Interaction between IDH1 WT and calmodulin and its implications for glioblastoma cell growth and migration

Isocitrate dehydrogenase (IDH) mutations are found in low-grade gliomas, and the product of the IDH mutant (MT), 2-hydroxyglutarate (2-HG), is the first known oncometabolite. However, the roles of the IDH wild type (WT) in high-grade glioblastoma, which rarely has the IDH mutation, are still unknown. To investigate possible pathways related to IDH WT in gliomas, we carried out bioinformatics analysis, and found that IDH1 has several putative calmodulin (CaM) binding sites. Pull-down and quantitative dissociation constant (Kd) measurements using recombinant proteins showed that IDH1 WT indeed binds to CaM with a higher affinity than IDH1 R132H MT. This biochemical interaction was demonstrated also in the cellular environment by immunoprecipitation with glioblastoma cell extracts. A synthetic peptide for the suggested binding region interfered with the interaction between CaM and IDH1, confirming the specificity of the binding. Direct binding between the synthetic peptide and CaM was observed in an NMR binding experiment, which additionally revealed that the peptide initially binds to the C-lobe of CaM. The physiological meaning of the CaM-IDH1 WT binding was shown with trifluoperazine (TFP), a CaM antagonist, which disrupted the binding and inhibited survival and migration of glioblastoma cells with IDH1 WT. As CaM signaling is activated in glioblastoma, our results suggest that IDH1 WT may be involved in the CaM-signaling pathway in the tumorigenesis of high-grade gliomas. (C) 2020 Elsevier Inc. All rights reserved.Peer reviewe

2-Cys Peroxiredoxins: Emerging Hubs Determining Redox Dependency of Mammalian Signaling Networks

Mammalian cells have a well-defined set of antioxidant enzymes, which includes superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins. Peroxiredoxins are the most recently identified family of antioxidant enzymes that catalyze the reduction reaction of peroxides, such as H2O2. In particular, typical 2-Cys peroxiredoxins are the featured peroxidase enzymes that receive the electrons from NADPH by coupling with thioredoxin and thioredoxin reductase. These enzymes distribute throughout the cellular compartments and, therefore, are thought to be broad-range antioxidant defenders. However, recent evidence demonstrates that typical 2-Cys peroxiredoxins play key signal regulatory roles in the various signaling networks by interacting with or residing near a specific redox-sensitive molecule. These discoveries help reveal the redox signaling landscape in mammalian cells and may further provide a new paradigm of therapeutic approaches based on redox signaling

GPX8 regulates clear cell renal cell carcinoma tumorigenesis through promoting lipogenesis by NNMT

Background Clear cell renal cell carcinoma (ccRCC), with its hallmark phenotype of high cytosolic lipid content, is considered a metabolic cancer. Despite the implication of this lipid-rich phenotype in ccRCC tumorigenesis, the roles and regulators of de novo lipid synthesis (DNL) in ccRCC remain largely unexplained. Methods Our bioinformatic screening focused on ccRCC-lipid phenotypes identified glutathione peroxidase 8 (GPX8), as a clinically relevant upstream regulator of DNL. GPX8 genetic silencing was performed with CRISPR-Cas9 or shRNA in ccRCC cell lines to dissect its roles. Untargeted metabolomics, RNA-seq analyses, and other biochemical assays (e.g., lipid droplets staining, fatty acid uptake, cell proliferation, xenograft, etc.) were carried out to investigate the GPX8s involvement in lipid metabolism and tumorigenesis in ccRCC. The lipid metabolic function of GPX8 and its downstream were also measured by isotope-tracing-based DNL flux measurement. Results GPX8 knockout or downregulation substantially reduced lipid droplet levels (independent of lipid uptake), fatty acid de novo synthesis, triglyceride esterification in vitro, and tumor growth in vivo. The downstream regulator was identified as nicotinamide N-methyltransferase (NNMT): its knockdown phenocopied, and its expression rescued, GPX8 silencing both in vitro and in vivo. Mechanically, GPX8 regulated NNMT via IL6-STAT3 signaling, and blocking this axis suppressed ccRCC survival by activating AMPK. Notably, neither the GPX8-NNMT axis nor the DNL flux was affected by the von Hippel Lindau (VHL) status, the conventional regulator of ccRCC high lipid content. Conclusions Taken together, our findings unravel the roles of the VHL-independent GPX8-NNMT axis in ccRCC lipid metabolism as related to the phenotypes and growth of ccRCC, which may be targeted for therapeutic purposes. Graphical abstractThe research was supported by the Basic Science Research Program (grant NRF-2018R1A3B1052328 to S.P.) funded by the Ministry of Science, Information and Communication Technology, by Future Planning through the National Research Foundation, and by the Basic Science Research Program through the National Research Foundation (NRF-2020R1I1A1A01073124 to J-M.K.) funded by the Ministry of Education of Korea

