Biology subjects

Takada, S.

Publications and source records attributed to Takada, S..

7 recordsLinked to original sources

bioRxiv2018

mitoNEET Regulates Mitochondrial Iron Homeostasis Interacting with Transferrin Receptor

Iron is an essential trace element for regulation of redox and mitochondrial function, and then mitochondrial iron content is tightly regulated in mammals. We focused on a novel protein localized at the outer mitochondrial membrane. Immunoelectron microscopy revealed transferrin receptor (TfR) displayed an intimate relationship with the mitochondria, and mass spectrometry analysis also revealed mitoNEET interacted with TfR in vitro. Moreover, mitoNEET was endogenously coprecipitated with TfR in the heart, which indicates that mitoNEET also interacts with TfR in vivo. We generated mice with cardiac-specific deletion of mitoNEET (mitoNEET-knockout). Iron contents in isolated mitochondria were significantly increased in mitoNEET-knockout mice compared to control mice. Mitochondrial reactive oxygen species (ROS) were higher, and mitochondrial maximal capacity and reserve capacity were significantly decreased in mitoNEET-knockout mice, which was consistent with cardiac dysfunction evaluated by echocardiography. The complex formation of mitoNEET with TfR may regulate mitochondrial iron contents via an influx of iron. A disruption of mitoNEET could thus be involved in mitochondrial ROS production by iron overload in the heart.

cell biology↗

bioRxiv2018

Chromatin remodelers couple inchworm motion with twist-defect formation to slide nucleosomal DNA

ATP-dependent chromatin remodelers control genome organization by repositioning, ejecting, or editing nucleosomes, activities that confer them essential regulatory roles on gene expression and DNA replication. Here, we investigate the mechanism of active DNA sliding by remodelers, which underlies most remodeling functions, via molecular dynamics simulations of the Snf2 remodeler in complex with a nucleosome. During its inchworm motion driven by ATP consumption, the remodeler overwrites the original nucleosome energy landscape via steric and electrostatic interactions to induce sliding of nucleosomal DNA. Repositioning is initiated at the remodeler binding location via the generation of twist defects, which then spontaneously propagate to complete sliding throughout the entire nucleosome. We also reveal how remodeler mutations and DNA sequence control active nucleosome repositioning. These results offer a mechanistic understanding of chromatin remodeling important for the interpretation of experimental studies.

biophysics↗

bioRxiv2018

Dynamic and Structural Modeling of the Specificity in Protein-DNA Interactions Guided by Binding Assay and Structure Data

How transcription factors (TFs) recognize their DNA sequences is often investigated complementarily by high-throughput protein binding assays and by structural biology experiments. The former quantifies the specificity of TF binding sites for numerous DNA sequences, often represented as the position-weight-matrix (PWM). The latter provides mechanistic insights into the interactions via the protein-DNA complex structures. However, these two types of data are not readily integrated. Here, we propose and test a new modeling method that incorporates the PWM with complex structure data. Based on pre-tuned coarse-grained models for proteins and DNAs, we model the specific protein-DNA interactions, PWMcos, in terms of an orientation-dependent potential function, which enables us to perform molecular dynamics simulations at unprecedentedly large scales. We show that the PWMcos model reproduces subtle specificity in the protein-DNA recognition. During the target search in genomic sequences, TF moves on highly rugged landscapes and occasionally flips on DNA depending on the sequence. The TATA-binding protein exhibits two remarkably distinct binding modes, of which frequencies differ between TATA-containing and TATA-less promoters. The PWMcos is general and can be applied to any protein-DNA interactions given their PWMs and complex structure data are available.

biophysics↗

bioRxiv2018

Endoderm Morphogenesis Reveals Integration of Distinct Processes in the Development and Evolution of Pharyngeal Arches

