Hiroaki Konishi

d-Dopachrome tautomerase is a candidate for key proteins to protect the rat liver damaged by carbon tetrachloride

Carbon tetrachloride (CCl4) is known to induce liver damage. Animal experiments with CCl4 injections have revealed many findings, especially mechanisms of liver damage and liver regeneration. Recently, proteomic approaches have been introduced in various studies to evaluate the quantitative and qualitative changes in the comprehensive proteome level. The aim of this research is to elucidate the key protein for liver damage, liver protection and liver regeneration by using proteomic techniques. 50 % (v/v) CCl4 in corn oil was administered intraperitoneally to adult male rats at a dose of 4ml/kg body weight. Approximately 24h after the injection, the liver was removed and extracted proteins were analyzed with cleavable isotope coded affinity tag (cICAT) reagents, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). A twelvefold increase in D-dopachrome tautomerase (DDT) was indicated. This enzyme has been reported to be involved in the biosynthesis of melanin, an antioxidant. According to the histological analysis, melanin levels were increased in un-damaged hepatocytes of CCl4-treated rats. These results suggest that the increase in DDT is a response to liver damage, accelerates melanin biosynthesis and protects the liver from oxidative stress induced by CCl4.

Distinct functions of human MVB12A and MVB12B in the ESCRT-I dependent on their posttranslational modifications

ESCRT-I, which mediates the sorting of ubiquitinated cargo protein from the plasma membrane to the endosomal vesicle, comprises a heterotetramer of TSG101 (Vps23), Vps28, Vps37 and MVB12 protein. In humans, the structurally similar subtypes MVB12A and MVB12B are subunits of ESCRT-I. However, no functional description of these proteins has been described. Here we show the differing effects of tyrosine phosphorylation and ubiquitination of both MVB12 proteins on their respective functions. As noted in our previous study, Tyr204 phosphorylation of MVB12A in response to epidermal growth factor (EGF) stimulation affects binding to CD2AP, which regulates the amounts of EGF receptor bound to ESCRT-I. Strikingly, ubiquitination of Lys264 and Lys290 of MVB12B was induced and led to the instability and inclusion of MVB12B in COS-7 cells. These ubiquitinations increased upon EGF stimulation, which was regulated by the phosphorylations of Tyr241 and Tyr243 of MVB12B. Furthermore, MVB12A was also involved in the aggregation-prone proteins of MVB12B. These results suggest that the expression of MVB12B may be normally suppressed through the ubiquitin-proteasome pathway that simultaneously regulates the fate of MVB12A and the functions of ESCRT-I.

Potential risk factors of persistent low back pain developing from mild low back pain in urban Japanese workers.

Study Design Two-year, prospective cohort data from the Japan epidemiological research of occupation-related back pain study in urban settings were used for this analysis. Objective To examine the association between aggravated low back pain and psychosocial factors among Japanese workers with mild low back pain. Summary of Background Data Although psychosocial factors are strongly indicated as yellow flags of low back pain (LBP) leading to disability, the association between aggravated LBP and psychosocial factors has not been well assessed in Japanese workers. Methods At baseline, 5,310 participants responded to a self-administered questionnaire including questions about individual characteristics, ergonomic work demands, and work-related psychosocial factors (response rate: 86.5%), with 3,811 respondents completing the 1-year follow-up questionnaire. The target outcome was aggravation of mild LBP into persistent LBP during the follow-up period. Incidence was calculated for the participants with mild LBP during the past year at baseline. Logistic regression was used to explore risk factors associated with persistent LBP. Results Of 1,675 participants who had mild LBP during the preceding year, 43 (2.6%) developed persistent LBP during the follow-up year. Multivariate analyses adjusted for individual factors and an ergonomic factor found statistically significant or almost significant associations of the following psychosocial factors with persistent LBP: interpersonal stress at work [adjusted odds ratio (OR): 1.96 and 95% confidence interval (95%CI): 1.00–3.82], job satisfaction (OR: 2.34, 95%CI: 1.21–4.54), depression (OR: 1.92, 95%CI: 1.00–3.69), somatic symptoms (OR: 2.78, 95%CI: 1.44–5.40), support from supervisors (OR: 2.01, 95%CI: 1.05–3.85), previous sick-leave due to LBP (OR: 1.94, 95%CI: 0.98–3.86) and family history of LBP with disability (OR: 1.98, 95%CI: 1.04–3.78). Conclusions Psychosocial factors are important risk factors for persistent LBP in urban Japanese workers. It may be necessary to take psychosocial factors into account, along with physical work demands, to reduce LBP related disability.

