8-Nitro-cGMP Attenuates the Interaction between SNARE Complex

Nov 6, 2017 - and Complexin through S‑Guanylation of SNAP-25 ... complexin (cplx), which binds to the SNARE complex with a high affinity. Pull-down ...
0 downloads 0 Views 3MB Size
Download PDF
Letter pubs.acs.org/chemneuro

8‑Nitro-cGMP Attenuates the Interaction between SNARE Complex and Complexin through S‑Guanylation of SNAP-25 Yusuke Kishimoto,† Kohei Kunieda,†,‡ Atsushi Kitamura,† Yuki Kakihana,† Takaaki Akaike,§ and Hideshi Ihara*,† †

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Department of Protein Factory, Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan § Department of Environmental Health Science and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan

Downloaded via UNIV OF SUNDERLAND on November 6, 2018 at 21:27:40 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

ABSTRACT: 8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is the second messenger in nitric oxide/ reactive oxygen species redox signaling. This molecule covalently binds to protein thiol groups, called S-guanylation, and exerts various biological functions. Recently, we have identified synaptosomal-associated protein 25 (SNAP-25) as a target of S-guanylation, and demonstrated that S-guanylation of SNAP25 enhanced SNARE complex formation. In this study, we have examined the effects of S-guanylation of SNAP25 on the interaction between the SNARE complex and complexin (cplx), which binds to the SNARE complex with a high affinity. Pull-down assays and coimmunoprecipitation experiments have revealed that S-guanylation of Cys90 in SNAP-25 attenuates the interaction between the SNARE complex and cplx. In addition, blue native-PAGE followed by Western blot analysis revealed that the amount of cplx detected at a high molecular weight decreased upon 8-nitro-cGMP treatment in SHSY5Y cells. These results demonstrated for the first time that S-guanylation of SNAP-25 attenuates the interaction between the SNARE complex and cplx. KEYWORDS: 8-nitro-cGMP, S-guanylation, SNAP-25, SNARE complex, complexin, exocytosis

N

hippocampus.8 NADPH oxidase (Nox) subunits deficit mice show impaired LTP and hippocampus-dependent memory.9 Furthermore, superoxide generated by Nox in activated microglia induces long-term depression (LTD) in the hippocampus.10 These studies demonstrated the physiological relevance of ROS in brain function. Previously, we discovered 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) in the process of NO/ROS redox signaling.6,11,12 This molecule covalently binds to protein thiol groups (S-guanylation) and exerts various biological functions, such as cytoprotection,4,13 cell senescence,14,15 and autophagy.16 Some target proteins of this post-translational modification (PTM) have been reported previously (e.g., HRas,14,15 mitochondrial heat shock protein 60,17 cGMPdependent protein kinase,18 and Kelch-like ECH-associated protein 1 (Keap1)12,13). We have found that synaptosomalassociated protein 25 (SNAP-25), a member of the soluble Nethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, was S-guanylated by 8-nitro-cGMP, and S-

itric oxide (NO) is a gasotransmitter that is involved in both physiological and pathological functions, including synaptic plasticity and neurodegenerative disorders in the nervous system.1 Typically, NO exerts its multiple functions through two mechanisms.2 One is the NO/guanosine 3′,5′cyclic monophosphate (cGMP) pathway; NO, generated by NO synthase (NOS), activates soluble guanylate cyclase to generates cGMP, which activates cGMP dependent protein kinase.2 The other is the pathway involving chemical modification of biomolecules by NO or peroxynitrite which is generated by the interaction of NO and reactive oxygen species (ROS).2 Although NOSs are considered to be NO-producing enzymes, they produce not only NO but also superoxide by the uncoupling reaction,3−5 the levels of which are regulated by several mechanisms, such as phosphorylation4 and splicing.3 ROS are well-known toxicants that damage DNA, protein, and lipids.6 However, recent studies demonstrated that ROS also work as redox signaling molecules in certain conditions.6 NO and ROS from NOSs were both involved in the modulation of redox signaling.3,4 Moreover, ROS play an important role in the synaptic plasticity formations.7 Superoxide produced via calcium influx through the N-methyl-D-aspartate (NMDA) receptor is necessary for long-term potentiation (LTP) in the © 2017 American Chemical Society

Received: September 18, 2017 Accepted: November 6, 2017 Published: November 7, 2017 217