The vertebrate pharyngeal arches (PAs) are established by a combination of two styles of segmentation; the most anterior 2 PAs are simultaneously but the others are sequentially formed. However, the mechanism underlying their coexistence is unclear. Here, we show that the simultaneous and sequential segmentation discretely proceeded, respectively, but were finally integrated at the second PP (PP2), by dynamic morphogenesis of pharyngeal endoderm in the zebrafish. The coordination of these 2 distinct processes appears to be common in the PA development of many vertebrates, in which specific developmental defects posterior to the PP2 are caused by mutations of particular genes or perturbation of retinoic acid signaling. Surprisingly, comparative analysis of PA segmentation showed that the combinatorial styles of PA development is present in shark but not in lamprey, suggesting that PA segmentation was modified in the stem gnathostomes corresponding to the drastic pharyngeal innovations, such as PA2-derived opercular.

developmental biology↗

bioRxiv2017

Reconstruction of atomistic structures from coarse-grained models for protein-DNA complexes

While coarse-grained (CG) simulations have widely been used to accelerate structure sampling of large biomolecular complexes, they are unavoidably less accurate and thus the reconstruction of all-atom (AA) structures and the subsequent refinement is of desire. In this study we developed an efficient method to reconstruct AA structures from sampled CG protein-DNA complex models, which attempts to model protein-DNA interface accurately. First we developed a method to reconstruct atomic details of DNA structures from a 3-site per nucleotide CG model, which uses a DNA fragment library. Next, for the protein-DNA interface, we referred to the sidechain orientations in the known structure of the target interface when available. The other parts are modeled by existing tools. We confirmed the accuracy of the protocol in various aspects including the structure deviation in the self-reproduction, the base pair reproducibility, atomic contacts at the protein-DNA interface, and feasibility of the posterior AA simulations.

biophysics↗

bioRxiv2017

Sequence-dependent nucleosome sliding in rotation-coupled and uncoupled modes revealed by molecular simulations

While nucleosome positioning on eukaryotic genome play important roles for genetic regulation, molecular mechanisms of nucleosome positioning and sliding along DNA are not well understood. Here we investigated thermally-activated spontaneous nucleosome sliding mechanisms developing and applying a coarse-grained molecular simulation method that incorporates both long-range electrostatic and short-range hydrogen-bond interactions between histone octamer and DNA. The simulations revealed two distinct sliding modes depending on the nucleosomal DNA sequence. A uniform DNA sequence showed frequent sliding with one base pair step in a rotation-coupled manner, akin to screw-like motions. On the contrary, a strong positioning sequence, the so-called 601 sequence, exhibits rare, abrupt transitions of five and ten base pair steps without rotation. Moreover, we evaluated the importance of hydrogen bond interactions on the sliding mode, finding that strong and weak bonds favor respectively the rotation-coupled and -uncoupled sliding movements.\n\nAuthor summaryNucleosomes are fundamental units of chromatin folding consisting of double-stranded DNA wrapped [~]1.7 times around a histone octamer. By densely populating the eukaryotic genome, nucleosomes enable efficient genome compaction inside the cellular nucleus. However, the portion of DNA occupied by a nucleosome can hardly be accessed by other DNA-binding proteins, obstructing fundamental cellular processes such as DNA replication and transcription. DNA compaction and access by other proteins can simultaneously be achieved via the dynamical repositioning of nucleosomes, which can slide along the DNA sequence. In this study, we developed and used coarse-grained molecular dynamics simulations to reveal the molecular details of nucleosome sliding. We find that the sliding mode is highly dependent on the underlying DNA sequence. Specifically, a sequence with a strong nucleosome positioning signal slides via large jumps by five and ten base pairs, preserving the optimal DNA bending profile. On the other hand, uniform sequences without the positioning signal slide via a screw-like motion of DNA, one base pair at the time. These results show that sequence has a large effect not only on the formation of nucleosomes, but also on the kinetics of repositioning.

biophysics↗

bioRxiv2016

Fetal therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells

Myelomeningocele (MMC) is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth. No effective treatment has been reported to date. Only surgical repairs have reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs) generated from a patient with Down syndrome (AF-T21-iPSCs) and twin-twin transfusion syndrome (AF-TTTS-iPSCs) to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a novel protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology.

cell biology↗