A Brain-specific Grb2-associated Regulator of Extracellular Signal-regulated Kinase (Erk)/Mitogen-activated Protein Kinase (MAPK) (GAREM) Subtype, GAREM2, Contributes to Neurite Outgrowth of Neuroblastoma Cells by Regulating Erk Signaling

Background: We previously identified GAREM1 as a downstream adaptor of the EGF receptor. Results: GAREM2 is a brain-specific GAREM subtype that is also tyrosine-phosphorylated and binds Grb2. Conclusion: GAREM2 is a regulator of neurite outgrowth of neuroblastoma cells in the presence of IGF-1. Significance: This study demonstrates the biological function of the GAREM family proteins, including their expression and subcellular localization. Grb2-associated regulator of Erk/MAPK1 (GAREM) is an adaptor molecule in the EGF-mediated signaling pathway. GAREM is expressed ubiquitously in human organs and cultured cells. Two GAREM homologues are encoded by the human genome. Therefore, previously identified GAREM is named GAREM1. Here we characterized a new subtype of GAREM, GAREM2, that is specifically expressed in the mouse, rat, and human brain. Three GAREM2 tyrosines (Tyr-102, Tyr-429, and Tyr-551) are phosphorylated upon EGF stimulation and are necessary for binding to Grb2. Furthermore, GAREM2 and Shp2 regulate Erk activity in EGF-stimulated cells. These characteristics are similar to those of GAREM1. GAREM2 is expressed in some neuroblastoma cell lines and is also tyrosine-phosphorylated and bound to Grb2 after treatment with EGF. Eventually, GAREM2 regulates Erk activation in the presence of EGF or insulin like growth factor 1. GAREM2 also regulates insulin-like growth factor 1-induced neuronal differentiation of the SH-SY5Y neuroblastoma cell line. Although the structure and function of both GAREM subtypes are similar, GAREM1 is recruited into the nucleus and GAREM2 is not. Nuclear localization of GAREM1 might be controlled by a GAREM1-specific nuclear localization sequence and 14-3-3ϵ binding. The N-terminal 20 amino acids of GAREM1 make up its nuclear localization sequence that is also a 14-3-3ϵ binding site. The GAREM family is a new class of adaptor molecules with subtype-specific biological functions.

A Ca2+-dependent Mechanism of Neuronal Survival Mediated by the Microtubule-associated Protein p600

Background: The microtubule-associated protein p600 forms a complex with the Ca2+ sensor calmodulin. Results: Knockdown of p600 or specific disruption of the Ca2+-dependent calmodulin/p600 binding triggers neuronal death following Ca2+ elevation. Conclusion: p600 is required for survival of hippocampal neurons following glutamate-induced Ca2+ elevation. Significance: This novel role for p600 in Ca2+ signaling and neuronal death has broad relevance to the study of neurodegeneration. In acute and chronic neurodegeneration, Ca2+ mishandling and disruption of the cytoskeleton compromise neuronal integrity, yet abnormalities in the signaling roles of cytoskeletal proteins remain largely unexplored. We now report that the microtubule-associated protein p600 (also known as UBR4) promotes neuronal survival. Following depletion of p600, glutamate-induced Ca2+ influx through NMDA receptors, but not AMPA receptors, initiates a degenerative process characterized by endoplasmic reticulum fragmentation and endoplasmic reticulum Ca2+ release via inositol 1,4,5-trisphosphate receptors. Downstream of NMDA receptors, p600 associates with the calmodulin·calmodulin-dependent protein kinase IIα complex. A direct and atypical p600/calmodulin interaction is required for neuronal survival. Thus, p600 counteracts specific Ca2+-induced death pathways through regulation of Ca2+ homeostasis and signaling.

tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

The Detergent-Soluble Cytoplasmic Pool of Survivin Suppresses Anoikis and Its Expression Is Associated with Metastatic Disease of Human Colon Cancer

Survivin is a component of the chromosomal passenger complex (CPC) that is essential for accurate chromosome segregation. Interfering with the function of Survivin in mitosis leads to chromosome segregation errors and defective cytokinesis. Survivin contains a Baculovirus IAP Repeat (BIR) and therefore was originally classified as inhibitor of apopotosis protein (IAP), yet its role in apoptosis after cellular stress remains largely unknown. We demonstrate here, that Survivin predominantly suppresses anoikis, a form of programmed cell death induced by loss of cellular adhesion to extracellular matrix. Interestingly, cells ectopically overexpressing EGFP-Survivin showed after loss of cell-matrix-interaction a decreased expression of IκB-α. Subsequent subcellular protein fractionation and immunoprecipitation experiments revealed that XIAP interacts with detergent-soluble Survivin which is known to cooperatively activate NF-κB signaling. Examination of the expression levels of detergent soluble Survivin in colorectal cancer cell lines and in colorectal cancerous tissues revealed that detergent soluble cytoplasmic Survivin levels correlated inversely with anoikis susceptibility in colorectal cancer. Therefore, the detergent soluble cytoplasmic Survivin might be a promising predictive biomarker for lymph node and distant metastases of colorectal cancer. We conclude that an anti-apoptotic function of detergent-soluble Survivin in interphase cells experiencing anoikis is mediated at least via XIAP/IκB-α/NF-κB signaling.

Assessment of psychosocial risk factors for the development of non-specific chronic disabling low back pain in Japanese workers—findings from the Japan epidemiological research of Occupation-related Back pain (JOB) study

To investigate the associations between psychosocial factors and the development of chronic disabling low back pain (LBP) in Japanese workers. A 1u2005yr prospective cohort of the Japan Epidemiological Research of Occupation-related Back Pain (JOB) study was used. The participants were office workers, nurses, sales/marketing personnel, and manufacturing engineers. Self-administered questionnaires were distributed twice: at baseline and 1u2005yr after baseline. The outcome of interest was the development of chronic disabling LBP during the 1u2005yr follow-up period. Incidence was calculated for the participants who experienced disabling LBP during the month prior to baseline. Logistic regression was used to assess risk factors for chronic disabling LBP. Of 5,310 participants responding at baseline (response rate: 86.5%), 3,811 completed the questionnaire at follow-up. Among 171 eligible participants who experienced disabling back pain during the month prior to baseline, 29 (17.0%) developed chronic disabling LBP during the follow-up period. Multivariate logistic regression analysis implied reward to work (not feeling rewarded, OR: 3.62, 95%CI: 1.17–11.19), anxiety (anxious, OR: 2.89, 95%CI: 0.97–8.57), and daily-life satisfaction (not satisfied, ORs: 4.14, 95%CI: 1.18–14.58) were significant. Psychosocial factors are key to the development of chronic disabling LBP in Japanese workers. Psychosocial interventions may reduce the impact of LBP in the workplace.

Role of a tyrosine phosphorylation of SMG-9 in binding of SMG-9 to IQGAP and the NMD complex.

SMG-9 is a component of the NMD complex, a heterotetramer that also includes SMG-1 and SMG-8 in the complex. SMG-9 was also originally identified as a tyrosine-phosphorylated protein but the role of the phosphorylation is not yet known. In this study, we determined that IQGAP protein, an actin cytoskeleton modifier acts as a binding partner with SMG-9 and this binding is regulated by phosphorylation of SMG-9 at Tyr-41. SMG-9 is co-localized with IQGAP1 as a part of the process of actin enrichment in non-stimulated cells, but not in the EGF-stimulated cells. Furthermore, an increase in the ability of SMG-9 to bind to SMG-8 occurs in response to EGF stimulation. These results suggest that tyrosine phosphorylation of SMG-9 may play a role in the formation of the NMD complex in the cells stimulated by the growth factor.

Inhibitory effect of SPE-39 due to tyrosine phosphorylation and ubiquitination on the function of Vps33B in the EGF-stimulated cells

Vps33B and SPE‐39 colocalize by fluorescence microscopy (View interaction)