Biology subjects

Takada, S.

Publications and source records attributed to Takada, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Heterodimers of functionally divergent ARF-GEF paralogues prevented by self-interacting dimerisation domain

Functionally divergent paralogs of homomeric proteins do not form potentially deleterious heteromers, which requires distinction between self and non-self (Hochberg et al., 2018; Marchant et al, 2019; Marsh and Teichmann, 2015). In Arabidopsis, two ARF guanine-nucleotide exchange factors (ARF-GEFs) related to mammalian GBF1, named GNOM and GNL1, can mediate coatomer complex (COPI)-coated vesicle formation in retrograde Golgi-endoplasmic reticulum (ER) traffic (Geldner et al., 2003; Richter et al., 2007; Teh and Moore, 2007). Unlike GNL1, however, GNOM is also required for polar recycling of endocytosed auxin efflux regulator PIN1 from endosomes to the plasma membrane. Here we show that these paralogues form homodimers constitutively but no heterodimers. We also address why and how GNOM and GNL1 might be kept separate. These paralogues share a common domain organisation and each N-terminal dimerisation (DCB) domain can interact with the complementary fragment ({Delta}DCB) of its own and the other protein. However, unlike self-interacting DCBGNOM (Grebe et al., 2000; Anders et al., 2008), DCBGNL1 did not interact with itself nor DCBGNOM. DCBGNOM removal or replacement with DCBGNL1, but not disruption of cysteine bridges that stabilise DCB-DCB interaction, resulted in GNOM-GNL1 heterodimers which impaired developmental processes such as lateral root formation. We propose precocious self-interaction of the DCBGNOM domain as a mechanism to preclude formation of fitness-reducing GNOM-GNL1 heterodimers.

plant biology↗

bioRxiv2021

modeling DNA opening in the eukaryotic transcription initiation complexes

Recently, the molecular mechanisms of transcription initiation have been intensively studied. Especially, the cryo-electron microscopy revealed atomic structure details in key states in the eukaryotic transcription initiation. Yet, the dynamic processes of the promoter DNA opening in the pre-initiation complex remain obscured. In this study, based on the three cryo-electron microscopic yeast structures for the closed, open, and initially transcribing complexes, we performed multiscale molecular dynamics (MD) simulations to model structures and dynamic processes of DNA opening. Combining coarse-grained and all-atom MD simulations, we first obtained the atomic model for the DNA bubble in the open complexes. Then, in the MD simulation from the open to the initially transcribing complexes, we found a previously unidentified intermediate state which is formed by the bottleneck in the fork loop 1 of Pol II: The loop opening triggered the escape from the intermediate, serving as a gatekeeper of the promoter DNA opening. In the initially transcribing complex, the non-template DNA strand passes a groove made of the protrusion, the lobe, and the fork of Rpb2 subunit of Pol II, in which several positively charged and highly conserved residues exhibit key interactions to the non-template DNA strand. Author SummaryTranscription is fundamental phenomenon in all species, and its regulation of the initiation process is important. In eukaryotes, multiple proteins, which is not only RNA polymerase but also transcription factors, assemble on the promotor to form the complex. After that, this complex open the part of DNA duplex, leading to forming single-stranded region for transcription. Previous study, including structural analysis and biochemical experiments, obtained the information about single-stranded region, but the structure is incomplete, so the details of the mechanism remains unclear. In this study, by the calculation using structural information about the protein-DNA complex, we obtained the structure of the complex with complete single-stranded region. As the result of the calculation, it found that the size of single-stranded region was fluctuated, which is related the sequence of the region. Furthermore, we found the intermediate state during the extended process of single-stranded region, which has not been reported in previous study. In this state, we found that the particular region of RNA polymerase is relevant to the extension of single-stranded region. These founding may contribute the understanding about the regulation of transcription initiation.

biophysics↗

bioRxiv2021

Fine-tuning of β-catenin in thymic epithelial cells is required for postnatal T cell development

