Biology subjects

Kashiwagi, K.

Publications and source records attributed to Kashiwagi, K..

9 recordsLinked to original sources

bioRxiv2022

Disruption of Smarce1, a component of the SWI/SNF chromatin remodeling complex, decreases nucleosome stability in mouse embryonic stem cells and impairs differentiation

The SWI/SNF chromatin remodeling complex consists of more than 10 component proteins that form a large protein complex of > 1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem (ES) cells. Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ES cells, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested an ectopic genomic distribution of the SWI/SNF complex, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ES cell differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

molecular biology↗

bioRxiv2021

Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 s and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3. SignificanceLight-driven chloride pumps have been identified in various species, including archaea and marine flavobacteria. The function of ion transportation controllable by light is utilized for optogenetics tools in neuroscience. Chloride pumps differ among species, in terms of amino acid homology and structural similarity. Our time-resolved crystallographic studies using X-ray free electron lasers reveal the molecular mechanism of halide ion transfer in a light-driven chloride pump from a marine flavobacterium. Our data indicate a common mechanism in chloride pumping rhodopsins, as compared to previous low temperature trapping studies of chloride pumps. These findings are significant not only for further improvements of optogenetic tools but also for a general understanding of the ion pumping mechanisms of microbial rhodopsins.

molecular biology↗

bioRxiv2021

A specific eIF4A paralog facilitates LARP1-mediated translation repression during mTORC1 inhibition

Eukaryotic translation initiation factor (eIF) 4A -- a DEAD-box RNA-binding protein -- plays an essential role in translation initiation. Two mammalian eIF4A paralogs, eIF4A1 and eIF4A2, have been assumed to be redundant because of their high homology, and the difference in their functions has been poorly understood. Here, we show that eIF4A1, but not eIF4A2, enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA-immunoprecipitation sequencing (RIP-Seq) of the two eIF4A paralogs revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTOR inhibition. This biased interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that the deletion of EIF4A1, but not EIF4A2, rendered the translation of TOP mRNAs resistant to mTOR inactivation. Moreover, eIF4A1 enhances the affinity between TOP mRNAs and LARP1 and thus ensures stronger translation repression upon mTORC1 inhibition. Our data show that the distinct protein interactions of these highly homologous translation factor paralogs shape protein synthesis during mTORC1 inhibition and provide a unique example of the repressive role of a universal translation activator.

molecular biology↗

bioRxiv2021

METTL18-mediated histidine methylation on RPL3 modulates translation elongation for proteostasis maintenance

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a substrate for the modification. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traverse on tyrosine codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the {tau}-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation tuning. Indeed, genome-wide ribosome profiling revealed suppressed ribosomal translocation at tyrosine codons by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a "ribosome code" that ensures proteome integrity in cells.

molecular biology↗

bioRxiv2021

eIF2B-capturing viral protein NSs suppresses the integrated stress response

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(P)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotides on unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. Our cryo-electron microscopy (cryo-EM) analysis revealed that SFSV NSs binds to the -subunit of eIF2B in a competitive manner with eIF2(P). Together with SFSV NSs, eIF2B exhibits normal nucleotide exchange activity even in the presence of eIF2(P). A genome-wide ribosome profiling analysis clarified that SFSV NSs in human cultured cells attenuates the ISR. Furthermore, SFSV NSs exhibited neuroprotective effects against the ISR-inducing stress. Since the ISR inhibition is beneficial in various neurological disease models, SFSV NSs is promising as a therapeutic ISR inhibitor.

molecular biology↗

bioRxiv2020

ISRIB blunts the integrated stress response by allosterically antagonising the inhibitory effect of phosphorylated eIF2 on eIF2B

The small molecule ISRIB antagonises the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(P). ISRIB and eIF2(P) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor (GEF) for eIF2. We have found that ISRIB-mediated acceleration of eIF2B activity in vitro is observed preferentially in the presence of eIF2(P) and is attenuated by mutations that desensitise eIF2B to the inhibitory effects of eIF2(P). ISRIBs efficacy as an ISR inhibitor in cells also depends on presence of eIF2(P). Cryo-EM showed that engagement of both eIF2B regulatory sites by two eIF2(P) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2 during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(P) and ISRIB reciprocally opposed each others binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

biochemistry↗

bioRxiv2020

Aberrant claudin-6-adhesion signal promotes endometrial cancer progression via estrogen receptor α

Cell adhesion proteins not only maintain tissue integrity but also possess signaling abilities to organize diverse cellular events in physiological and pathological processes; however, the underlying mechanism remains obscure. Among cell adhesion molecules, the claudin (CLDN) family often possesses aberrant expression in various cancers, but the biological relevance and molecular basis have not yet been established. Here, we show that high CLDN6 expression promotes endometrial cancer progression and represents the poor prognostic marker. The second extracellular domain and Y196/200 of CLDN6 were required to recruit and activate Src-family kinases (SFKs) and to stimulate malignant phenotypes. Importantly, we demonstrate that the CLDN6/SFK/PI3K-dependent AKT and SGK (serum- and glucocorticoid-regulated kinase) signalings target Ser518 in the human estrogen receptor and ligand-independently activate target genes in endometrial cancer cells, resulting in cancer development. The identification of this machinery highlights regulation of the transcription factors by cell adhesion to advance tumor progression.

cancer biology↗

bioRxiv2020

Different Proteins Mediate Step-wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

Archaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that in Thermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out of Thermoplasma acidophilum and Pyrobaculum calidifontis cells revealed 10-nm fibers and 30-40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist in T. acidophilum and T. kodakarensis but not in P. calidifontis or Sulfolobus solfataricus. In vitro reconstitution showed that, in T. acidophilum, the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa in the T. acidophilum chromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on the P. calidifontis chromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, including T. acidophilum, P. calidifontis, and T. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

microbiology↗

bioRxiv2018

Structural Basis for eIF2B Inhibition in Integrated Stress Response

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers methionylated initiator tRNA to the ribosome in a GTP-dependent manner. In contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor eIF2B, which leads to a deficiency of active eIF2 and resultant global translation repression. To unveil the mechanism by which the eIF2 phosphorylation status regulates the eIF2B nucleotide exchange activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. Intriguingly, the unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active \"productive\" and nucleotide exchange-inactive \"nonproductive\" modes, respectively. The nonproductive-mode phosphorylated eIF2, extending from one of the two eIF2B \"central cavities\", not only prevents nucleotide exchange on itself, but also sterically prevents unphosphorylated eIF2 from productively binding on the other central cavity of eIF2B, which explains how phosphorylated eIF2 inhibits eIF2B.\n\nOne Sentence SummaryA drastic change in the binding mode of eIF2 to eIF2B induces translational control in stress.

molecular biology↗