DOI: 10.1021/acschemneuro.7b00363 ACS Chem. Neurosci. 2018, 9, 217−223

Letter

ACS Chemical Neuroscience

Figure 1. Cell permeability of 8-nitro-cGMP in SH-SY5Y cells. SH-SY5Y cells were treated with 8-nitro-cGMP for 3 h. (A) S-Guanylated proteins in SH-SY5Y cells were analyzed by Western blotting using anti-S-guanylated protein antibody. (B) 8-Nitro-cGMP and S-guanylated proteins in SHSY5Y cells were analyzed by immunohistochemical analysis. SH-SY5Y cells treated with 8-nitro-cGMP were stained using anti-8-nitro-cGMP and anti-S-guanylated protein antibodies. DIC: differential interference contrast microscope. Scale bars: 100 μm. (C) 8-Nitro-cGMP in SH-SY5Y cell was analyzed by LC-MS/MS analysis. 8-Nitro-cGMP in SH-SY5Y cell was measured by LC-MS/MS using a stable-isotope dilution method. Representative LC-MS/MS chromatograms are shown here.

with a high affinity but not to monomeric SNAP-25, syntaxin, and VAMP2.24 Cplx binding to the SNARE complex is an important process of priming and exocytosis.24 In addition, Cplx enhances SNARE complex oligomerization, which is required for exocytosis.25 Besides, cplx plays important roles in brain functions.26,27 For instance, cplx I-knockout mice show motor incoordination; a reduction in grooming, rearing, and exploratory behavior; and social behavior deficit.26,27 However, molecular mechanism regulating the cplx function has not been fully understood. To date, the effect of 8-nitro-cGMP on the interaction between the SNARE complex and exocytosis regulatory proteins has not been clarified. In this study, we have investigated the effect of 8-nitro-cGMP on the interaction between the SNARE complex and cplx to clarify the roles of 8nitro-cGMP in exocytosis in neurons. We revealed that Sguanylation of SNAP-25 by 8-nitro-cGMP attenuates the interaction between the SNARE complex and cplx.

guanylation of Cys90 in SNAP-25 enhances the formation of the SNARE complex.19 Thus, 8-nitro-cGMP is expected to function as an important mediator of NO/ROS signaling in the nervous system. However, the molecular mechanism for regulation of SNAP-25 by 8-nitro-cGMP is not yet well understood. In the process of exocytosis of neurotransmitters, the SNARE complex is the key component to catalyze membrane fusion.20 The SNARE complex is composed of three SNARE proteins (SNAP-25, syntaxin, and vesicle-associated membrane protein 2 (VAMP2)).20 Exocytosis of neurotransmitters requires multistep reactions including “docking” and “priming,” and then Ca2+ influx triggers membrane fusion.21 PTM of SNAP-25 has some neuropathophysiological significance. The residue substitutions of phosphorylation sites resulted in an increase in anxiety-related behavior in mice22 and physical stress for mice enhances SNAP-25 phosphorylation.23 Exocytosis of neurotransmitters is modulated not only by SNARE proteins but also by exocytosis-modulatory proteins, including synaptotagmins, mammalian uncoordinated-18 (Munc18), and complexin (cplx).20,21 Cplx is an exocytosisregulatory protein that binds to the ternary SNARE complex

■

RESULTS AND DISCUSSION Cell permeability of 8-nitro-cGMP in vascular smooth muscle cells has been reported.12 Here, we confirmed whether 8-nitro218

DOI: 10.1021/acschemneuro.7b00363 ACS Chem. Neurosci. 2018, 9, 217−223

Letter

ACS Chemical Neuroscience

Figure 2. Effects of 8-nitro-cGMP on interaction between SNARE complex and externally added GST-cplx in synaptosomes and SH-SY5Y cells. Effects of 8-nitro-cGMP on interaction between SNARE complex and externally added GST-cplx in synaptosomes and SH-SY5Y cells were analyzed by pull down assay. (A) Rat synaptosomes were treated with 8-nitro-cGMP (0, 10, or 100 μM). SNAP-25 pulled-down by GST-cplx was analyzed by Western blotting using anti-FLAG-tag antibody. (B) Graph shows bands intensity of pulled-down SNAP-25, normalized with input data and presented as a percent of control (mean ± SEM; n = 4). One-way ANOVA with Tukey’s multiple comparison post hoc test was used for statistical analysis. *P < 0.05. (C) SH-SY5Y cells transfected with FLAG-tagged SNAP-25 (wild-type or C90A mutant) were treated with 8-nitro-cGMP (0, 10, or 100 μM). FLAG-tagged SNAP-25 pulled-down by GST-cplx was analyzed by Western blotting using anti-FLAG-tag antibody. (D) Graph shows bands intensity of pulled-down FLAG-tagged SNAP-25 normalized with input data and presented as a percent of control (mean ± SEM; n = 4). Student’s t test and one-way ANOVA with Tukey’s multiple comparison post hoc test were used for statistical analysis. *P < 0.05, **P < 0.01 compared to the each control, ##P < 0.01 compared to the wild-type treated with 100 μM 8-nitro-cGMP.