In the thymus, the thymic epithelium provides a microenvironment essential for the development of functionally competent and self-tolerant T cells. Previous findings showed that modulation of Wnt/{beta}-catenin signaling in thymic epithelial cells (TECs) disrupts embryonic thymus organogenesis. However, the role of {beta}-catenin in TECs for postnatal T cell development remains to be elucidated. Here, we analyzed gain-of-function (GOF) and loss-of-function (LOF) of {beta}-catenin highly specific in TECs. We found that GOF of {beta}-catenin in TECs results in severe thymic dysplasia and T cell deficiency beginning from the embryonic period. By contrast, LOF of {beta}-catenin in TECs reduces the number of cortical TECs and thymocytes modestly and only postnatally. These results indicate that fine-tuning of {beta}-catenin expression within a permissive range is required for TECs to generate an optimal microenvironment to support postnatal T cell development.

immunology↗

bioRxiv2021

Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation

In FoF1-ATP synthase, proton translocation through Fo drives rotation of the c-subunit oligomeric ring relative to the a-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c-subunits contribute to proton release to and proton uptake from the a-subunit. However, no studies have demonstrated cooperativity among c-subunits toward FoF1-ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring c-ring with various combinations of wild-type and cE56D, enabled by genetically fused single-chain c-ring. ATP synthesis and proton pump activities were decreased by a single cE56D mutation and further decreased by double cE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. Simulations revealed that prolonged proton uptake in mutated c-subunits is shared between two c-subunits, explaining the cooperation observed in biochemical assays.

biochemistry↗

bioRxiv2021

Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the so-called hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown, potentially due to its conformational plasticity. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. The conformational change of the hinge domain might be essential for the dsDNA binding regulation and play important roles in condensin-mediated DNA-loop extrusion.

biophysics↗

bioRxiv2021

Rigid-Body Fitting to Atomic Force Microscopy Images for Inferring Probe Shape and Biomolecular Structure

Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhAC, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available. Author SummaryObservation of functional dynamics of individual biomolecules is important to understand molecular mechanisms of cellular phenomena. High-speed (HS) atomic force microscopy (AFM) is a powerful tool that enables us to visualize the real-time dynamics of working biomolecules under near-physiological conditions. However, the information available by the AFM images is limited to the two-dimensional surface shape detected via the force to the probe. While the surface information is affected by the shape of the probe tip, the probe shape itself cannot be directly measured before each AFM measurement. To overcome this problem, we have developed a computational method to simultaneously infer the probe tip shape and the molecular placement from an AFM image. We show that our method successfully estimates the effective AFM tip shape and visualizes a structure with a more accurate placement. The estimation of a molecular placement with the correct probe tip shape enables us to obtain more insights into functional dynamics of the molecule from HS-AFM images.

biophysics↗

bioRxiv2021

DNA translocase repositions a nucleosome by the lane-switch mechanism

Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone core complexes and forms nucleosomes, translocases inevitably encounter nucleosomes. Previous studies have shown that a histone core complex repositions upstream (downstream) when SP6RNA or T7 RNA polymerase (bacterial exonuclease, RecBCD) partially unwraps nucleosomal DNA. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identify the lane-shift mechanism for downstream nucleosome repositioning via coarse-grained molecular dynamics simulations, which we validated by restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding region (lane) to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have crucial implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling. SIGNIFICANCEEukaryotic chromosomes are composed of repeating subunits termed nucleosomes. Thus, proteins that translocate along the chromosome, DNA translocases, inevitably collide with nucleosomes. Previous studies revealed that a translocase repositions a nucleosome upstream or downstream upon their collision. Though the molecular mechanisms of the upstream repositioning have been extensively studied, that of downstream repositioning remains elusive. In this study, we performed coarse-grained molecular dynamics simulations, proposed the lane-shift mechanism for downstream repositioning, and validated this mechanism by restriction enzyme digestion assays and deep sequencing assays. This mechanism has broad implications for how translocases deal with nucleosomes for their functions.

