Biology subjects

Ogata, K.

Publications and source records attributed to Ogata, K..

4 recordsLinked to original sources

bioRxiv2021

IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay

Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1{beta} or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1{beta} or TLR stimulus dynamically induced the formation of Regnase-1-{beta}-transducin repeat-containing protein ({beta}TRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. Strikingly, both interactions occur in a mutually exclusive manner, underscoring the importance of modulating Regnase-1s activity. Additionally, we show that in IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 are known to be required for the recognition by {beta}TRCP and proteasome-mediated degradation. 14-3-3 stabilizes Regnase-1 but abolishes its activity by inhibiting Regnase-1-mRNA association. Furthermore, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.

immunology↗

bioRxiv2021

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which in vitro kinase reaction is used to generate the targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. Over 7,000 tyrosine phosphorylation sites were quantified from several tens of {micro}g of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner. MotivationSensitivity for detecting phosphopeptides with a particular phosphorylation motif is limited, especially for tyrosine phosphopeptides.

systems biology↗

bioRxiv2020

Structural basis of the regulation of normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2

Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression by antagonizing the repressive effect of polycomb-group proteins. Aberrant upregulation of H3K36me2, either by overexpression or point mutations of NSD2/MMSET, an H3K36 dimethyltransferase, is found in various cancers, including multiple myeloma. To understand the mechanism underlying its regulation, here we report the cryo-electron microscopy structure of the catalytic fragment of NSD2 bound to the nucleosome at 2.8 [A] resolution. The nucleosomal DNA is partially unwrapped at superhelix location +5.5, facilitating the access of NSD2 to H3K36. NSD2 interacts with DNA and H2A along with H3. The autoinhibitory loop of NSD2 changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed two oncogenic mutations, E1099K and T1150A, to aberrantly activate NSD2 by increasing its catalytic turnover but not the nucleosome affinity. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing the catalytic turnover. Our analyses would guide the development of specific inhibitors of NSD2 for the treatment of various cancers.

biochemistry↗

bioRxiv2020

Effect of Phosphorylation on the Collision Cross Sections of Peptide Ions in Ion Mobility Spectrometry

The insertion of ion mobility spectrometry (IMS) between LC and MS can improve peptide identification in both proteomics and phosphoproteomics by providing structural information that is complementary to LC and MS, because IMS separates ions on the basis of differences in their shapes and charge states. However, it is necessary to know how phosphate groups affect the peptide collision cross sections (CCS) in order to accurately predict phosphopeptide CCS values and to maximize the usefulness of IMS. In this work, we systematically characterized the CCS values of 4,433 pairs of mono-phosphopeptide and corresponding unphosphorylated peptide ions using trapped ion mobility spectrometry (TIMS). Nearly one-third of the mono-phosphopeptide ions evaluated here showed smaller CCS values than their unphosphorylated counterparts, even though phosphorylation results in a mass increase of 80 Da. Significant changes of CCS upon phosphorylation occurred mainly in structurally extended peptides with large numbers of basic groups, possibly reflecting intramolecular interactions between phosphate and basic groups.

biochemistry↗