cGMP penetrated the membrane of SH-SY5Y neuroblastoma cells by Western blotting, immunohistochemical analysis (IHC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (Figure 1). Western blot analysis revealed that 8-nitro-cGMP treatment of SH-SY5Y cells increased the amount of S-guanylated proteins in the cell lysates that contains both cytosolic and membrane proteins (Figure 1A). Next, we performed IHC to detect the levels of 8nitro-cGMP and S-guanylated proteins in SH-SY5Y cells treated with 8-nitro-cGMP. As shown in Figure 1B, we observed 8-nitro-cGMP-dependent elevation of intracellular 8-nitro-cGMP and S-guanylated protein levels in the SH-SY5Y cells. Furthermore, we analyzed the concentration of intracellular 8-nitro-cGMP in SH-SY5Y cells by LC-MS/MS using a stable-isotope dilution method. As shown in Figure 1C, 8-nitrocGMP was detected at the same retention time as was 8-nitroc[13C10]GMP in the lysate from 8-nitro-cGMP-treated cells. The concentration of 8-nitro-cGMP was determined as 6.74 ± 0.10 pmol/mg protein estimated from 8-nitro-c[13C10]GMP. The concentration of 8-nitro-cGMP in the brains of mice has been reported to be approximately 3 pmol/mg protein;19 thus, the intracellular 8-nitro-cGMP concentration in SH-SY5Y cells treated with 100 μM 8-nitro-cGMP is not very different from that under physiological conditions. Taken together, our results

indicate that exogenously added 8-nitro-cGMP penetrates the cell membrane of SH-SY5Y cells and reacts with the cytoplasmic proteins. We have previously reported that the S-guanylation of SNAP-25 enhances the formation of the SNARE complex,19 but its effect on the interaction between the SNARE complex and cplx has not been clarified. Here, we investigated the effect of 8-nitro-cGMP on the interaction between the SNARE complex and cplx. First, to examine the interaction of cplx with the SNARE complex, we performed a pull-down assay. SNARE complexes in the solubilized synaptosome (Figure 2A and B) and cells (Figure 2C and D) were bound to exogenously added cplx conjugated with glutathione S-transferase (GST) beads. The amount of pull-downed SNAP-25 was analyzed by Western blotting (Figure 2). Initially, we predicted that the amount of SNAP-25 harvested by cplx in the pull-down assay would be increased by 8-nitro-cGMP treatment, because our previous study showed that 8-nitro-cGMP enhances SNARE complex formation.19 Unexpectedly, the amount of SNAP-25 in the rat synaptosome bound to exogenously added GST-cplx was significantly decreased by 8-nitro-cGMP treatment (Figure 2). We previously reported that the main target of Sguanylation of SNAP-25 is the cysteine 90 in this protein.19 Therefore, to examine whether the effect of 8-nitro-cGMP 219

DOI: 10.1021/acschemneuro.7b00363 ACS Chem. Neurosci. 2018, 9, 217−223

Letter

ACS Chemical Neuroscience

Figure 3. Effects of 8-nitro-cGMP on interaction between SNARE complex and endogenously expressing V5-tagged cplx in SH-SY5Y cells. Effects of 8-nitro-cGMP on interaction between SNARE complex and endogenously expressing V5-tagged cplx in SH-SY5Y cells were analyzed by co-IP assay. (A) SH-SY5Y cells cotransfected with FLAG-tagged SNAP-25 (wild-type or C90A mutant) and V5-tagged cplx were treated with 8-nitro-cGMP (0, 10, or 100 μM). Immunoprecipitated FLAG-tagged SNAP-25 and coimmunoprecipitated V5-tagged cplx were analyzed by Western blotting using anti-FLAG-tag and anti-V5-tag antibodies, respectively. (B) Graph shows bands intensity of coimmunoprecipitated V5-tagged cplx normalized with immnoprecipitated FLAG-tagged SNAP-25 and presented as a percent of control (mean ± S.E.M.; n = 6). Student’s t test and Kruskal−Wallis test with Dunn’s multiple comparison post hoc test were used for statistical analysis. *P < 0.05 compared to the control, ##P < 0.01 compared to the wildtype treated with 100 μM 8-nitro-cGMP.