Cost-of-Illness Trends Associated with Thyroid Disease in Korea

BackgroundThe purpose of this study is to analyze the scale of and trends associated with the cost-of-illness of thyroid disease in Korea at 2-year intervals during the last 10 years for which data are available.MethodsCost-of-illness was estimated in terms of direct and indirect costs. Direct costs include direct medical costs due to hospitalization, outpatient and pharmacy sectors, transportation, and care-giver costs. Indirect costs include future income loss due to premature death and loss of productivity as a result of absence from work.ResultsThe cost-of-illness of thyroid disease in Korea was estimated at 224.2 billion won in 2002, 303.4 billion won in 2004, 400.3 billion won in 2006, 570.4 billion won in 2008, and 762.2 billion won in 2010. For example, the cost-of-illness of thyroid disease in 2010 was 3.4 times greater compared to 2002. The direct cost of the total cost-of-illness was 69.7%, which accounted for the highest proportion of costs. Cost-of-illness for individuals between the ages of 30 and 50 accounted for the greatest share of costs.ConclusionThe cost-of-illness of thyroid disease was relatively large in economically active age groups, and demonstrated a very rapid growth rate compared to other major diseases in Korea. Therefore, we suggest nationwide recognition of the importance of prevention and management of thyroid disease and prioritization of the management of thyroid disease among current and future health promotion policies in Korea

In Vitro Toxicity Screening of Fifty Complex Mixtures in HepG2 Cells

To develop the risk prediction technology for mixture toxicity, a reliable and extensive dataset of experimental results is required. However, most published literature only provides data on combinations containing two or three substances, resulting in a limited dataset for predicting the toxicity of complex mixtures. Complex mixtures may have different mode of actions (MoAs) due to their varied composition, posing difficulty in the prediction using conventional toxicity prediction models, such as the concentration addition (CA) and independent action (IA) models. The aim of this study was to generate an experimental dataset comprising complex mixtures. To identify the target complex mixtures, we referred to the findings of the HBM4EU project. We identified three groups of seven to ten components that were commonly detected together in human bodies, namely environmental phenols, perfluorinated compounds, and heavy metal compounds, assuming these chemicals to have different MoAs. In addition, a separate mixture was added consisting of seven organophosphate flame retardants (OPFRs), which may have similar chemical structures. All target substances were tested for cytotoxicity using HepG2 cell lines, and subsequently 50 different complex mixtures were randomly generated with equitoxic mixtures of EC10 levels. To determine the interaction effect, we calculated the model deviation ratio (MDR) by comparing the observed EC10 with the predicted EC10 from the CA model, then categorized three types of interactions: antagonism, additivity, and synergism. Dose–response curves and EC values were calculated for all complex mixtures. Out of 50 mixtures, none demonstrated synergism, while six mixtures exhibited an antagonistic effect. The remaining mixtures exhibited additivity with MDRs ranging from 0.50 to 1.34. Our experimental data have been formatted to and constructed for the database. They will be utilized for further research aimed at developing the combined CA/IA approaches to support mixture risk assessment

Heteronuclear NMR As a 4‑in‑1 Analytical Platform for Detecting Modification-Specific Signatures of Therapeutic Insulin Formulations

