Language：English   Publishing type：Research paper (scientific journal)  

Extracellular vesicles (EVs) play important roles in intercellular communication in various biological events. In particular, EVs released from cancer cells have attracted special attention. Although it has been reported that cancer-associated glycosphingolipids play important roles in the enhancement of malignant properties of cancer cells, the presence, behavior, and roles of glycosphingolipids in EVs have not been elucidated. Recently, we reported crucial roles of EVs expressing gangliosides, GD2, and/or GD3 in the enhancement of cancer properties in malignant melanomas and gliomas. However, how EVs containing cancer-associated glycosphingolipids play their roles has not been reported to date. Here, we studied spatio-temporal mechanisms for GD3/GD2-containing EVs released from gliomas in the actions toward target cells. Proteome analyses of EVs with/without GD3/GD2 revealed an equally high concentration of integrin isoforms in both GD3/GD2+ and GD3/GD2- EVs. PKH26-labeled EVs attached, invaded, and distributed to/in the target cells within 1 h. GD3/GD2 formed molecular complexes with integrins on EVs as elucidated by immunoprecipitation/immunoblotting and immunocytostaining. The addition of antibodies reactive with GD3, GD2, or integrins resulted in the suppression of the enhancing effects of EVs in the cell adhesion assay. The addition of GD3/GD2 + EVs to GD3/GD2- cells clearly increased the phosphorylation levels of the PDGF receptor, FAK, and Erk1/2 in immunoblotting, suggesting GD3/GD2+ EVs activate the signaling pathway in the target cells within 15 min after addition. Anti-ganglioside antibodies clearly blocked signaling with EVs. In conclusion, EVs released from GD3/GD2-expressing glioma cells enhance cancer phenotypes and malignant signals via the cluster formation of integrins and GD3/GD2 on EVs, leading to the regulation of the cancer microenvironment.

DOI： 10.3390/ijms252312752

Open Access

Scopus

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acschembio.4c00432

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Background

Thyroid hormones (TH) regulate the basal metabolic rate through their receptors THRα and THRβ. TH activates lipid metabolism via THRβ, however, an excess amount of TH can lead to tachycardia, bone loss, and muscle wasting through THRα. In recent years, TH analogs that selectively bind to THRβ have gained attention as new agents for treating dyslipidemia and obesity, which continue to pose major challenges to public health worldwide.

Methods

We developed a TH analog, ZTA-261, by modifying the existing THRβ-selective agonists GC-1 and GC-24. To determine the THRβ-selectivity of ZTA-261, an in vitro radiolabeled TH displacement assay was conducted. ZTA-261 was intraperitoneally injected into a mouse model of high-fat diet-induced obesity, and its effectiveness in reducing body weight and visceral fat, and improving lipid metabolism was assessed. In addition, its toxicity in the liver, heart, and bone was evaluated.

Results

ZTA-261 is more selective towards THRβ than GC-1. Although ZTA-261 is less effective in reducing body weight and visceral fat than GC-1, it is as effective as GC-1 in reducing the levels of serum and liver lipids. These effects are mediated by the same pathway as that of T₃, a natural TH, as evidenced by similar changes in the expression of TH-induced and lipid metabolism-related genes. The bone, cardiac, and hepatotoxicity of ZTA-261 are significantly lower than those of GC-1.

Conclusions

ZTA-261, a highly selective and less toxic THRβ agonist, has the potential to be used as a drug for treating diseases related to lipid metabolism.