followed by Western blotting to analyze the size and amount of cplx and SNAP-25-containing protein complexes in SH-SY5Y cells (Figure 4). BN-PAGE is the method to analyze protein complexes and allows isolation of protein complexes while preserving their native conformation.28 Recently, this method was utilized for analyzing the SNARE complex with cplx and other SNARE modulatory proteins in the human brain of individuals with schizophrenia.29 In this study, we applied the BN-PAGE to analyze the SNARE complex, including modulatory proteins such as cplx in the SH-SY5Y cells transfected with FLAG-tagged SNAP-25 and V5-tagged cplx. To our knowledge, this is the first report to analyze the SNARE complex formation and interaction of cplx in the cultured cells by BN-PAGE. As shown in Figure 4A, SNAP-25 immunoreactive bands of various sizes were observed and 8-nitro-cGMP increased the amount of high-molecular-mass complexes containing SNAP-25 (Figure 4B), consistent with our previous finding, indicating that 8-nitro-cGMP enhances SNARE complex formation and the amount of SDS-resistant SNARE complexes.19 In addition, 8-nitro-cGMP decreased the amount of V5-tagged cplx detected at a molecular mass of approximately 1000 kDa, wherein one SNAP-25 immunoreactive band was detected (Figure 4C, D). Because our co-IP analysis in this study revealed that 8-nitro-cGMP reduced the interaction between SNAP-25 and cplx in SH-SY5Y cells (Figure 3), V5-tagged cplx detected at a high molecular mass was presumed to be associated with the SNARE complex. Taking the facts mentioned above into considerations, our results strongly suggest that 8-nitro-cGMP increases the amount of SNAP-25 containing large complex, on the other hand, it decreases the affinity of cplx with the SNARE complex at high molecular mass. Cplx has been well-known to regulate neurotransmitter release at the synapse by binding to the SNARE complex with a high affinity.24 However, the molecular mechanism that regulates the affinity between cplx and the SNARE complex has not been fully understood. PTMs of cplx and SNARE proteins are candidate mechanism for regulating the affinity between cplx and the SNARE complex. Indeed, it has

depends on the cysteine 90 S-guanylation of SNAP-25, we performed the pull-down assay using SH-SY5Y cells-transfected with FLAG-tagged SNAP-25 (wild-type or cysteine 90 alanine point mutant (C90A)) (Figure 2C, D). The substitution of cysteine 90 to alanine in SNAP-25 did not affect the binding of GST-cplx to SNARE complex (data not shown). The amount of FLAG-tagged SNAP-25 bound to GST-cplx decreased upon 8-nitro-cGMP treatment in the SNAP-25 wild-type transfected cells; on the other hand, the decrease was suppressed in SNAP25 C90A mutant transfected cells (Figure 2C, D). It has been reported that cplx binds to the SNARE complex with a high affinity but does not bind to the SNAP-25 monomer. Hence, the SNAP-25 detected here is a portion of the SNARE complex. These results suggest that the S-guanylation of SNAP25 at C90 by 8-nitro-cGMP critically attenuates the interaction between the SNARE complex and exogenously added cplx. In the pull-down assay, there is the possibility that the endogenously expressed cplx that has already bound to the SNARE complex inhibits the interaction between the SNARE complex and exogenously added cplx. In the next study, to examine whether 8-nitro-cGMP attenuates the interaction between the SNARE complex and endogenously expressing cplx in living cells, we performed coimmunoprecipitation (coIP) of SNAP-25 and cplx followed by Western blot analysis using SH-SY5Y cells cotransfected with FLAG-tagged SNAP-25 (wild-type or C90A mutant) and V5-tagged cplx (Figure 3). 8Nitro-cGMP treatment significantly decreased the amount of V5-tagged cplx coimmunoprecipitated by the anti-FLAG-tag antibody in SH-SY5Y cells cotransfected with FLAG-tagged SNAP-25 wild-type and V5-tagged cplx (Figure 3). On the other hand, the decrease was suppressed in SNAP-25 C90A mutant-transfected cells (Figure 3). These results indicate that 8-nitro-cGMP attenuates the interaction between the SNARE complex and not only exogenously added cplx but also endogenously expressing cplx in the living cells through Sguanylation of SNAP-25 at C90. In the next study, to confirm the affinity attenuation between the SNARE complex and cplx by another way, we performed blue native (BN)-polyacrylamide gel electrophoresis (PAGE) 220

DOI: 10.1021/acschemneuro.7b00363 ACS Chem. Neurosci. 2018, 9, 217−223

Letter

ACS Chemical Neuroscience

Figure 4. Effects of 8-nitro-cGMP on SNARE complex formation and association of V5-tagged cplx in SH-SY5Y cells. Effects of 8-nitro-cGMP on SNARE complex formation and association of V5-tagged cplx in SH-SY5Y cells were analyzed by BN-PAGE. (A, C) SH-SY5Y cells transfected with V5-tagged cplx were treated with 8-nitro-cGMP (0, 10, or 100 μM). Proteins were separated by BN-PAGE followed by Western blotting using antiSNAP-25 (A) and anti-V5-tag antibodies (C). (B, D) Graph shows bands intensity of SNAP-25 at high molecular weight (higher than 150 kDa, indicated by bracket (B)) and V5-tagged cplx detected at approximately 1,000 kDa (indicated by arrow (D)). Data were presented as a percent of control (mean ± SEM; n = 9). Kruskal−Wallis test with Dunn’s multiple comparison post hoc test were used for statistical analysis. *P < 0.05, ***P < 0.001 compared to the control.

