Kou Motani, Noriko Saito-Tarashima, Kohei Nishino, Shunya Yamauchi, Noriaki Minakawa and Hidetaka Kosako : The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER, Cell Reports, Vol.41, No.12, 111868, 2022.
(Summary)
STING, an endoplasmic reticulum (ER)-resident receptor for cyclic di-nucleotides (CDNs), is essential for innate immune responses. Upon CDN binding, STING moves from the ER to the Golgi, where it activates downstream type-I interferon (IFN) signaling. General cargo proteins exit from the ER via concentration at ER exit sites. However, the mechanism of STING concentration is poorly understood. Here, we visualize the ER exit sites of STING by blocking its transport at low temperature or by live-cell imaging with the cell-permeable ligand bis-SATE-2'F-c-di-dAMP, which we have developed. After ligand binding, STING forms punctate foci at non-canonical ER exit sites. Unbiased proteomic screens and super-resolution microscopy show that the Golgi-resident protein ACBD3/GCP60 recognizes and concentrates ligand-bound STING at specialized ER-Golgi contact sites. Depletion of ACBD3 impairs STING ER-to-Golgi trafficking and type-I IFN responses. Our results identify the ACBD3-mediated non-canonical cargo concentration system that drives the ER exit of STING.
(Keyword)
Ligands / Proteomics / Membrane Proteins / Endoplasmic Reticulum / Golgi Apparatus / Interferon Type I / Protein Transport
Kohei Nishino, Harunori Yoshikawa, Kou Motani and Hidetaka Kosako : Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics., Journal of Proteome Research, 2022.
(Summary)
Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.
Ariel Pradipta, Miwa Sasai, Kou Motani, Su Ji Ma, Youngae Lee, Hidetaka Kosako and Masahiro Yamamoto : killing program requires Irgm2 but not its microbe vacuolar localization., Life Science Alliance, Vol.4, No.7, e202000960, 2021.
Kou Motani and Hidetaka Kosako : BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING)., The Journal of Biological Chemistry, Vol.295, No.32, 11174-11183, 2020.
(Summary)
Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. Global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull-down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only non-biotinylated peptides are identified, which cannot be distinguished from peptides of non-specifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.
Kohki Kido, Satoshi Yamanaka, Shogo Nakano, Kou Motani, Souta Shinohara, Akira Nozawa, Hidetaka Kosako, Sohei Ito and Tatsuya Sawasaki : AirID, a Novel Proximity Biotinylation Enzyme, for Analysis of Protein-Protein Interactions, eLife, Vol.9, No.e54983, 2020.
(Summary)
Proximity biotinylation based on BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4 complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.
Kou Motani and Hidetaka Kosako : Phosphoproteomic identification and functional characterization of protein kinase substrates by 2D-DIGE and Phos-tag PAGE, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1867, 57-61, 2019.
(Summary)
Protein phosphorylation is one of the most common post-translational modifications in eukaryotes and can regulate diverse properties of proteins. Protein kinases are encoded by more than 500 genes in higher eukaryotes and play central roles in various cellular signaling pathways. Consequently, genetic abnormalities of protein kinases have been implicated in many diseases. To fully understand the complex phosphorylation-mediated signaling networks, it is important to globally identify and functionally characterize in vivo substrates of individual protein kinases. Advances in electrophoresis-based phosphoproteomic technologies such as two-dimensional difference gel electrophoresis (2D-DIGE) following immobilized metal affinity chromatography (IMAC) and phosphate-affinity Phos-tag PAGE have enabled efficient and detailed analysis of protein kinase substrates. Here, we describe physiological functions of the newly identified substrates of several disease-related protein kinases including ERK, PKD and PINK1.
Takeshi Terabayashi, Katsuhiro Hanada, Kou Motani, Hidetaka Kosako, Mami Yamaoka, Toshihide Kimura and Toshimasa Ishizaki : Baicalein disturbs the morphological plasticity and motility of breast adenocarcinoma cells depending on the tumor microenvironment, Genes to Cells, Vol.23, No.6, 466-479, 2018.