DOI： 10.1038/s43856-024-00574-z

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Other Link： https://www.nature.com/articles/s43856-024-00574-z

DOI： 10.1101/2024.07.07.602439

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Language：English   Publisher：Japanese Society for Biomedical Mass Spectrometry  

<p>Liquid biopsies mainly analyze nucleic acids and proteins in the free-state or extracellular vesicles (EVs) in non-solid biological samples, primarily blood. Collecting and processing liquid biopsy samples is challenging due to the large volume of samples and reagents and the need for special equipment. In a previous study, we reported a method for enriching free nucleic acids using a polyamine solution that is effective for liquid biopsy. We also investigated the reactivity of this method for EV recovery in cell culture supernatants using mass spectrometry. Samples were prepared from the cell line NCI-N87 supernatants after 48 h of culture in a serum-free medium. For comparison, samples were treated using a solution containing polyamines (PA method) or ultracentrifugation (UC method). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the single-pot solid-phase-enhanced sample-preparation (SP3) method revealed differences between the two methods in the total ion chromatogram of the sample. However, the results of the Gene Ontology (GO) analysis showed that both methods achieved the best enrichment in GO terms related to EV. In addition, the volcano plot analysis revealed that proteins suggested to exist in EVs were distributed in areas consistent with both methods. These results indicated that the PA method can recover EV proteins in liquid samples, and their comprehensive analysis is possible using the SP3 method.</p>

DOI： 10.24508/mms.2024.06.006

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. Microalbuminuria is currently the most common non-invasive biomarker for the early diagnosis of DN. However, renal structural damage may have advanced when albuminuria is detected. In this study, we sought biomarkers for early DN diagnosis through proteomic analysis of urinary extracellular vesicles (uEVs) from type 2 diabetic model rats and normal controls. Isocitrate dehydrogenase 1 (IDH1) was significantly increased in uEVs from diabetic model rats at the early stage despite minimal differences in albuminuria between the groups. Calorie restriction significantly suppressed the increase in IDH1 in uEVs and 24-hour urinary albumin excretion, suggesting that the increase in IDH1 in uEVs was associated with the progression of DN. Additionally, we investigated the origin of IDH1-containing uEVs based on their surface sugar chains. Lectin affinity enrichment and immunohistochemical staining showed that IDH1-containing uEVs were derived from proximal tubules. These findings suggest that the increase in IDH1 in uEVs reflects pathophysiological alterations in the proximal tubules and that IDH1 in uEVs may serve as a potential biomarker of DN in the proximal tubules.

DOI： 10.1096/fj.202400371R

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Language：English   Publishing type：Research paper (scientific journal)  

The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production with reduced biohazard concerns in future biological endeavors.

DOI： 10.1038/s41467-024-48056-z

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<jats:p>Lipid droplets (LDs) are lipid storage organelles in plant leaves and seeds. Seed LD proteins are well known, and their functions in lipid metabolism have been characterized; however, many leaf LD proteins remain to be identified. We therefore isolated LDs from leaves of the leaf LD–overaccumulating mutant <jats:italic>high sterol ester 1</jats:italic> (<jats:italic>hise1</jats:italic>) of <jats:italic>Arabidopsis thaliana</jats:italic> by centrifugation or co-immunoprecipitation. We then performed LD proteomics by mass spectrometry and identified 3,206 candidate leaf LD proteins. In this study, we selected 31 candidate proteins for transient expression assays using a construct encoding the candidate protein fused with green fluorescent protein (GFP). Fluorescence microscopy showed that MYOSIN BINDING PROTEIN14 (MYOB14) and two uncharacterized proteins localized to LDs labeled with the LD marker. Subcellular localization analysis of MYOB family members revealed that MYOB1, MYOB2, MYOB3, and MYOB5 localized to LDs. LDs moved along actin filaments together with the endoplasmic reticulum. Co-immunoprecipitation of myosin XIK with MYOB2-GFP or MYOB14-GFP suggested that LD-localized MYOBs are involved in association with the myosin XIK–LDs. The two uncharacterized proteins were highly similar to enzymes for furan fatty acid biosynthesis in the photosynthetic bacterium <jats:italic>Cereibacter sphaeroides</jats:italic>, suggesting a relationship between LDs and furan fatty acid biosynthesis. Our findings thus reveal potential molecular functions of LDs and provide a valuable resource for further studies of the leaf LD proteome.</jats:p>

