Tissue-specific roles of GCN2 in aging and autosomal dominant retinitis pigmentosa.

The organisms have the capacity to sense and adapt to their surroundings for their life in a dynamic environment. In response to amino acid starvation, cells activate a rectifying physiological program, termed the integrated stress response (ISR), to restore cellular homeostasis. General controlled non-repressed (GCN2) kinase is a master regulator of the ISR and modulates protein synthesis in response to amino acid starvation. We previously established the GCN2/ATF4/4E-BP pathway in development and aging. Here, we investigated the tissue-specific roles of GCN2 upon dietary restriction of amino acid in a Drosophila model. The knockdown of GCN2 in the gut and fat body, an energy sensing organ in Drosophila, abolished the beneficial effect of GCN2 in lifespan extension upon dietary restriction of amino acids. Proteome analysis in an autosomal dominant retinitis pigmentosa (ADRP) model showed that dietary restriction of amino acids regulates the synthesis of proteins in several pathways, including mitochondrial translation, mitochondrial gene expression, and regulation of biological quality, and that gcn2-mutant flies have reduced levels of these mitochondria-associated proteins, which may contribute to retinal degeneration in ADRP. These results indicate that the tissue-specific regulation of GCN2 contributes to normal physiology and ADRP progression.

Synthetic Extracellular Matrix of Polyvinyl Alcohol Nanofibers for Three-Dimensional Cell Culture.

An ideal extracellular matrix (ECM) replacement scaffold in a three-dimensional cell (3D) culture should induce in vivo-like interactions between the ECM and cultured cells. Highly hydrophilic polyvinyl alcohol (PVA) nanofibers disintegrate upon contact with water, resulting in the loss of their fibrous morphology in cell cultures. This can be resolved by using chemical crosslinkers and post-crosslinking. A crosslinked, water-stable, porous, and optically transparent PVA nanofibrous membrane (NM) supports the 3D growth of various cell types. The binding of cells attached to the porous PVA NM is low, resulting in the aggregation of cultured cells in prolonged cultures. PVA NMs containing integrin-binding peptides of fibronectin and laminin were produced to retain the blended peptides as cell-binding substrates. These peptide-blended PVA NMs promote peptide-specific cell adherence and growth. Various cells, including epithelial cells, cultured on these PVA NMs form layers instead of cell aggregates and spheroids, and their growth patterns are similar to those of the cells cultured on an ECM-coated PVA NM. The peptide-retained PVA NMs are non-stimulatory to dendritic cells cultured on the membranes. These peptide-retaining PVA NMs can be used as an ECM replacement matrix by providing in vivo-like interactions between the matrix and cultured cells.

The Function of Drosophila USP14 in Endoplasmic Reticulum Stress and Retinal Degeneration in a Model for Autosomal Dominant Retinitis Pigmentosa.

Endoplasmic reticulum (ER) stress and its adaptive cellular response, the unfolded protein response (UPR), are involved in various diseases including neurodegenerative diseases, metabolic diseases, and even cancers. Here, we analyzed the novel function of ubiquitin-specific peptidase 14 (USP14) in ER stress. The overexpression of Drosophila USP14 protected the cells from ER stress without affecting the proteasomal activity. Null Hong Kong (NHK) and alpha-1-antitrypsin Z (ATZ) are ER-associated degradation substrates. The degradation of NHK, but not of ATZ, was delayed by USP14. USP14 restored the levels of rhodopsin-1 protein in a Drosophila model for autosomal dominant retinitis pigmentosa and suppressed the retinal degeneration in this model. In addition, we observed that proteasome complex is dynamically reorganized in response to ER stress in human 293T cells. These findings suggest that USP14 may be a therapeutic strategy in diseases associated with ER stress.