progress to clarify the neurophysiological role of S-guanylation of SNAP25 by 8-nitro-cGMP. In conclusion, 8-nitro-cGMP treatment attenuates the interaction between the SNARE complex and both exogenously added and endogenously expressing cplx. Moreover, cplx was detected at a high molecular mass in SH-SY5Y cells, and it was released upon 8-nitro-cGMP treatment. Taken together, our results from a pull-down assay, co-IP, and BN-PAGE strongly suggest that 8-nitro-cGMP attenuates the interaction between the SNARE complex and cplx through S-guanylation at C90 in SNAP-25. Our findings for 8-nitro-cGMP, which is generated through NO/ROS signaling, described above, provide new insights into NO/ROS redox signaling in neurotransmission. Because the binding of cplx to the SNARE complex is an important process in exocytosis, there is a possibility that 8nitro-cGMP regulates exocytosis, neurotransmission, and brain functions.

previously been reported that cplx is phosphorylated at ser115 in the rat brain and that phosphorylated cplx exhibits enhanced SNARE complex binding.30 The authors speculated that the phosphorylation may provide a new route for modulating fast neurotransmitter release.30 Here, we demonstrated for the first time that the cplx binding to the SNARE complex was modulated by PTM (S-guanylation) of SNAP-25. The Sguanylation of SNAP-25 attenuates the interaction between cplx and the SNARE complex. The S-guanylation of SNAP-25 may be involved in neurotransmitter release. Our present studies unclarified the neurophysiological meaning of the regulation of affinity changing between the SNARE complex and cplx by 8-nitro-cGMP. Previously, it was reported that attenuation of the interaction of the SNARE complex with cplx in squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis.25 Thus, 8-nitro-cGMP might regulate neurophysiological function by regulating SNARE complex formation and the affinity between the SNARE complex and cplx. Further work is in 221

DOI: 10.1021/acschemneuro.7b00363 ACS Chem. Neurosci. 2018, 9, 217−223

Letter

ACS Chemical Neuroscience

■

Guidelines for Animal Experimentation of Osaka Prefecture University. Synaptosome or SH-SY5Y cells transfected with FLAGtagged SNAP-25 were treated with 8-nitro-cGMP for one or 3 h, respectively. Purified GST-cplx bound to GSH-conjugated magnetic beads was incubated with the lysates for 3 h at room temperature. After washing, proteins bound to beads were eluted with SDS sample buffer and analyzed by Western blotting. Coimmunoprecipitation (Co-IP). SH-SY5Y cells cotransfected with FLAG-tagged SNAP-25 and V5-tagged cplx were treated with 8nitro-cGMP for 3 h. Supernatants after centrifugation of cell lysates were incubated with anti-DDDDK-tag antibody-conjugated agarose for 1 h at room temperature. After washing, proteins bound to beads were eluted with SDS sample buffer. FLAG-tagged SNAP-25 and V5tagged cplx were analyzed by Western blotting. Blue-Native (BN)-PAGE. Native SNARE complex associated with cplx was analyzed by BN-PAGE as previously described29 with minor modifications. In brief, sample preparation was carried out using NativePAGE Sample Prep Kit (Thermo) according to the manufacture instructions. 8-Nitro-cGMP-treated SH-SY5Y cells-transfected with V5-tagged cplx was lysed with lysis buffer containing NativePAGE 1× sample buffer, 0.5% TritonX-100, 10% glycerol and protease inhibitors. Just after adding one-fourth volume of 0.5% CBB-G250 to samples, BN-PAGE was performed using 4% upper 4−16% gradient separation Bis-Tris polyacrylamide gel. After BN-PAGE, the separation gels were soaked in transfer buffer containing 0.1% SDS. followed by transfer buffer containing 0.02% SDS for 10 min at room temperature, respectively. Proteins were transfer to PVDF membrane, CBB was removed by washing with methanol. V5-tagged cplx and SNAP-25 was detected by Western blotting. Statistical Analysis. The data presented as the mean ± SE of individual experiments performed at least three times. Statistical significance was determined by one-way analysis of variance (ANOVA), Tukey’s multiple comparison post hoc test using the GraphPad Prism Software (GraphPad Software, La Jolla, CA).