(Summary)
During tumor invasion, cancer cells change their morphology and mode of migration based on communication with the surrounding environment. Numerous studies have indicated that paracrine interactions from non-neoplastic cells impact the migratory and invasive properties of cancer cells. Thus, these interactions are potential targets for anticancer therapies. In this study, we showed that the flavones member baicalein suppresses the motility of breast cancer cells that is promoted by paracrine interactions. First, we identified laminin-332 (LN-332) as a principle paracrine factor in conditioned medium from mammary epithelium-derived MCF10A cells that regulates the morphology and motility of breast adenocarcinoma MDA-MB-231 cells. Then, we carried out a morphology-based screen for small compounds, which showed that baicalein suppressed the morphological changes and migratory activity of MDA-MB-231 cells that were induced by conditioned medium from MCF10A cells and LN-332. We also found that baicalein caused narrower and incomplete lamellipodia formation in conditioned medium-treated MDA-MB-231 cells, although actin dynamics downstream of Rho family small GTPases were unaffected. These results suggest the importance of mammary epithelial cells in the cancer microenvironment promoting the migratory activity of breast adenocarcinoma cells and show a novel mechanism through which baicalein inhibits cancer cell motility.
Kou Motani and Hidetaka Kosako : Activation of stimulator of interferon genes (STING) induces ADAM17-mediated shedding of the immune semaphorin SEMA4D., The Journal of Biological Chemistry, Vol.293, No.20, 7717-7726, 2018.
(Summary)
Stimulator of interferon genes (STING) is an endoplasmic reticulum-resident membrane protein that mediates cytosolic pathogen DNA-induced innate immunity and inflammatory responses in host defenses. STING is activated by cyclic di-nucleotides and is then translocated to the Golgi apparatus, an event that triggers STING assembly with the downstream enzyme TANK-binding kinase 1 (TBK1). This assembly leads to the phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3), which in turn induces expression of type-I interferon (IFN) and chemokine genes. STING also mediates inflammatory responses independently of IRF3, but these molecular pathways are largely unexplored. Here, we analyzed the RAW264.7 macrophage secretome to comprehensively identify proinflammatory factors released into the extracellular medium upon STING activation. In total, we identified 1299 proteins in macrophage culture supernatants, of which 23 were significantly increased after STING activation. These proteins included IRF3-dependent cytokines, as well as previously unknown targets of STING, such as the immune semaphorin SEMA4D/CD100, which possesses proinflammatory cytokine-like activities. Unlike for canonical cytokines, the expression of the SEMA4D gene was not up-regulated. Instead, upon STING activation, membrane-bound SEMA4D was cleaved into a soluble form, suggesting the presence of a post-translational shedding machinery. Importantly, the SEMA4D shedding was blocked by TMI-1, an inhibitor of the sheddase ADAM metallopeptidase domain 17 (ADAM17) but not by the TBK1 inhibitor BX795. These results suggest that STING activates ADAM17 and that this activation produces soluble proinflammatory SEMA4D independently of the TBK1/IRF3-mediated transcriptional pathway.
Hidetaka Kosako and Kou Motani : Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE, Methods in Molecular Biology, Vol.1487, 137-149, 2017.
Kou Motani, S Ito and S Nagata : DNA-Mediated Cyclic GMP-AMP Synthase-Dependent and -Independent Regulation of Innate Immune Responses, The Journal of Immunology, Vol.194, No.10, 4914-4923, 2015.
