Biology subjects

Kosako, H.

Publications and source records attributed to Kosako, H..

23 records · Page 2Linked to original sources

bioRxiv2022

Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

Upon infection with Toxoplasma gondii, host cells produce immune-related GTPases (IRGs) to kill the parasite. T. gondii counters this response by releasing ROP18 kinase, which inactivates IRG GTPases and inhibits their recruitment to the T. gondii parasitophorous vacuole (PV). However, the molecular mechanisms of this process are entirely unknown. Here we report the atomic structures of Irgb6 with a phosphomimetic mutation by ROP18. The mutant has lower GTPase activity and is not recruited to the PV membrane (PVM). The crystal structure shows the mutant exhibit a distinct conformation from the physiological nucleotide-free form, thus preventing GTPase cycling. This change allosterically modifies the conformation of the membrane-binding interface, preventing physiological PVM-binding. Docking simulation of PI5P also supports the impaired binding of the mutant to PVM. We thus demonstrate the structural basis for T. gondii escape from host cell-autonomous defense, and provide a structural model for regulating enzymatic activity by phosphorylation.

microbiology↗

bioRxiv2022

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides using Tamavidin 2-REV for BioID and Cell Surface Proteomics

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the low abundance of protein biotinylation in cells required large amounts of cellular proteins to detect enough biotinylated peptides. Here we optimized the workflow for efficient enrichment and identification of biotinylated peptides. The most efficient recovery was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and the optimal temperature and salt concentration for elution. Using the optimized workflow, over 2-fold more biotinylated peptides were identified with higher purity from RAW264.7 macrophages expressing TurboID-fused STING. In addition, sequential digestion with Glu-C and trypsin led to the identification of biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon EGF stimulation. This workflow would be useful not only for BioID and cell surface proteomics but also for various other applications based on protein biotinylation.

biochemistry↗

bioRxiv2021

OTUD1 deubiquitylase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways

Deubiquitylating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-{kappa}B activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-{kappa}B pathway by hydrolysing K63-linked ubiquitin chains from NF-{kappa}B signalling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-{kappa}B activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. The N-terminal intrinsically disordered region of OTUD1, which contains an EGTE motif, is indispensable for KEAP1-binding and NF-{kappa}B suppression. OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-/--mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

cell biology↗

bioRxiv2020

Peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation

Most of peroxisomal matrix proteins including a hydrogen peroxide (H2O2)-decomposing enzyme, catalase, are imported in a peroxisome-targeting signal type-1 (PTS1)-dependent manner. However, little is known about regulation of the membrane-bound protein import machinery. Here, we report that Pex14, a central component of the protein translocation complex in peroxisomal membrane, is phosphorylated in response to oxidative stresses such as H2O2 in mammalian cells. H2O2-induced phosphorylation of Pex14 at Ser232 suppresses peroxisomal import of catalase in vivo and selectively decreases formation of PTS1 receptor Pex5-mediated ternary complex of Pex14 with catalase in vitro. Phosphomimetic Pex14-S232D mutant elevates the level of cytosolic catalase, but not canonical PTS1-proteins, conferring higher cell resistance to H2O2. We thus suggest that H2O2-induced phosphorylation of Pex14 spatiotemporally regulates peroxisomal import of catalase, functioning in counteracting action against oxidative stress by the increase of cytosolic catalase.

cell biology↗

bioRxiv2019

Quantitative proteomics revealed a tight correlation between mitotic phosphorylation/dephosphorylation and structural properties of substrate domains

Protein phosphorylation plays a critical role in the regulation and progression of mitosis. More than 10,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4,100 and >2,200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.\n\nSignificance StatementProgression of mitosis is tightly regulated by protein phosphorylation/dephosphorylation. Although proteomic studies have identified tens of thousands of phosphosites in mitotic cells, the roles of them remain to be answered. We approached this question from the viewpoint of the higher-order structure of phosphosites. Quantitative proteomics and bioinformatic analyses revealed that more than 70% of mitotic dephosphorylation events occurred in non-structured regions. Non-structured regions of cellular proteins are attracting considerable attention in terms of their involvement in dynamic rearrangements of intracellular membrane-less organelles and protein assembly/disassembly processes. Our results suggest the possibility that a vast amount of mitosis-associated dephosphorylation/phosphorylation at non-structured regions plays a role in regulating the dynamic assembly/disassembly of intracellular architectures and organelles such as chromosomes and nucleolus.

cell biology↗