METHODS

Materials. SH-SY5Y cells were kindly supplied by Dr. Hidemitsu Nakajima (Osaka Prefecture University). The anti-8-nitro-cGMP antibody (clone 1G6),12 anti-S-guanylated protein antibody,12 and anti-SNAP-25 antibody (clone BR05)19 were prepared as previously described. pcDNA3.1/nV5-DEST, LR reaction kit, and NativePAGE Kit were from Thermo Fisher Scientific (Waltham, MA). Glutathione (GSH) sepharose, polyvinylidene difluoride (PVDF) membrane, and peroxidase (POD)-conjugated secondary antibodies were from GE Healthcare (Buckinghamshire, England, UK). Dulbecco’s modified Eagle’s medium (D-MEM) and Hoechst33258 were purchased from Wako Pure Chemical (Osaka, Japan). Penicillin−streptomycin (PS) solution, protease inhibitor cocktail, Blocking One, and anti-V5-tag antibody were from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS), MagneGST glutathione particles, polyethylenimine (PEI)-Max, nitrocellulose membrane, chemiluminescence reagent, anti-FLAG-tag antibody, Block Ace, Can Get Signal Solutions, HiLyte Fluor 555- and 647-conjugated secondary antibodies, mounting medium, and anti-DDDDK-tag antibody agarose were obtained from Biosera (Nuaille, France), Promega (Madison, WI), Polysciences (Warrington, PA), Merck Millipore (Darmstadt, Germany), SigmaAldrich (St. Louis, MO), Snow Brand Milk Products (Tokyo, Japan), TOYOBO (Osaka, Japan), AnaSpec (Fremont, CA), SeraCare Life Sciences (Milford, MA), and Medical and Biological Laboratories (Nagoya, Japan), respectively. All other chemicals and reagents were from common suppliers and were of the highest grade commercially available. Plasmids and Protein Expression. Expression plasmid containing rat SNAP-25-B wild-type and C90A mutant cDNA (SNAP-25-B/ pcDNA3.2/nFLAG-DEST) were constructed as previously described.19 The human cplx-I entry vector (FLJ 92108AAAF) was kindly supplied by Dr. Naoki Goshima (National Institute of Advanced Industrial Science and Technology). Cplx-I was subcloned into pcDNA3.1/nV5-DEST or 5′-FLAG-GST-tagged destination vectors (5FG-DEST) (kindly supplied by Dr. Goshima) by LR reaction. GST-tagged cplx-I was produced by a wheat germ cell-free protein expression system (CellFree Science; Ehime, Japan), followed by purification using glutathione sepharose (GE Healthcare). Cell Culture, Transfection, and 8-Nitro-cGMP Treatment. SHSY5Y cells were cultured in D-MEM supplemented with 10% FBS and 1% PS, and incubated in humidified atmosphere with 5% CO2 at 37 °C. Cells were transfected with plasmids using PEI-Max, incubated for 24 h, and then treated with 8-nitro-cGMP for 3 h.19 Western Blot Analysis. Cells were lysed with phosphate buffered saline (PBS) containing 1% TritonX-100 and protease inhibitor cocktail. The cell lysates were subjected to SDS-PAGE, followed by Western blotting. Immunoreactive bands were detected using chemiluminescence reagent and luminescent image analyzer LAS1000 (Fujifilm, Tokyo, Japan). Immunocytochemistry. Immunocytochemistry was performed as described previously.12,19 Briefly, after fixation, permeabilization, and blocking, the cells were incubated with primary antibodies overnight at 4 °C, and then with fluorescent-conjugated secondary antibodies and Hoechst 33258. The images were captured and processed by using OLYMPUS FV1200 IX83 microscope (OLYMPUS, Tokyo, Japan). LC-MS/MS Analysis. 8-Nitro-cGMP in the SH-SY5Y cells were measured by LC-MS/MS using a stable-isotope dilution method as previously described.13,19 In brief, cells were lysed in methanol containing 1 μM 8- nitro-c[13C10]GMP and 5 mM NEM. After centrifugation, the supernatants were dried, resolved in 0.1% formic acid, and injected to high performance liquid chromatography (HPLC) system (Waters, Milford, MA). Samples were separated by reverse-phase HPLC and analyzed with electrospray ionization triple quadrupole mass spectrometer (Xevo TQD; Waters,). The observed ion masses (parent → daughter ions) were m/z 391 → 151 and m/z 401 → 156 for endogenous 8-nitro-cGMP and spiked 8-nitroc[13C10]GMP, respectively. Pull-Down Assay. Synaptosome was prepared as described previously.19 This study was performed in accordance with the

■

AUTHOR INFORMATION

Corresponding Author

*Mailing address: #608 Bldg. C10, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan. Tel: +81-72-254-9753. E-mail: [email protected]. ORCID

Takaaki Akaike: 0000-0002-0623-1710 Hideshi Ihara: 0000-0002-8971-5138 Author Contributions