(Summary)
Cytoplasmic DNA activates cyclic GMP-AMP synthase (cGAS) to produce cyclic 2'-5'3'-5'GMP-AMP dinucleotide (2'5 'cGAMP). The binding of 2'5'cGAMP to an adaptor protein, stimulator of IFN genes (STING), activates a transcription factor, IFN regulatory factor 3, leading to the induction of IFN and chemokine gene expression. In this study, we found that the 2'5'cGAMP-dependent STING activation induced highly upregulated CXCL10 gene expression. Formation of a distinct STING dimer, which was detected by native PAGE, was induced by 2'5'cGAMP, but not 3'-5'3'-5'cGAMP. Analysis of DNase II(-/-) mice, which constitutively produce IFN-β and CXCL10, showed the accumulation of 2'5'cGAMP in their fetal livers and spleens, suggesting that the undigested DNA accumulating in DNase II(-/-) cells may have leaked from the lysosomes into the cytoplasm. The DNase II(-/-) mouse embryonic fibroblasts produced 2'5'cGAMP in a cGAS-dependent manner during apoptotic cell engulfment. However, cGAS deficiency did not impair the STING-dependent upregulation of CXCL10 in DNase II(-/-) mouse embryonic fibroblasts that was induced by apoptotic cell engulfment or DNA lipofection. These results suggest the involvement of a cGAS-independent additional DNA sensor(s) that induces the STING-dependent activation of innate immunity.
Q Wang, R Imamura, Kou Motani, H Kushiyama, S Nagata and T Suda : Pyroptotic cells externalize eat-me and release find-me signals and are efficiently engulfed by macrophages, International Immunology, Vol.25, 363-372, 2013.
(Summary)
Pathogenic intracellular bacteria often hijack macrophages for their propagation. The infected macrophages release IL-1β and IL-18 and simultaneously commit suicide, which is called pyroptosis; both responses require caspase-1. Here, we found that pyroptotic cells induced by microbial infection were efficiently engulfed by human monocytic THP-1-cell-derived macrophages or mouse peritoneal macrophages. This engulfment was inhibited by the D89E mutant of milk fat globule (MFG) epidermal growth factor (EGF) factor 8 (MFG-E8; a phosphatidylserine-binding protein) that has been shown previously to inhibit phosphatidylserine-dependent engulfment of apoptotic cells by macrophages, suggesting that the engulfment of pyroptotic cells by macrophages was also phosphatidylserine dependent. Using a pair of cell lines that respectively exhibited pyroptosis or apoptosis after muramyl dipeptide treatment, we showed that both pyroptotic and apoptotic cells bound to a T-cell immunoglobulin and mucin domain-containing 4 (Tim4; another phosphatidylserine-binding protein)-coated plate, whereas heat-killed necrotic cells did not, indicating that phosphatidylserine was externalized in pyroptosis and apoptosis but not in accidental necrosis. Macrophages engulfed apoptotic cells most efficiently, followed by pyroptotic and then heat-killed necrotic cells. Pyroptotic cells also released a macrophage attractant(s), 'find-me' signal, whose activity was diminished by apyrase that degrades nucleoside triphosphate to nucleoside monophosphate. Heat-killed necrotic cells and pyroptotic cells released ATP much more efficiently than apoptotic cells. These results suggest that pyroptotic cells, like apoptotic cells, actively induce phagocytosis by macrophages using 'eat-me' and find-me signals. Based on these results, a possible role of coordinated induction of pyroptosis and inflammatory cytokine production is discussed.
Kou Motani, H Kushiyama, R Imamura, T Kinoshita, T Nishiuchi and T Suda : Caspase-1 induces apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-mediated necrosis independently of its catalytic activity, The Journal of Biological Chemistry, Vol.286, 33963-33972, 2011.
(Summary)
The adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), connects pathogen/danger sensors such as NLRP3 and NLRC4 with caspases and is involved in inflammation and cell death. We have found that ASC activation induced caspase-8-dependent apoptosis or CA-074Me (cathepsin B inhibitor)-inhibitable necrosis depending on the cell type. Unlike necroptosis, another necrotic cell death, ASC-mediated necrosis, was neither RIP3-dependent nor necrostatin-1-inhibitable. Although acetyl-YVAD-chloromethylketone (Ac-YVAD-CMK) (caspase-1 inhibitor) did not inhibit ASC-mediated necrosis, comprehensive gene expression analyses indicated that caspase-1 expression coincided with the necrosis type. Furthermore, caspase-1 knockdown converted necrosis-type cells to apoptosis-type cells, whereas exogenous expression of either wild-type or catalytically inactive caspase-1 did the opposite. Knockdown of caspase-1, but not Ac-YVAD-CMK, suppressed the monocyte necrosis induced by Staphylococcus and Pseudomonas infection. Thus, the catalytic activity of caspase-1 is dispensable for necrosis induction. Intriguingly, a short period of caspase-1 knockdown inhibited IL-1β production but not necrosis, although longer knockdown suppressed both responses. Possible explanations of this phenomenon are discussed.