Detecting possible modifications of therapeutic proteins is a critical element of the quality control of protein drugs. Typically, a number of techniques are used to evaluate different modifications of therapeutic protein formulations. Using heteronuclear NMR spectroscopy, we show that the difference between various insulin formulations can be detected “as is” with little pretreatment and quickly. As an application to the quality control of insulin formulations, the NMR approach was compared with four different analytical methods: with reverse phase high pressure liquid chromatography (HPLC) (for mutations), with size exclusion chromatography (for oligomerization), with electrophoresis (for denaturation), and with mass spectrometry (for deamidation). All of the results showed that this single NMR method can provide the specific signatures for each modification and information that is at least equivalent to that offered by the conventional analytical methods. Importantly, NMR could yield information at each amino acid residue level which no other technique provided. The suggested NMR method, then, can be considered to be a facile and effective means of evaluating therapeutic protein formulations in a multifaceted way

Revisiting Platinum-Based Anticancer Drugs to Overcome Gliomas

Although there are many patients with brain tumors worldwide, there are numerous difficulties in overcoming brain tumors. Among brain tumors, glioblastoma, with a 5-year survival rate of 5.1%, is the most malignant. In addition to surgical operations, chemotherapy and radiotherapy are generally performed, but the patients have very limited options. Temozolomide is the most commonly prescribed drug for patients with glioblastoma. However, it is difficult to completely remove the tumor with this drug alone. Therefore, it is necessary to discuss the potential of anticancer drugs, other than temozolomide, against glioblastomas. Since the discovery of cisplatin, platinum-based drugs have become one of the leading chemotherapeutic drugs. Although many studies have reported the efficacy of platinum-based anticancer drugs against various carcinomas, studies on their effectiveness against brain tumors are insufficient. In this review, we elucidated the anticancer effects and advantages of platinum-based drugs used in brain tumors. In addition, the cases and limitations of the clinical application of platinum-based drugs are summarized. As a solution to overcome these obstacles, we emphasized the potential of a novel approach to increase the effectiveness of platinum-based drugs

Highly conserved protein Rv1211 in Mycobacterium tuberculosis is a natively unfolded protein that binds to a calmodulin antagonist, trifluoperazine

Rv1211 is a conserved hypothetical protein in Mycobacterium tuberculosis and is required for the growth and pathogenesis of the bacteria. The protein has been suggested as a calmodulin-like calcium-binding protein with an EF-hand motif and as a target of trifluoperazine, a calmodulin antagonist in eukaryotes that inhibits mycobacterial growth. Here, we expressed the recombinant protein of Rv1211 and per -formed structural and biochemical studies of Rv1211 and its interaction with Ca2 thorn or trifluoperazine. Surprisingly, Rv1211 exhibited an elution property typical of a natively unfolded protein. Subsequent circular dichroism experiments with temperature elevation and trifluoroethanol treatment showed that Rv1211 has unfolded structure. Additional NMR experiment confirmed the unfolded state of the protein and further showed that it does not bind to Ca2 + . Still, Rv1211 did bind to trifluoperazine, as evidenced by the two-dimensional NMR spectra of N-15-labeled Rv1211. However, there were no peak shifts upon binding, showing that Rv1211 retained its unfolded state even after the trifluoperazine binding. The residues involved in the binding were clustered in the C-terminal region, as identified by the sequence assignment. Isothermal titration calorimetry showed that the Kd of trifluoperazine-Rv1211 binding is 41 mM and that the stoichiometry is 1 : 2 (Rv1211: trifluoperazine). Our results argue against the sug-gestion of Rv1211 as a Ca2+ -binding calmodulin-like protein, and show that Rv1211 isa natively unfolded protein that binds to trifluoperazine. In addition, our results suggest the evidence of the "Fuzziness " in the Rv1211-trifluoperazine interaction that differs from the conventional binding-induced folding of natively unfolded proteins. (C) 2022 Elsevier Inc. All rights reserved.N