DOI： 10.3389/fpls.2024.1331479

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Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 μM, 1 μM, and 2 μM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 μM) as a substrate (18.1, 14.9, and 6.43 μM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

DOI： 10.1016/j.jchromb.2024.123993

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Language：English   Publisher：Japanese Proteomics Society  

<p>Analysis of the peptides and proteins synthesized in a cell-free translation system containing either the natural tRNA extract, the hybrid-SL tRNA set, or the hybrid-SL tRNA set minus one chimeric tRNA using LC-MS/MS.</p>

DOI： 10.14889/jpdm.2024.0022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Abstract

Desmoid tumors (DTs), also called desmoid‐type fibromatoses, are locally aggressive tumors of mesenchymal origin. In the present study, we developed a novel mouse model of DTs by inducing a local mutation in the Ctnnb1 gene, encoding β‐catenin in PDGFRA‐positive stromal cells, by subcutaneous injection of 4‐hydroxy‐tamoxifen. Tumors in this model resembled histologically clinical samples from DT patients and showed strong phosphorylation of nuclear SMAD2. Knockout of SMAD4 in the model significantly suppressed tumor growth. Proteomic analysis revealed that SMAD4 knockout reduced the level of Cysteine‐and‐Glycine‐Rich Protein 2 (CSRP2) in DTs, and treatment of DT‐derived cells with a TGF‐β receptor inhibitor reduced CSRP2 RNA levels. Knockdown of CSRP2 in DT cells significantly suppressed their proliferation. These results indicate that the TGF‐β/CSRP2 axis is a potential therapeutic target for DTs downstream of TGF‐β signaling.

DOI： 10.1111/cas.16037

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Language：English   Publishing type：Research paper (scientific journal)  

Cancer cachexia is a complex metabolic disorder accounting for ~20% of cancer-related deaths, yet its metabolic landscape remains unexplored. Here, we report a decrease in B vitamin-related liver enzymes as a hallmark of systemic metabolic changes occurring in cancer cachexia. Metabolomics of multiple mouse models highlights cachexia-associated reductions of niacin, vitamin B6, and a glycine-related subset of one-carbon (C1) metabolites in the liver. Integration of proteomics and metabolomics reveals that liver enzymes related to niacin, vitamin B6, and glycine-related C1 enzymes dependent on B vitamins decrease linearly with their associated metabolites, likely reflecting stoichiometric cofactor-enzyme interactions. The decrease of B vitamin-related enzymes is also found to depend on protein abundance and cofactor subtype. These metabolic/proteomic changes and decreased protein malonylation, another cachexia feature identified by protein post-translational modification analysis, are reflected in blood samples from mouse models and gastric cancer patients with cachexia, underscoring the clinical relevance of our findings.

DOI： 10.1038/s41467-023-41952-w

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acschembio.3c00046

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Language：English   Publishing type：Research paper (scientific journal)  

Stomata are pores in the leaf epidermis of plants and their opening and closing regulate gas exchange and water transpiration. Stomatal movements play key roles in both plant growth and stress responses. In recent years, small molecules regulating stomatal movements have been used as a powerful tool in mechanistic studies, as well as key players for agricultural applications. Therefore, the development of new molecules regulating stomatal movement and the elucidation of their mechanisms have attracted much attention. We herein describe the discovery of 2,6-dihalopurines, AUs, as a new stomatal opening inhibitor, and their mechanistic study. Based on biological assays, AUs may involve in the pathway related with plasma membrane H+-ATPase phosphorylation. In addition, we identified leucine-rich repeat extensin proteins (LRXs), LRX3, LRX4 and LRX5 as well as RALF, as target protein candidates of AUs by affinity based pull down assay and molecular dynamics simulation. The mechanism of stomatal movement related with the LRXs-RALF is an unexplored pathway, and therefore further studies may lead to the discovery of new signaling pathways and regulatory factors in the stomatal movement.

