Biology subjects

Miyazawa, T.

Publications and source records attributed to Miyazawa, T..

6 recordsLinked to original sources

bioRxiv2026

Acute Metabolic Switch by High-Dose Dehydroascorbic Acid: Rapid TCA Cycle Depletion and Early Signature of Methionine Trap within 1 Hour in KRAS-Mutant Cells

Dehydroascorbic acid (DHA), the oxidized form of vitamin C, exhibits specific toxicity against KRAS-mutant cancer cells; however, the early metabolic reprogramming mechanisms leading to cell death remain incompletely understood. In this study, we reveal the non-linear, dose-dependent response of the intracellular metabolome during the ultra-early phase of 1-hour DHA exposure. Under 1 mM DHA exposure, an early significant accumulation of the primary catabolite erythrulose (p = 0.030) and moderate consumption of glutamate (p = 0.0086) were observed, while core TCA cycle homeostasis and cell viability were preserved. In contrast, under 10 mM DHA exposure, an acute and profound alteration of the metabolic profile was observed. Along with the significant accumulation of glycerate, indicative of a glycolytic blockade, and the terminal peroxidation product oxalate, core TCA cycle intermediates such as -ketoglutarate and malate showed rapid depletion (Log2FC < -2.0, p < 10-7). Furthermore, coupled with this acute energy depletion, a statistically significant accumulation of free methionine (Log2FC = 1.14, p = 0.011), which was below the limit of detection in the control, was identified alongside profound depletions of glutamate (p < 0.001) and cysteine (p = 0.0027). These results strongly suggest that the initial response to high-dose DHA is not merely a linear accumulation of oxidative stress, but rather an "abrupt metabolic switch" characterized by rapid TCA cycle dysfunction followed by the early signature of a methionine trap.

cancer biology↗

bioRxiv2024

Establishment of a Chikungunya virus pseudotype system strictly dependent on viral protein expression

Chikungunya virus (CHIKV) is an enveloped RNA virus that causes Chikungunya fever in humans. It is classified into the arboviruses (arthropod-borne viruses) and is transmitted by mosquitoes. Therefore, mosquitoes can replicate many types of cells derived from mammals or insects. In this study, we tried to establish the widely useable Chikungunya virus pseudotype-system adapting various viral species, and we demonstrated the production of Chikungunya pseudotype virus baring the envelope protein from two different viral families, Coronaviridae or Rhabdoviridae i.e., severe acute respiratory syndrome coronavirus 2 spike protein (CoV-2-S) or vesicular stomatitis virus glycoprotein (VSV-G), respectively. We found that the capsid protein of Chikungunya virus is not always necessary in the formation of Chikungunya virus-based pseudotypes, but that the capsid protein increases the efficiency of expression of the sub-genomic RNA which codes the labeled genes. Our established pseudotype virus-producing system supplied a sufficient titer of virions for application to most virological experiments that showed more than 104 focus forming units (FFU)/ml. The pseudotype infections were strictly dependent on compatibility between the viral envelope protein and its receptor and there was no false-positive background infection. Our established pseudotype virus system can be used as a robust platform to study various virus infections and for screening and in-depth evaluation of neutralizing antibodies and antiviral agents.

molecular biology↗

bioRxiv2023

Crystal structures reveal the framework of cis-acyltransferase modular polyketide synthases

Although the domains of cis-acyltransferase (cis-AT) modular polyketide synthases (PKSs) have been understood at atomic resolution for over a decade, the domain-domain interactions responsible for the architectures and activities of these giant molecular assembly lines remain largely uncharacterized. The multimeric structure of the 6{beta}6 fungal fatty acid synthase (FAS) provides 6 equivalent reaction chambers for its acyl carrier protein (ACP) domains to shuttle carbon building blocks and the growing acyl chain between surrounding, oriented enzymatic domains. The presumed homodimeric oligomerization of cis-AT assembly lines is insufficient to provide similar reaction chambers; however, the crystal structure of a ketosynthase (KS)+AT didomain presented here and three already reported show an interaction between the AT domains appropriate for lateral multimerization. This interaction was used to construct a framework for the pikromycin PKS from its KS, AT, and docking domains that contains highly-ordered reaction chambers. Its AT domains also mediate vertical interactions, both with upstream KS domains and downstream docking domains.

biochemistry↗

bioRxiv2022

Dynamic evolution of retroviral envelope genes in egg-laying mammalian genomes

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino-acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

genomics↗

bioRxiv2022

Monotreme-specific conserved proteins derived from retroviral reverse transcriptase

Endogenous retroviruses (ERVs) have played an essential role in the evolution of mammals. Many ERV-derived genes are reported in the therians that are involved in placental development. However, the contribution of the ERV-derived genes in monotremes, which are oviparous mammals, remains to be uncovered. Here, we conducted a comprehensive search for possible ERV-derived genes in platypus and echidna genomes and identified three reverse transcriptase-like genes named RTOM1, 2, and 3. They were found to be clustered in the GRIP2 intron. Phylogenetic analysis revealed that RTOM1, 2, and 3 are strongly conserved between these species, and they were generated by tandem duplications before the divergence of platypus and echidna. The RTOM transcripts were specifically expressed in the testis, suggesting the physiological importance of RTOM genes. This is the first study reporting monotreme-specific de novo gene candidates derived from ERVs, which provides new insights into the unique evolution of monotremes.

genomics↗

bioRxiv2021

An ancient retroviral RNA element hidden in mammalian genomes and its involvement in co-opted retroviral gene regulation

Retroviruses utilize multiple unique RNA elements to control several aspects of RNA processing, such as splicing, subcellular export, and translation. However, it is mostly unclear whether such functional RNA elements are present in endogenous retroviruses (ERVs), many of which were inserted into the host genomes millions of years ago. Previously, in human ERV-derived syncytin-1 gene, we found a cis-acting RNA element named SPRE that enhances its protein expression. In this study, we found a 17-nt common sequence in SPRE of syncytin-1 and another ERV-derived gene, syncytin-2, and the sequence is confirmed to be essential for the expression of the proteins. We detected the sequences of SPRE-like elements in 41 ERV families. Though the SPRE-like elements were not found in currently prevailing (i.e. exogenous) viral sequences, more than thousands of copies of the elements were found in several mammalian genomes, suggesting the ancient integration and propagation of the SPRE-harboring retroviruses in mammalian lineages. Indeed, other mammalian ERV-derived genes: mac-syncytin-3 of macaque, syncytin-Ten1 of tenrec, and syncytin-Car1 of Carnivora contain the SPRE-like elements, and we validated their function for efficient protein expression by in vitro assays. A reporter assay revealed that the enhancement of gene expression by SPRE depended on reporter genes. Moreover, the mutation in SPRE did not affect the gene expression in codon-optimized syncytin-2. However, the same mutation in SPRE impaired the gene expression in wild-type syncytin-2, suggesting that the SPRE dependency of Syncytin-2 expression is due to the negative factors such as inefficient codon frequency or repressive elements within the coding sequence. These results provide new implications that ERVs harbor unique RNA elements involved in the regulation of ERV-derived genes.

molecular biology↗