Takashi Uehara

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide.

Hydrogen sulfide (H2S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H2S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H2S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H2S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H2S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

Participation of metabotropic glutamate receptors in pentetrazol-induced kindled seizure

Purpose:u2002 The present study was undertaken to clarify the effects of (RS)‐1‐aminoindan‐1,5‐dicarboxylic acid (AIDA), a metabotropic glutamate receptor (mGluR) 1 antagonist, (2R,4R)‐4‐aminopyrrolidine‐2,4‐dicarboxylate ((2R,4R)‐APDC), a mGluR2/3 agonist, and L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4), a mGluR4/8 agonist, on pentetrazol‐induced kindled seizures.

Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2u2009μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

The ECS(SPSB) E3 ubiquitin ligase is the master regulator of the lifetime of inducible nitric-oxide synthase.

The ubiquitin-proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C-Cullin-5-SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70-interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime.

Screening systems for the identification of S-nitrosylated proteins.

S-nitrosylation is a well-characterized reaction involving the covalent binding of nitric oxide (NO) to cysteine residues (Cys) in a protein. Similar to protein phosphorylation, S-nitrosylation is a post-translational modification involved in the regulation of a large number of intracellular functions and signaling events. Moreover, like phosphorylation, S-nitrosylation is precisely regulated in time and space. A procedure known as the biotin-switch method that specifically detects S-nitrosylated proteins (SNO-P) was recently developed by Snyders group. They found that many proteins are substrates for NO, and several groups have attempted to identify other SNO-P by improving this method. In this review, we describe the SNO-P identified using modified versions of the biotin-switch method.

Correlation Between Attenuation of Protein Disulfide Isomerase Activity Through S-Mercuration and Neurotoxicity Induced by Methylmercury

AbstractMethylmercury (MeHg), an environmental pollutant, causes neuronal death via endoplasmic reticulum (ER) stress; however, the precise mechanism is not fully understood. The aim of this study was to elucidate the possible mechanism of MeHg-induced neurotoxicity. Treatment with MeHg resulted in a loss of cell viability in a concentration-dependent manner accompanying the expression of ER stress marker genes in human neuroblastoma SH-SY5Y cells. We next attempted to identify a target protein for MeHg in the ER. MeHg covalently modified protein disulfide isomerase (PDI), which is important for disulfide bond formation in nascent proteins in the ER lumen. S-Nitrosylation of the catalytic domains of PDI by nitric oxide was attenuated up to 50xa0% by a MeHg challenge in cells. The MeHg-modified C-terminal catalytic domain in PDI was detected by nMALDI-TOF/MS. Furthermore, treatment with MeHg significantly attenuated the enzymatic activity of PDI. Taken together, these observations suggest that MeHg results in ER stress and following the unfolded protein response pathway via ER dysfunction due to S-mercuration of the C-terminus of PDI.

Regulation of Histone Deacetylase 6 Activity via S-Nitrosylation.

Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles via S-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

Polysulfide Na 2 S 4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through formation of sulfur adducts

Electrophiles can activate redox signal transduction pathways, through actions of effector molecules (e.g., kinases and transcription factors) and sensor proteins with low pKa thiols that are covalently modified. In this study, we investigated whether 1,4-naphthoquinone (1,4-NQ) could affect the phosphatase and tensin homolog (PTEN)–Akt signaling pathway and persulfides/polysulfides could modulate this adaptive response. Simultaneous exposure of primary mouse hepatocytes to Na2S4 and 1,4-NQ markedly decreased 1,4-NQ-mediated cell death and S-arylation of cellular proteins. Modification of cellular PTEN during exposure to 1,4-NQ was also blocked in the presence of Na2S4. 1,4-NQ, at up to 10u2009µM, increased phosphorylation of Akt and cAMP response element binding protein (CREB). However, at higher concentrations, 1,4-NQ inhibited phosphorylation of both proteins. These bell-shaped dose curves for Akt and CREB activation were right-shifted in cells treated with both 1,4-NQ and Na2S4. Incubation of 1,4-NQ with Na2S4 resulted in formation of 1,4-NQ–S–1,4-NQ-OH. Unlike 1,4-NQ, authentic 1,4-NQ-S-1,4-NQ-OH adduct had no cytotoxicity, covalent binding capability nor ability to activate PTEN-Akt signaling in cells. Our results suggested that polysulfides, such as Na2S4, can increase the threshold of 1,4-NQ for activating PTEN–Akt signaling and cytotoxicity by capturing this electrophile to form its sulfur adducts.

A Retrospective Study of the Effects of Oncology Pharmacist Participation in Treatment on Therapeutic Outcomes and Medical Costs

Specialist oncology pharmacists are being trained in Japan to assist cancer treatment teams. These specialized pharmacists address patients physical and mental problems in pharmacist-managed cancer care clinics, actively participate in formulating treatment policies, and are beneficial in offering qualitative improvements to patient services and team medical care. However, the effect of outpatient treatment by oncology pharmacists on therapeutic outcomes and medical costs is still unknown. A retroactive comparative analysis of the treatment details and clinical course was conducted among three groups of patients: patients who underwent adjuvant chemotherapy managed by a gynecologic oncologist only (S arm), patients managed by a non-oncologist (general practice gynecologist) only (NS arm), and patients managed by both a non-oncologist and a specialist oncology pharmacist (NS+Ph arm). The medical cost per course was significantly lower for patients in the NS+Ph arm than for those in the other two arms. Surprisingly, the outpatient treatment rate in the NS+Ph arm was overwhelmingly high. The involvement of an oncology pharmacist did not make a significant difference in therapeutic outcomes such as recurrence rate and survival. The participation of oncology pharmacists in the management of cancer patients undergoing chemotherapy enables safe outpatient treatment and also reduces medical costs.

Functional analysis of juxta- and intra-membrane domains of murine APP by genome editing in Neuro2a cells.

Amyloid-β precursor protein (APP) correlates with the pathogenesis of certain brain diseases, such as Alzheimer disease (AD). APP is cleaved by several enzymes to produce APP metabolites, including the amyloid beta peptide (Aβ), which accumulates in the brain of AD patients. However, the exact functions of APP metabolites remain elusive. In this study, using genome editing technology, we mutated juxta- and intra-membrane domains of murine APP in the mouse neuroblastoma cell line, Neuro2a. We identified several clones that expressed characteristic patterns of APP metabolites. Mutations in juxta- (deletion 673A), and intra-membrane (deletion 705-6LM) domains of APP, decreased overall levels of APP metabolites or decreased the level of α-secretase-cleaved carboxy-terminal fragment (αCTF), respectively. APP is known to influence neuronal differentiation; therefore, we used theses clones to dissect the function of APP metabolites during neuronal differentiation. One clone (CA), which expressed reduced levels of both FL-APP and αCTF, showed increased expression of the neuronal marker, β3-tubulin, and enhanced retinoic acid (RA)-induced neurite outgrowth. In contrast, a clone that expressed FL-APP, but was devoid of αCTF (CE), showed comparable expression of β3-tubulin and neurite outgrowth compared with normal Neuro2a cells. These data indicate that FL-APP is a suppressor of neurite outgrowth. Our data suggest a novel regulatory function of juxta- and intra-membrane domains on the metabolism and function of APP.