👤 Mikako Shirouzu

Notch signaling is a fundamental signal that regulates morphogenesis and cell differentiation during the embryonic period, and it plays a crucial role in macrophage differentiation. Macrophage-mediated inflammation promotes atherosclerosis from the initial lesion formation to acute thrombotic complications in advanced plaques. However, their role in atherosclerosis remains unclear. We herein focused on the Notch ligand Delta-like ligand 1 (Dll1), and examined its role in the pathobiology of atherosclerosis. In Apoe Dll1 blockade suppressed both initial lesion development and plaque vulnerability compared with lesions in mice treated with non-immune IgG. Dll1 Ab decreased lipid accumulation in advanced lesions and increased the collagen content. In ex vivo cultured macrophages, the blockade of Dll1-Notch signaling by Dll1 blocking antibodies suppressed the mRNA expression of Tnf and the release of activated matrix metalloproteinase 9, which increased plaque vulnerability. In contrast, the stimulation of Dll1-Notch by recombinant Dll1 induced Il1b, Il6, and Tnf expression in macrophages, as well as NF-κB activation. An exploratory transcriptome analysis of atherosclerotic arteries suggested that Dll1-Notch signaling regulates the expression of genes associated with inflammation and mitosis. These results indicate that Dll1 promotes the pathobiology of atherosclerosis from the initial lesion development to plaque destabilization in advanced atherosclerotic lesions. Show less

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca Show less

The Dedicator Of CytoKinesis (DOCK) family of atypical guanine nucleotide exchange factors activates the Rho family GTPases Rac and/or Cdc42 through DOCK homology region 2 (DHR-2). Previous structural analyses of the DHR-2 domains of DOCK2 and DOCK9 have shown that they preferentially bind Rac1 and Cdc42, respectively; however, the molecular mechanism by which DHR-2 distinguishes between these GTPases is unclear. Here we report the crystal structure of the Cdc42-bound form of the DOCK7 DHR-2 domain showing dual specificity for Rac1 and Cdc42. The structure revealed increased substrate tolerance of DOCK7 at the interfaces with switch 1 and residue 56 of Cdc42. Furthermore, molecular dynamics simulations showed a closed-to-open conformational change in the DOCK7 DHR-2 domain between the Cdc42- and Rac1-bound states by lobe B displacement. Our results suggest that lobe B acts as a sensor for identifying different switch 1 conformations and explain how DOCK7 recognizes both Rac1 and Cdc42. Show less