K Tabata, H Sakai, R Nakajima, R Saya-Nishimura, Kou Motani, S Okano, Y Shibata, Y Abiko and T Suzuki : Acute application of cisplatin affects methylation status in neuroblastoma cells, Oncology Reports, Vol.25, 1655-1660, 2011.
(Summary)
The pharmacological mechanism of the anti-cancer effect of cisplatin is well known to be DNA intercalation, but the direct or indirect effects of cisplatin on protein expression in cancer cells remain to be explained. In this study, we used a proteomic approach to clarify the early impact of cisplatin on protein expression. In a 2-dimensional gel electrophoresis proteomic experiment, the application of cisplatin for 24 h increased the expression of four proteins and decreased the levels of one protein in neuroblastoma IMR-32 cells. Levels of S-adenosyl-L-homocysteine hydrolase, a key enzyme in methylation metabolism, were increased the most. Therefore, we examined the methylation status of histone proteins. Histone H3K9 methylation was reduced by the application of cisplatin for 24 h. These results suggest that acute cisplatin treatment alters methylation status. Thus, these data can help clarify the unknown pharmacological mechanisms of cisplatin, including the anticancer effect, adverse effects and/or the mechanism of drug resistance.
Kou Motani, K Kawase, R Imamura, T Kinoshita, H Kushiyama and T Suda : Activation of ASC induces apoptosis or necrosis, depending on the cell type, and causes tumor eradication, Cancer Science, Vol.101, 1822-1827, 2010.
M Hasegawa, R Imamura, Kou Motani, T Nishiuchi, N Matsumoto, T Kinoshita and T Suda : Mechanism and repertoire of ASC-mediated gene expression, The Journal of Immunology, Vol.182, No.12, 7655-7662, 2009.
(Summary)
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor molecule that mediates inflammatory and apoptotic signals. Although the role of ASC in caspase-1-mediated IL-1beta and IL-18 maturation is well known, ASC also induces NF-kappaB activation and cytokine gene expression in human cells. In this study, we investigated the molecular mechanism and repertoire of ASC-induced gene expression in human cells. We found that the specific activation of ASC induced AP-1 activity, which was required for optimal IL8 promoter activity. ASC activation also induced STAT3-, but not STAT1-, IFN-stimulated gene factor 3- or NF-AT-dependent reporter gene expression. The ASC-mediated AP-1 activation was NF-kappaB-independent and primarily cell-autonomous response, whereas the STAT3 activation required NF-kappaB activation and was mediated by a factor that can act in a paracrine manner. ASC-mediated AP-1 activation was inhibited by chemical or protein inhibitors for caspase-8, caspase-8-targeting small-interfering RNA, and p38 and JNK inhibitors, but not by a caspase-1 inhibitor, caspase-9 or Fas-associated death domain protein (FADD) dominant-negative mutants, FADD- or RICK-targeting small-interfering RNAs, or a MEK inhibitor, indicating that the ASC-induced AP-1 activation is mediated by caspase-8, p38, and JNK, but does not require caspase-1, caspase-9, FADD, RICK, or ERK. DNA microarray analyses identified 75 genes that were induced by ASC activation. A large proportion of them was related to transcription (23%), inflammation (21%), or cell death (16%), indicating that ASC is a potent inducer of inflammatory and cell death-related genes. This is the first report of ASC-mediated AP-1 activation and the repertoire of genes induced downstream of ASC activation.
Kou Motani, K Tabata, Y Kimura, S Okano, Y Shibata, Y Abiko, H Nagai, T Akihisa and T Suzuki : Proteomic analysis of apoptosis induced by xanthoangelol, a major chalcone constituent of Angelica keiskei, in neuroblastoma, Biological & Pharmaceutical Bulletin, Vol.31, No.4, 618-626, 2008.