Y.K., K.K., T.A., and H.I. designed the research. Y.K., K.K., and A.K. performed experiments and analysis and interpreted the data. H.I. conducted the experiments. Y.K. and H.I. wrote the manuscript. Funding

This work was supported in part by a Grant-in-Aid for a Grantin-Aid for Scientific Research A (25253020 to T.A.), a Grant-inAid for Scientific Research B (16H04674 to H.I.), a Grant-inAid for Challenging Exploratory Research (16K15208 to T.A. and 16K13089 to H.I.), and a Grant-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Area) (26111008 to T.A. and 26111011 to H.I.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Dr. Hidemitsu Nakajima (Osaka Prefecture University) for the kind gift of the cells and Dr. Naoki Goshima 222

DOI: 10.1021/acschemneuro.7b00363 ACS Chem. Neurosci. 2018, 9, 217−223

Letter

ACS Chemical Neuroscience

redox-based mitochondrial signaling. Antioxid. Redox Signaling 20, 295−307. (18) Akashi, S., Ahmed, K. A., Sawa, T., Ono, K., Tsutsuki, H., Burgoyne, J. R., Ida, T., Horio, E., Prysyazhna, O., Oike, Y., Rahaman, M. M., Eaton, P., Fujii, S., and Akaike, T. (2016) Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein S-Guanylation. Biochemistry 55, 751−761. (19) Kunieda, K., Tsutsuki, H., Ida, T., Kishimoto, Y., Kasamatsu, S., Sawa, T., Goshima, N., Itakura, M., Takahashi, M., Akaike, T., and Ihara, H. (2015) 8-Nitro-cGMP enhances SNARE complex formation through S-guanylation of Cys90 in SNAP25. ACS Chem. Neurosci. 6, 1715−1725. (20) Brunger, A. T. (2005) Structure and function of SNARE and SNARE-interacting proteins. Q. Rev. Biophys. 38, 1−47. (21) Jahn, R., and Fasshauer, D. (2012) Molecular machines governing exocytosis of synaptic vesicles. Nature 490, 201−207. (22) Watanabe, S., Yamamori, S., Otsuka, S., Saito, M., Suzuki, E., Kataoka, M., Miyaoka, H., and Takahashi, M. (2015) Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice. Epilepsy Res. 115, 30−44. (23) Yamamori, S., Sugaya, D., Iida, Y., Kokubo, H., Itakura, M., Suzuki, E., Kataoka, M., Miyaoka, H., and Takahashi, M. (2014) Stressinduced phosphorylation of SNAP-25. Neurosci. Lett. 561, 182−187. (24) Mohrmann, R., Dhara, M., and Bruns, D. (2015) Complexins: small but capable. Cell. Mol. Life Sci. 72, 4221−4235. (25) Tokumaru, H., Umayahara, K., Pellegrini, L. L., Ishizuka, T., Saisu, H., Betz, H., Augustine, G. J., and Abe, T. (2001) SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis. Cell 104, 421−432. (26) Drew, C. J., Kyd, R. J., and Morton, A. J. (2007) Complexin 1 knockout mice exhibit marked deficits in social behaviours but appear to be cognitively normal. Hum. Mol. Genet. 16, 2288−2305. (27) Glynn, D., Drew, C. J., Reim, K., Brose, N., and Morton, A. J. (2005) Profound ataxia in complexin I knockout mice masks a complex phenotype that includes exploratory and habituation deficits. Hum. Mol. Genet. 14, 2369−2385. (28) Wittig, I., Braun, H. P., and Schagger, H. (2006) Blue native PAGE. Nat. Protoc. 1, 418−428. (29) Ramos-Miguel, A., Beasley, C. L., Dwork, A. J., Mann, J. J., Rosoklija, G., Barr, A. M., and Honer, W. G. (2015) Increased SNARE protein-protein interactions in orbitofrontal and anterior cingulate cortices in schizophrenia. Biol. Psychiatry 78, 361−373. (30) Shata, A., Saisu, H., Odani, S., and Abe, T. (2007) Phosphorylated synaphin/complexin found in the brain exhibits enhanced SNARE complex binding. Biochem. Biophys. Res. Commun. 354, 808−813.

(National Institute of Advanced Industrial Science and Technology) for kindly gifting us the plasmids.

