A Glycoengineered Interferon-ß Mutein (R27T) Generates Prolonged Signaling by an Altered Receptor-Binding Kinetics.

The glycoengineering approach is used to improve biophysical properties of protein-based drugs, but its direct impact on binding affinity and kinetic properties for the glycoengineered protein and its binding partner interaction is unclear. Type I interferon (IFN) receptors, composed of IFNAR1 and IFNAR2, have different binding strengths, and sequentially bind to IFN in the dominant direction, leading to activation of signals and induces a variety of biological effects. Here, we evaluated receptor-binding kinetics for each state of binary and ternary complex formation between recombinant human IFN-ß-1a and the glycoengineered IFN-ß mutein (R27T) using the heterodimeric Fc-fusion technology, and compared biological responses between them. Our results have provided evidence that the additional glycan of R27T, located at the binding interface of IFNAR2, destabilizes the interaction with IFNAR2 via steric hindrance, and simultaneously enhances the interaction with IFNAR1 by restricting the conformational freedom of R27T. Consequentially, altered receptor-binding kinetics of R27T in the ternary complex formation led to a substantial increase in strength and duration of biological responses such as prolonged signal activation and gene expression, contributing to enhanced anti-proliferative activity. In conclusion, our findings reveal N-glycan at residue 25 of R27T is a crucial regulator of receptor-binding kinetics that changes biological activities such as long-lasting activation. Thus, we believe that R27T may be clinically beneficial for patients with multiple sclerosis.

Generation of med28 specific monoclonal antibodies.

Med28 plays a role in transcription, signal transduction, and cell proliferation. The overexpression of med28 is associated with tumor progression in in vitro and in vivo models. Recently it has been reported that the elevated expression of med28 is associated with poor outcome in women with breast cancer. The expression level of med28 in in vitro and in vivo was examined by using anti-rabbit polyclonal antibody in previous reports. In this study, we report for the first time the generation and characterization of four monoclonal antibodies against med28 through immunoblotting, immunofluorescence microscopy, immunoprecipitation, and immunohistochemical analyses. These antibodies will be useful in detecting med28 in in vitro and in vivo.

SMAD4 suppresses AURKA-induced metastatic phenotypes via degradation of AURKA in a TGFß-independent manner.

UNLABELLED: SMAD4 has been suggested to inhibit the activity of the WNT/ß-catenin signaling pathway in cancer. However, the mechanism by which SMAD4 antagonizes WNT/ß-catenin signaling in cancer remains largely unknown. Aurora A kinase (AURKA), which is frequently overexpressed in cancer, increases the transcriptional activity of ß-catenin/T-cell factor (TCF) complex by stabilizing ß-catenin through the inhibition of GSK-3ß. Here, SMAD4 modulated AURKA in a TGFß-independent manner. Overexpression of SMAD4 significantly suppressed AURKA function, including colony formation, migration, and invasion of cell lines. In addition, SMAD4 bound to AURKA induced degradation of AURKA by the proteasome. A luciferase activity assay revealed that the transcriptional activity of the ß-catenin/TCF complex was elevated by AURKA, but decreased by SMAD4 overexpression. Moreover, target gene analysis showed that SMAD4 abrogated the AURKA-mediated increase of ß-catenin target genes. However, this inhibitory effect of SMAD4 was abolished by overexpression of AURKA or silencing of AURKA in SMAD4-overexpressed cells. Meanwhile, the SMAD4-mediated repression of AURKA and ß-catenin was independent of TGFß signaling because blockage of TGFßR1 or restoration of TGFß signaling did not prevent suppression of AURKA and ß-catenin signaling by SMAD4. These results indicate that the tumor-suppressive function of SMAD4 is mediated by downregulation of ß-catenin transcriptional activity via AURKA degradation in a TGFß-independent manner. IMPLICATIONS: SMAD4 interacts with AURKA and antagonizes its tumor-promoting potential, thus demonstrating a novel mechanism of tumor suppression.

Low SP1 expression differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.

Specificity protein 1 (SP1) is an essential transcription factor that regulates multiple cancer-related genes. Because aberrant expression of SP1 is related to cancer development and progression, we focused on SP1 expression in gastric carcinoma and its correlation with disease outcomes. Although patient survival decreased as SP1 expression increased (P<0.05) in diffuse-type gastric cancer, the lack of SP1 expression in intestinal-type gastric cancer was significantly correlated with poor survival (P<0.05). The knockdown of SP1 in a high SP1-expressing intestinal-type gastric cell line, MKN28, increased migration and invasion but decreased proliferation. Microarray data in SP1 siRNA-transfected MKN28 revealed that the genes inhibiting migration were downregulated, whereas the genes negatively facilitating proliferation were increased. However, both migration and invasion were decreased by forced SP1 expression in a low SP1-expressing intestinal-type gastric cell line, AGS. Unlike the intestinal-type, in a high SP1-expressing diffuse-type gastric cell line, SNU484, migration and invasion were decreased by SP1 siRNA. In contrast to previous studies that did not identify differences between the 2 histological types, our results reveal that low expression of SP1 is involved in cancer progression and metastasis and differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.