Biology subjects

Nakai, Y.

Publications and source records attributed to Nakai, Y..

4 recordsLinked to original sources

bioRxiv2025

Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

ZIP14/SLC39A14, a membrane-bound metal transporter, is essential for systemic metal homeostasis and has been implicated in inflammatory and metabolic disorders, including cancer-associated cachexia. Despite its biological and therapeutic significance, no selective inhibitors have been identified. Here, we identify 1-phenyl-8-(2-phenylethyl)-1,3,8-triazaspiro[4.5]decan-4-one (PPTD) as the first selective small-molecule inhibitor of ZIP14. PPTD efficiently blocks ZIP14-mediated uptake of zinc, iron, manganese, and cadmium, while sparing the closely related transporter ZIP8/SLC39A8. Mechanistically, PPTD binds specifically to a pocket formed at the dimer interface of ZIP14, as revealed by AlphaFold3 structural prediction, ligand-interaction profiling, structure-activity analyses, and site-directed mutagenesis, providing direct evidence for a targeted inhibition mechanism. ZIP14-driven metal influx promotes reactive oxygen species and lipid peroxidation, leading to cytotoxicity, which PPTD effectively reverses. In vivo, PPTD ameliorates major features of cancer cachexia in mice, including weight loss, reduced survival, muscle wasting, impaired locomotor activity, and disease progression. PPTD thus provides both a chemical probe to dissect ZIP14 function and a potential therapeutic candidate for cancer cachexia, establishing a foundation for the development of therapies targeting ZIP14-mediated metal dysregulation.

biochemistry↗

bioRxiv2025

SpyRing-Mediated Cyclization of TEV Protease, Guided by AlphaFold, Improves Thermostability

Cyclization is a promising strategy to enhance protein stability, but its applicability is often limited by structural constraints such as the distance between terminal regions. Here, we report the rational design and characterization of a cyclized Tobacco Etch Virus protease (cTEVp) using the SpyRing system, which enables covalent cyclization through SpyTag/SpyCatcher-mediated isopeptide bond formation. We applied this approach to a widely used engineered TEVp variant (L56V, S135G, S219V, {Delta}238-242) and employed AlphaFold structure prediction to optimize linker length and domain positioning. Despite the ~40 [A] separation between the N- and C-termini of native TEVp, AlphaFold modeling suggested that the fused SpyTag and SpyCatcher domains can adopt a favorable configuration for intramolecular cyclization. The resulting cTEVp exhibited proteolytic activity comparable to the non-cyclized TEVp, indicating that structural constraint via SpyRing-mediated cyclization did not impair enzymatic function. Importantly, cTEVp displayed significantly improved thermostability relative to its non-cyclized counterpart, as demonstrated by higher retention of soluble enzyme and residual activity following heat treatment at 50{degrees}C. Our findings validate the effectiveness of SpyRing-mediated cyclization in improving TEVp stability and highlight the utility of computationally guided cyclization as a generalizable strategy for engineering thermally resilient proteins. This study establishes a framework that integrates structure prediction and rational protein engineering, contributing to the development of robust biocatalysts for synthetic biology and industrial biotechnology applications. Table of Contents (TOC) / Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/646106v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@4efc7eorg.highwire.dtl.DTLVardef@1a2eab8org.highwire.dtl.DTLVardef@e00b2forg.highwire.dtl.DTLVardef@fe78a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Progressive spreading of DNA methylation in the GSTP1 promoter CpG island across transitions from precursors to invasive prostate cancer

Glutathione S-transferase pi 1 (GSTP1) is lowly expressed in normal prostate luminal cells and becomes induced in most proliferative inflammatory atrophy lesions (PIA). GSTP1 becomes silenced in prostatic intraepithelial neoplasia (PIN) and prostate adenocarcinoma (CaP) via cytosine-phospho-guanine (CpG) island promoter hypermethylation. However, GSTP1 methylation patterns in PIA and PIN, and their relationship to patterns in CaP are poorly understood. We used bisulfite genomic sequencing to examine patterns of GSTP1 promoter CpG island methylation in laser capture microdissected benign, PIA, PIN, and CaP regions from 32 subjects that underwent radical prostatectomy. We analyzed 908 sequence clones across 24 normal epithelium, 37 PIA, 18 PIN, and 23 CaP regions, allowing assessment of 34,863 CpG sites with allelic phasing. Normal and PIA lesions were mostly unmethylated with 0.52 and 1.3% of total CpG sites methylated, respectively. PIN and CaP lesions had greater methylation with 24% and 51% of total CpG sites methylated, respectively. The degree of GSTP1 methylation showed progression from PIA << PIN < CaP. PIN lesions showed more partial methylation compared to CaP lesions. Partially methylated lesions were enriched for methylation changes at AP1 and SP1 transcription factor binding sites. These results demonstrate that methylation density in the GSTP1 CpG island in PIN was intermediate relative to that in normal prostate epithelium/PIA and CaP lesions. These results are consistent with gradual spreading of DNA methylation centered at the SP1/AP1 transcription factor binding sites in precursor lesions, with subsequent spreading of methylation across the entire CpG island in transition to CaP.

cancer biology↗

bioRxiv2019

Identification of bacteria involved in the decomposition of lignocellulosic biomass treated with cow rumen fluid by metagenomic analysis

Previously, pretreatment of plant biomass using rumen fluid systems was developed to decompose cell wall. However, microbes which involved in plant cell wall decomposition in this system have not been identified, because the conditions of this system are different from the in situ rumen environment. We investigated the bacteria involved in the decomposition of cellulose and hemicellulose in a waste paper with the rumen pretreatment system using shotgun metagenomic analysis with next generation sequencing. After pretreatment of waste paper, about a half of the cellulose and hemicellulose content was decomposed. Genes encoding for cellulase and hemicellulase were mainly found to belonging to Ruminococcus, Clostridium, and Exiguobacterium. This study shows that Clostridium and Exiguobacterium, which have not been identified as predominant genus involved in cellulose and hemicellulose decomposition, might be categorized as the main fibrolytic bacteria in this system.

microbiology↗