Structural basis for receptor-regulated SMAD recognition by MAN1.

Receptor-regulated SMAD (R-SMAD: SMAD1, SMAD2, SMAD3, SMAD5 and SMAD8) proteins are key transcription factors of the transforming growth factor-ß (TGF-ß) superfamily of cytokines. MAN1, an integral protein of the inner nuclear membrane, is a SMAD cofactor that terminates TGF-ß superfamily signals. Heterozygous loss-of-function mutations in MAN1 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. MAN1 interacts with MAD homology 2 (MH2) domains of R-SMAD proteins using its C-terminal U2AF homology motif (UHM) domain and UHM ligand motif (ULM) and facilitates R-SMAD dephosphorylation. Here, we report the structural basis for R-SMAD recognition by MAN1. The SMAD2-MAN1 and SMAD1-MAN1 complex structures show that an intramolecular UHM-ULM interaction of MAN1 forms a hydrophobic surface that interacts with a hydrophobic surface among the H2 helix, the strands ß8 and ß9, and the L3 loop of the MH2 domains of R-SMAD proteins. The complex structures also show the mechanism by which SMAD cofactors distinguish R-SMAD proteins that possess a highly conserved molecular surface.

Structural basis for transcriptional coactivator recognition by SMAD2 in TGF-ß signaling.

Transforming growth factor-ß (TGF-ß) proteins regulate multiple cellular functions, including cell proliferation, apoptosis, and extracellular matrix formation. The dysregulation of TGF-ß signaling causes diseases such as cancer and fibrosis, and therefore, understanding the biochemical basis of TGF-ß signal transduction is important for elucidating pathogenic mechanisms in these diseases. SMAD proteins are transcription factors that mediate TGF-ß signaling-dependent gene expression. The transcriptional coactivator CBP directly interacts with the MH2 domains of SMAD2 to activate SMAD complex-dependent gene expression. Here, we report the structural basis for CBP recognition by SMAD2. The crystal structures of the SMAD2 MH2 domain in complex with the SMAD2-binding region of CBP showed that CBP forms an amphiphilic helix on the hydrophobic surface of SMAD2. The expression of a mutated CBP peptide that showed increased SMAD2 binding repressed SMAD2-dependent gene expression in response to TGF-ß signaling in cultured cells. Disrupting the interaction between SMAD2 and CBP may therefore be a promising strategy for suppressing SMAD-dependent gene expression.

Active induction of in vivo microbubbles by acoustic radiation force at the bifurcation of blood vessel and its evaluation.

Alhough the development of drug delivery system using microbubbles and ultrasound is expected, because microbubbles diffuse in bloodstream, we have so far reported our attempts for active control of the microbubbles in flow by acoustic radiation force in order to increase local concentration of the microbubbles. However, there was no evidence that in vivo microbubbles act as similar as in vitro experiments, because there were limitations for reproduction of in vivo conditions. In this study, we have elucidated the relationship between brightness variation and microbubbles concentration in the suspension to estimate the absolute concentration in an invisible condition considering in vivo experiment. Then we conducted an experiment of active induction of microbubbles in a Y-form bifurcation of artificial blood vessel, where experimental conditions were with focused ultrasound, the central frequency of 5 MHz, flow velocity of 30 mm/s, and maximum sound pressure of 300 kPa-pp, respectively. Then we applied the conditions for active induction of in vivo microbubbles to compare with in vitro experiments. We used a bifurcation of blood vessel in an ear of a rabbit because the bifurcation shape in its blood vessel is visible. As the results of the experiment, the microbubbles concentration in the induced path was almost two times higher than that in the other path, which agrees with the results from in vitro experiments.