Kazuhisa Miyake, Ayuko Sakane, Ikuko Sagawa, Yoko Tomida, Jiro Kasahara and Takuya Sasaki : Actin Cytoskeletal Reorganization Function of JRAB/MICAL-L2 Is Fine-tuned by Intramolecular Interaction between First LIM Zinc Finger and C-terminal Coiled-coil Domains, Scientific Reports, Vol.9, No.1, 12794, 2019.
(Summary)
JRAB/MICAL-L2 is an effector protein of Rab13, a member of the Rab family of small GTPase. JRAB/MICAL-L2 consists of a calponin homology domain, a LIM domain, and a coiled-coil domain. JRAB/MICAL-L2 engages in intramolecular interaction between the N-terminal LIM domain and the C-terminal coiled-coil domain, and changes its conformation from closed to open under the effect of Rab13. Open-form JRAB/MICAL-L2 induces the formation of peripheral ruffles via an interaction between its calponin homology domain and filamin. Here, we report that the LIM domain, independent of the C-terminus, is also necessary for the function of open-form JRAB/MICAL-L2. In mechanistic terms, two zinc finger domains within the LIM domain bind the first and second molecules of actin at the minus end, potentially inhibiting the depolymerization of actin filaments (F-actin). The first zinc finger domain also contributes to the intramolecular interaction of JRAB/MICAL-L2. Moreover, the residues of the first zinc finger domain that are responsible for the intramolecular interaction are also involved in the association with F-actin. Together, our findings show that the function of open-form JRAB/MICAL-L2 mediated by the LIM domain is fine-tuned by the intramolecular interaction between the first zinc finger domain and the C-terminal domain.
Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells. Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA. The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter. Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.
Jun-ichi Kido, Mika Bandou, Yuka Hiroshima, Hiroyuki Iwasaka, Keisuke Yamada, Naoto Ohgami, Toshiyuki Namubu, Masatoshi Kataoka, Takenori Yamamoto, Yasuo Shinohara, Ikuko Sagawa and Toshihiko Nagata : Analysis of proteins in human gingival crevicular fluid by mass spectrometry, Journal of Periodontal Research, Vol.47, No.4, 488-499, 2012.
(Summary)
Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.
Taiki Kohiki, Yusuke Kato, Masaya Denda, Yusuke Nishikawa, Kazuko YORITA, Ikuko Sagawa, Tsubasa Inokuma, Akira Shigenaga, Kiyoshi Fukui and Akira Otaka : Development and application of novel protein labeling reagent "SEAL", Peptide Science 2018, 104, 2019.
Proceeding of International Conference:
1.
Taiki Kohiki, Yusuke Kato, Masaya Denda, Yusuke Nishikawa, Kazuko YORITA, Ikuko Sagawa, Tsubasa Inokuma, Akira Shigenaga, Kiyoshi Fukui and Akira Otaka : Development and application of novel protein labeling reagent "SEAL", 10th International Peptide Symposium, Kyoto, Dec. 2018.
2.
Yusuke Kato, Nobuo Maita, Taiki Kohiki, Sumire Kurosawa, Yusuke Nishikawa, Ikuko Sagawa, Masaya Denda, Tsubasa Inokuma, Yuji Shishido, Kazuko YORITA, Akira Shigenaga, Akira Otaka and Kiyoshi Fukui : Combined approach of computation and enzymology to investigate novel D-amino acid oxidase inhibitors, The 13th International Symposium of the Institute Network for Biomedical Sciences joint with the 3rd Symposium of the Inter-University Research Network for Trans-Omics Medicine and the 28th Hot Spring Harbor Symposium, Fukuoka, Oct. 2018.
3.
Yusuke Kato, Taiki Kohiki, Yusuke Nishikawa, Ikuko Sagawa, Masaya Denda, Tsubasa Inokuma, Yuji Shishido, Akira Shigenaga, Akira Otaka and Kiyoshi Fukui : Studies on binding molecules to D-amino acid oxidase using computational approaches, The 12th International Symposium of the Institute Network, Tokyo, Nov. 2017.