DOI： 10.1021/acschembio.2c00771

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/sscp.202200121

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for Cancer Research ({AACR})  

<jats:title>Abstract</jats:title>
<jats:p>Metastasis is responsible for the majority of deaths of cancer patients. However, mechanisms governing metastasis in colorectal cancer (CRC) remain largely unknown. Here we investigated how CRC cells acquire metastatic potential using a novel mouse model of CRC that spontaneously develops liver metastasis, generated by introducing sporadic mutations of Ctnnb1, Kras, Trp53, and Smad4 (CKPS) genes. Proteomic analyses revealed elevated expression of CRC stem cell markers ALCAM (CD166) and PROM1 (CD133) in CRC cells from the metastatic model compared with those from a non-metastatic model. Spleen-to-liver metastasis assays using CRC cells derived from the CKPS model (CKPS cells) demonstrated the functional importance of ALCAM and PROM1 in initiating metastasis. Genetic and pharmacological analyses using CKPS cells in 2D and spheroid culture revealed that expression of ALCAM and PROM1 is regulated positively and negatively by the cAMP/PKA/CREB and TGF-β/SMAD4 pathways, respectively. Consistently, phospho-CREB was expressed in both primary and metastatic lesions of CKPS mice and CRC patients, and knockout of CREB in CKPS cells reduced their spheroid-forming and metastasis-initiating abilities. Treatment with a CREB inhibitor potentiated the effect of irinotecan in suppressing liver metastasis by CKPS cells. These results reveal the essential roles of ALCAM and PROM1, as well as their upstream regulators, the cAMP/PKA/CREB and TGF-β/SMAD4 pathways, in maintaining the stemness and metastatic potential of CRC cells and indicate that CREB inhibition may be a potential therapeutic strategy against metastatic CRC.</jats:p>

DOI： 10.1158/0008-5472.can-22-1369

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Language：English   Publishing type：Research paper (scientific journal)  

Malignant mesothelioma (MM) is an aggressive tumor that typically develops after a long latency following asbestos exposure. Although mechanistic target of rapamycin complex 1 (mTORC1) activation enhances MM cell growth, the mTORC1 inhibitor everolimus has shown limited efficacy in clinical trials of MM patients. We explored the mechanism underlying mTORC1 activation in MM cells and its effects on cell proliferation and progression. Analysis of the expression profiles of 87 MMs from The Cancer Genome Atlas revealed that 40 samples (46%) displayed altered expression of RPTOR (mTORC1 component) and genes immediately upstream that activate mTORC1. Among them, we focused on RHEB and RHEBL1, which encode direct activators of mTORC1. Exogenous RHEBL1 expression enhanced MM cell growth, indicating that RHEB-mTORC1 signaling acts as a pro-oncogenic cascade. We investigated molecules that directly activate RHEBs, identifying SmgGDS as a novel RHEB-binding protein. SmgGDS knockdown reduced mTORC1 activation and inhibited the proliferation of MM cells with mTORC1 activation. Interestingly, SmgGDS displayed high binding affinity with inactive GDP-bound RHEBL1, and its knockdown reduced cytosolic RHEBL1 without affecting its activation. These findings suggest that SmgGDS retains GDP-bound RHEBs in the cytosol, whereas GTP-bound RHEBs are localized on intracellular membranes to promote mTORC1 activation. We revealed a novel role for SmgGDS in the RHEB-mTORC1 pathway and its potential as a therapeutic target in MM with aberrant mTORC1 activation. IMPLICATIONS: Our data showing that SmgGDS regulates RHEB localization to activate mTORC1 indicate that SmgGDS can be used as a new therapeutic target for MM exhibiting mTORC1 activation.

DOI： 10.1158/1541-7786.MCR-20-0637

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Language：English   Publisher：(一社)日本癌学会  

J-GLOBAL

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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