(Summary)
Neuroblastoma is the most common solid tumor in children. Despite aggressive chemotherapy, the prognosis of patients with advanced neuroblastoma is still very poor. Our recent study showed that xanthoangelol, a major chalcone constituent of the stem exudates of Angelica keiskei, induced caspase-3-dependent apoptosis in neuroblastoma cells. However, details of the mechanism underlying its apoptotic action are still unclear. Here we show that xanthoangelol triggers oxidative stress by generation of reactive oxygen species and induces apoptosis through release of cytochrome c and activation of caspase-9 in IMR-32 cells. Pretreatment with an antioxidant, vitamin E, prevented the increase of reactive oxygen species and apoptosis induced by xanthoangelol. Proteomic analysis using 2-dimensional electrophoresis and MALDI-TOF-MS revealed that DJ-1 protein was involved in xanthoangelol-induced apoptosis. DJ-1 responded to its oxidative stress status by being oxidized itself. Furthermore, DJ-1 was down-regulated by xanthoangelol, leading to loss of antioxidant function and acceleration of apoptosis. We also show that xanthoangelol has a cytotoxic effect on drug-resistant LA-N-1 and NB-39 cells as well as drug-sensitive IMR-32 and SK-N-SH cells. These findings suggest that xanthoangelol induces apoptosis by increasing reactive oxygen species and targeting DJ-1, and such mechanism may be an effective therapeutic approach for advanced neuroblastoma.
K Tabata, Kou Motani, N Takayanagi, R Nishimura, S Asami, Y Kimura, M Ukiya, D Hasegawa, T Akihisa and T Suzuki : Xanthoangelol, a major chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma and leukemia cells, Biological & Pharmaceutical Bulletin, Vol.28, No.8, 1404-1407, 2005.
(Summary)
Xanthoangelol, a major chalcone constituent of the stem exudates of Angelica keiskei, was evaluated for cell toxicity and apoptosis-inducing activity in human neuroblastoma (IMR-32) and leukemia (Jurkat) cells. Xanthoangelol concentration-dependently reduced the survival rates of both cell lines as revealed by the trypan blue exclusion test. Early apoptosis induced by 4 h incubation with xanthoangelol was detected using flow cytometry after double-staining with annexin V and propidium iodide (PI). Western blot analysis showed that xanthoangelol markedly reduced the level of precursor caspase-3 and increased the level of cleaved caspase-3, but Bax and Bcl-2 proteins were not affected. These results suggest that xanthoangelol induces apoptotic cell death by activatation of caspase-3 in neuroblastoma and leukemia cells through a mechanism that does not involve Bax/Bcl-2 signal transduction. Therefore, xanthoangelol may be applicable as an effective drug for treatment of neuroblastoma and leukemia.
Shunya Yamauchi, Noriko Saito-Tarashima, Kou Motani, Hidetaka Kosako and Noriaki Minakawa : Synthesis of cyclic dinucleotide analog enhanced membrane permeability, 15h International Symposium on Nanomedicine (ISNM2022), Dec. 2022.
2.
Kou Motani and Hidetaka Kosako : BioID-based Screening Of Biotinylation Sites Using Tamavidin 2-REV Globally Identifies Interactors Of Stimulator Of Interferon Genes (STING), ASCB | EMBO 2019 Meeting, Washington DC, USA,, Dec. 2019.
3.
Kou Motani and Hidetaka Kosako : BioID-Based Screening of Biotinylation Sites Globally Identifies STING Interactors, Keystone Symposia "Proteomics and its Application to Translational and Precision Medicine, Stockholm, Sweden,, Apr. 2019.
4.
Kou Motani, Mayumi Kajimoto and Hidetaka Kosako : Identification of the ATG8 family member GABARAPL2 as a novel TBK1 substrate, 12th International Symposium of the Institute Network, Nov. 2017.
Kou Motani, Noriko Saito-Tarashima, K Nishino, Shunya Yamauchi, Noriaki Minakawa and Hidetaka Kosako : ACBD3 forms specialized ER-Golgi contact sites to drive the ER exit of STING., The 17th International Symposium of the Institute Network, Kanazawa, Oct. 2022.