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Biology subjects

Nakashima, R.

Publications and source records attributed to Nakashima, R..

3 recordsLinked to original sources

Dietary lipids attenuate IGF-1-Akt and injure epithelial-endothelial injury program that accelerates obstructive lung disease

Background Altered lipid metabolism is increasingly implicated in chronic obstructive pulmonary disease (COPD), but it remains unclear how a pre-existing obstructive lung state modifies the response to systemic lipid excess. We investigated whether high-fat diet (HFD) amplifies COPD-relevant lung injury and examined epithelial and vascular programs associated with this response. Methods Male wild-type (WT) and beta-epithelial sodium channel-transgenic ({beta}ENaC-Tg) mice were fed control diet or HFD for 10-11 weeks. Lung structure and function, whole-lung transcriptomes, Akt-FOXO1 signaling, apoptosis-related responses, and pulmonary vascular profiles were assessed. Streptozotocin-induced insulin-deficient diabetes and pharmacological IGF-1 receptor inhibition were used as mechanistic comparators. Palmitate responses were examined in human bronchial epithelial cells, ENaC-hyperactive epithelial cells, and endothelial cells, including conditioned-medium transfer. HFD preconditioning was also evaluated in an elastase-induced emphysema model. Statistical analyses included unpaired two-tailed Student's t tests, one-way ANOVA with Tukey-Kramer or Dunnett multiple-comparison testing, Pearson correlation, and Benjamini-Hochberg correction for RNA-sequencing analyses. Results HFD produced similar increases in body weight, glycemia, and adiposity in WT and {beta}ENaC-Tg mice, while further increasing distal-airspace enlargement and reducing FEV0.1/FVC in {beta}ENaC-Tg mice. Lung transcriptomics revealed coordinated remodeling of lipid metabolic, PI3K-Akt, and vascular programs. HFD reduced Akt phosphorylation, increased FOXO1 and Fasl, and increased TUNEL-positive cells in epithelial regions. Palmitate attenuated IGF-1-induced Akt activation in bronchial epithelial cells, whereas IGF-1 receptor inhibition reproduced Akt suppression and apoptosis-related responses without fully reproducing the HFD phenotype. HFD preconditioning also increased elastase-induced airspace enlargement, accompanied by parallel upregulation of FOXO1 and TUNEL positivity. HFD reduced pulmonary CD34-positive vascular profiles, and palmitate activated endothelial cells directly and through conditioned media from ENaC-hyperactive epithelium. In men with airflow obstruction, hepatic steatosis coincided with lower percent-predicted FEV1. Conclusions Dietary lipid stress amplifies obstructive lung injury and engages complementary epithelial and vascular responses. Impaired epithelial IGF-1-Akt signaling and epithelial-endothelial crosstalk provide a mechanistic framework linking systemic metabolic stress to reduced resilience of the obstructive lung.

molecular biology↗

Structural basis of nucleotide selectivity in pyruvate kinase

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

biochemistry↗

Development of a cellular reporter assay to measure activity of MutSβ, a therapeutic target for Huntington's disease

Genetic modifiers of age of onset in Huntingtons disease (HD) provide compelling evidence that somatic expansion of the CAG repeats is a critical driver of pathogenesis and demonstrate that repeat instability is modulated by DNA mismatch repair (MMR). A component of this pathway, MutS{beta}, a heterodimer comprised of MSH2 and MSH3, has emerged as a potential target for small-molecule therapeutic intervention. However, a robust cellular assay to interrogate genetic and pharmacological modifiers of MutS{beta} has not been reported. We have repurposed and optimized a tetranucleotide reporter assay to measure MutS{beta} activity in MMR-competent cells. We show that repeat instability is modulated by MSH3 protein levels and by its ATPase activity. In addition, we show that an inhibitor of HDAC3 modulates repeat instability, demonstrating the utility of the assay for pharmacological studies.

neuroscience↗