biophysics↗

bioRxiv2021

The Kinetic Landscape of Nucleosome Assembly: A Coarse-Grained Molecular Dynamics Study

The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. We find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences.

biophysics↗

bioRxiv2020

The human brain vasculature shows a distinct expression pattern of SARS-CoV-2 entry factors

A large number of hospitalized COVID-19 patients show neurological symptoms such as ischemic- and hemorrhagic stroke as well as encephalitis, and SARS-CoV-2 can directly infect endothelial cells leading to endotheliitis across multiple vascular beds. These findings suggest an involvement of the brain- and peripheral vasculature in COVID-19, but the underlying molecular mechanisms remain obscure. To understand the potential mechanisms underlying SARS-CoV-2 tropism for brain vasculature, we constructed a molecular atlas of the expression patterns of SARS-CoV-2 viral entry-associated genes (receptors and proteases) and SARS-CoV-2 interaction partners in human (and mouse) adult and fetal brain as well as in multiple non-CNS tissues in single-cell RNA-sequencing data across various datasets. We observed a distinct expression pattern of the cathepsins B (CTSB) and -L (CTSL) - which are able to substitute for the ACE2 co-receptor TMPRSS2 - in the human vasculature with CTSB being mainly expressed in the brain vasculature and CTSL predominantly in the peripheral vasculature, and these observations were confirmed at the protein level in the Human Protein Atlas and using immunofluorescence stainings. This expression pattern of SARS-CoV-2 viral-entry associated proteases and SARS-CoV-2 interaction partners was also present in endothelial cells and microglia in the fetal brain, suggesting a developmentally established SARS-CoV-2 entry machinery in the human vasculature. At both the adult and fetal stages, we detected a distinct pattern of SARS-CoV-2 entry associated genes transcripts in brain vascular endothelial cells and microglia, providing a potential explanation for an inflammatory response in the brain endothelium upon SARS-CoV-2 infection. Moreover, CTSB was co-expressed in adult and fetal brain endothelial cells with genes and pathways involved in innate immunity and inflammation, angiogenesis, blood-brain-barrier permeability, vascular metabolism, and coagulation, providing a potential explanation for the role of brain endothelial cells in clinically observed (neuro)vascular symptoms in COVID-19 patients. Our study serves as a publicly available single-cell atlas of SARS-CoV-2 related entry factors and interaction partners in human and mouse brain endothelial- and perivascular cells, which can be employed for future studies in clinical samples of COVID-19 patients.

neuroscience↗

bioRxiv2020

Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother

High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement of the instruments, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie, compared to that mimics the raw AFM movie, and the Kalman filter result. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhAc and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.

biophysics↗

bioRxiv2020

The Particle Filter Method to Integrate High-Speed Atomic Force Microscopy Measurement with Biomolecular Simulations

The high-speed atomic force microscopy (HS-AFM) can observe structural dynamics of biomolecules at single-molecule level in real time near physiological condition, but its spatiotemporal resolution is limited. Complementarily, molecular dynamics (MD) simulations have higher spatiotemporal resolutions albeit with some artifact. Here, in order to integrate the HS-AFM data and coarse-grained (CG)-MD simulations, we develop a particle filter method, one of the sequential Bayesian data assimilation approaches. We tested the method in a twin experiment. We first made a reference HS-AFM movie from a CG-MD trajectory of a test molecule, a nucleosome, which serves as an "experimental measurement". Then, we performed the particle filter simulation with 512 particles that captured large-scale nucleosome structural dynamics compatible with the AFM movie. Comparing the particle filter simulations with 8 - 8192 particles, we found that the use of more particles consistently results in larger likelihood for the whole AFM movie. By comparing the likelihoods from different ionic concentrations and from different timescales, we found that the "true" concentration and timescale can be inferred as the largest likelihood of the whole AFM movie, but not that of each AFM image. The particle filter method provides a general approach to integrate the HS-AFM data with MD simulations.