■

REFERENCES

(1) Esplugues, J. V. (2002) NO as a signalling molecule in the nervous system. Br. J. Pharmacol. 135, 1079−1095. (2) Garthwaite, J. (2008) Concepts of neural nitric oxide-mediated transmission. Eur. J. Neurosci. 27, 2783−2802. (3) Ihara, H., Kitamura, A., Kasamatsu, S., Ida, T., Kakihana, Y., Tsutsuki, H., Sawa, T., Watanabe, Y., and Akaike, T. (2017) Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation. Biochem. J. 474, 1149−1162. (4) Kasamatsu, S., Watanabe, Y., Sawa, T., Akaike, T., and Ihara, H. (2014) Redox signal regulation via nNOS phosphorylation at Ser847 in PC12 cells and rat cerebellar granule neurons. Biochem. J. 459, 251− 263. (5) Stuehr, D., Pou, S., and Rosen, G. M. (2001) Oxygen reduction by nitric-oxide synthases. J. Biol. Chem. 276, 14533−14536. (6) Nishida, M., Kumagai, Y., Ihara, H., Fujii, S., Motohashi, H., and Akaike, T. (2016) Redox signaling regulated by electrophiles and reactive sulfur species. J. Clin. Biochem. Nutr. 58, 91−98. (7) Klann, E., and Thiels, E. (1999) Modulation of protein kinases and protein phosphatases by reactive oxygen species: implications for hippocampal synaptic plasticity. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 23, 359−376. (8) Knapp, L. T., and Klann, E. (2002) Role of reactive oxygen species in hippocampal long-term potentiation: contributory or inhibitory? J. Neurosci. Res. 70, 1−7. (9) Kishida, K. T., Hoeffer, C. A., Hu, D., Pao, M., Holland, S. M., and Klann, E. (2006) Synaptic plasticity deficits and mild memory impairments in mouse models of chronic granulomatous disease. Mol. Cell. Biol. 26, 5908−5920. (10) Zhang, J., Malik, A., Choi, H. B., Ko, R. W., Dissing-Olesen, L., and MacVicar, B. A. (2014) Microglial CR3 activation triggers longterm synaptic depression in the hippocampus via NADPH oxidase. Neuron 82, 195−207. (11) Sawa, T., Ihara, H., Ida, T., Fujii, S., Nishida, M., and Akaike, T. (2013) Formation, signaling functions, and metabolisms of nitrated cyclic nucleotide. Nitric Oxide 34, 10−18. (12) Sawa, T., Zaki, M. H., Okamoto, T., Akuta, T., Tokutomi, Y., Kim-Mitsuyama, S., Ihara, H., Kobayashi, A., Yamamoto, M., Fujii, S., Arimoto, H., and Akaike, T. (2007) Protein S-guanylation by the biological signal 8-nitroguanosine 3′,5′-cyclic monophosphate. Nat. Chem. Biol. 3, 727−735. (13) Fujii, S., Sawa, T., Ihara, H., Tong, K. I., Ida, T., Okamoto, T., Ahtesham, A. K., Ishima, Y., Motohashi, H., Yamamoto, M., and Akaike, T. (2010) The critical role of nitric oxide signaling, via protein S-guanylation and nitrated cyclic GMP, in the antioxidant adaptive response. J. Biol. Chem. 285, 23970−23984. (14) Ihara, H., Kasamatsu, S., Kitamura, A., Nishimura, A., Tsutsuki, H., Ida, T., Ishizaki, K., Toyama, T., Yoshida, E., Abdul Hamid, H., Jung, M., Matsunaga, T., Fujii, S., Sawa, T., Nishida, M., Kumagai, Y., and Akaike, T. (2017) Exposure to Electrophiles Impairs Reactive Persulfide-dependent Redox Signaling in Neuronal Cells. Chem. Res. Toxicol. 30, 1673. (15) Nishida, M., Sawa, T., Kitajima, N., Ono, K., Inoue, H., Ihara, H., Motohashi, H., Yamamoto, M., Suematsu, M., Kurose, H., van der Vliet, A., Freeman, B. A., Shibata, T., Uchida, K., Kumagai, Y., and Akaike, T. (2012) Hydrogen sulfide anion regulates redox signaling via electrophile sulfhydration. Nat. Chem. Biol. 8, 714−724. (16) Ito, C., Saito, Y., Nozawa, T., Fujii, S., Sawa, T., Inoue, H., Matsunaga, T., Khan, S., Akashi, S., Hashimoto, R., Aikawa, C., Takahashi, E., Sagara, H., Komatsu, M., Tanaka, K., Akaike, T., Nakagawa, I., and Arimoto, H. (2013) Endogenous nitrated nucleotide is a key mediator of autophagy and innate defense against bacteria. Mol. Cell 52, 794−804. (17) Rahaman, M. M., Sawa, T., Ahtesham, A. K., Khan, S., Inoue, H., Irie, A., Fujii, S., and Akaike, T. (2014) S-guanylation proteomics for 223

DOI: 10.1021/acschemneuro.7b00363 ACS Chem. Neurosci. 2018, 9, 217−223