 hold strong promise for the development of peptide-based immunotherapeutics that could be of great value in curbing TLR-related immune complications including RA.

This is the first report on the development of a covalently bone morphogenetic protein-2 (BMP2)-immobilized hydrogel that is suitable for osteogenic differentiation of human periodontal ligament stem cells (hPLSCs). O-propargyl-tyrosine (OpgY) was site-specifically incorporated into BMP2 to prepare BMP2-OpgY with an alkyne group. The engineered BMP2-OpgY exhibited osteogenic characteristics after in vitro osteogenic differentiation of hPLSCs, indicating the osteogenic ability of BMP2-OpgY. A methoxy polyethylene glycol-(polycaprolactone-(N3)) block copolymer (MC-N3) was prepared as an injectable in situ-forming hydrogel. BMP2 covalently immobilized on an MC hydrogel (MC-BMP2) was prepared quantitatively by a simple biorthogonal reaction between alkyne groups on BMP2-OpgY and azide groups on MC-N3 via a Cu(I)-catalyzed click reaction. The hPLSCs-loaded MC-BMP2 formed a hydrogel almost immediately upon injection into animals. In vivo osteogenic differentiation of hPLSCs in the MC-BMP2 formulation was confirmed by histological staining and gene expression analyses. Histological staining of hPLSC-loaded MC-BMP2 implants showed evidence of mineralized calcium deposits, whereas hPLSC-loaded MC-Cl or BMP2-OpgY mixed with MC-Cl, implants showed no mineral deposits. Additionally, MC-BMP2 induced higher levels of osteogenic gene expression in hPLSCs than in other groups. In conclusion, BMP2-OpgY covalently immobilized on MC-BMP2 induced osteogenic differentiation of hPLSCs as a noninvasive method for bone tissue engineering.

Background Immunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. Immunotoxins based on full-length antibodies are less studied, even though the fragment crystallizable (Fc) domain plays an important role in regulating the concentration of immunoglobulin G (IgG) in the serum and in antibody-mediated immune responses against pathogens. Results We devised a method to site-specifically conjugate IgG and another protein using a cysteine residue introduced into the IgG and a bio-orthogonally reactive unnatural amino acid incorporated into the other protein. The human epidermal growth factor receptor 2 (Her2)-targeting IgG, trastuzumab, was engineered to have an unpaired cysteine in the heavy chain, and an unnatural amino acid with the azido group was incorporated into an engineered Pseudomonas exotoxin A (PE24). The two protein molecules were conjugated site-specifically using a bifunctional linker having dibenzocyclooctyne and maleimide groups. Binding to Her2 and interaction with various Fc receptors of trastuzumab were not affected by the conjugation with PE24. The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines, which involved the inhibition of cellular protein synthesis due to the modification of elongation factor-2. Conclusions We constructed the site-specifically conjugated immunotoxin based on IgG and PE24, which induced target-specific cytotoxicity. To evaluate the molecule as a cancer therapeutic, animal studies are planned to assess tumor regression, half-life in blood, and in vivo immunogenicity. In addition, we expect that the site-specific conjugation method can be used to develop other antibody-protein conjugates for applications in therapeutics and diagnostics. Electronic supplementary material The online version of this article (10.1186/s13036-019-0188-x) contains supplementary material, which is available to authorized users.

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Here, we propose an experimental methodology based on femtosecond‐resolved fluorescence spectroscopy to measure the hydrogen (H)‐bond free energy of water at protein surfaces under isothermal conditions. A demonstration was conducted by installing a non‐canonical isostere of tryptophan (7‐azatryptophan) at the surface of a coiled‐coil protein to exploit the photoinduced proton transfer of its chromophoric moiety, 7‐azaindole. The H‐bond free energy of this biological water was evaluated by comparing the rates of proton transfer, sensitive to the hydration environment, at the protein surface and in bulk water, and it was found to be higher than that of bulk water by 0.4 kcal mol−1. The free‐energy difference is dominated by the entropic cost in the H‐bond network among water molecules at the hydrophilic and charged protein surface. Our study opens a door to accessing the energetics and dynamics of local biological water to give insight into its roles in protein structure and function.