biophysics↗

bioRxiv2020

Quantitative analyses reveal extracellular dynamics of Wnt ligands in Xenopus embryos

The mechanism of intercellular transport of Wnt ligands is still a matter of debate. Here, to better understand this issue, we examined distribution and dynamics of Wnt8 in Xenopus embryos. While Venus-tagged Wnt8 was found on the surfaces of cells close to Wnt-producing cells, we also detected its dispersal over distances of 15 cell diameters. A combination of fluorescence correlation spectroscopy and quantitative imaging revealed that only a small proportion of Wnt8 ligands diffuses freely, whereas most Wnt8 molecules are bound to cell surfaces. Fluorescence decay after photoconversion showed that Wnt8 ligands bound on cell surfaces decreased exponentially, suggesting a dynamic exchange of bound forms of Wnt ligands. Mathematical modelling based on this exchange recapitulates a graded distribution of bound, but not free, Wnt ligands. Based on these results, we propose that Wnt distribution in tissues is controlled by a dynamic exchange of its abundant bound and rare free populations.

developmental biology↗

bioRxiv2019

Lin28a/let-7 Pathway Modulates the Hox Code via Polycomb Regulation during Axial Patterning in Vertebrates

The body plan along the anteroposterior axis and regional identities are specified by the spatiotemporal expression of Hox genes. Multistep controls are required for their unique expression patterns; however, the molecular mechanisms behind the tight control of Hox genes are not fully understood. In this study, we demonstrated that the Lin28a/let-7 reciprocal regulatory pathway is critical for vertebral specification. Lin28a-/- mice exhibited homeotic transformations of vertebrae which were caused by the global dysregulation of posterior Hox genes. The accumulation of let-7-family microRNAs in Lin28a-/- mice resulted in the reduction of PRC1 occupancy at the Hox cluster loci by targeting Cbx2. Consistently, Lin28a loss in embryonic stem-like cells led to aberrant induction of posterior Hox genes, which was rescued by the knockdown of let-7-family microRNAs. These results suggest that Lin28/let-7 pathway is possibly involved in the modulation of the "Hox code" via Polycomb regulation during axial patterning in vertebrates.

developmental biology↗

bioRxiv2019

Correlation between increased atrial expression of genes related to fatty acid metabolism and autophag y in patients with chronic atrial fibrillation

Atrial metabolic disturbance contributes to the onset and development of atrial fibrillation (AF). Autophagy plays a role in maintaining the cellular energy balance. We examined whether the altered atrial expression of genes related to fatty acid metabolism is linked to that related to autophagy in chronic AF. Right atrial tissue was obtained during heart surgery from 51 patients with sinus rhythm (SR, n=38) or chronic AF (n=13). Preoperative fasting serum free-fatty-acid levels were significantly higher in the AF patients. The atrial gene expression of fatty acid binding protein 3 (FABP3), which is involved in the cells fatty acid uptake and intracellular fatty acid transport, was significantly increased in AF patients compared to SR patients; in the SR patients it was positively correlated with the right atrial diameter and intra-atrial EMD, parameters of structural and electrical atrial remodeling that was evaluated by an echocardiography. In contrast, the two groups atrial contents of diacylglycerol (DAG), a toxic fatty acid metabolite, were comparable. Importantly, the atrial gene expression of microtubule-associated protein light chain 3 (LC3) was significantly increased in the AF patients, and autophagy-related genes including LC3 were positively correlated with the atrial expression of FABP3. In conclusion, in chronic AF patients, the atrial expression of FABP3 was upregulated in association with autophagy-related genes without altered atrial DAG content. Our findings may support the hypothesis that dysregulated cardiac fatty acid metabolism contributes to the progression of AF and induction of autophagy has a cardioprotective effect against cardiac lipotoxicity in chronic AF.

molecular biology↗

bioRxiv2019

Flexible Fitting of Biomolecular Structures to Atomic Force Microscopy Images via Biased Molecular Simulations

Atomic force microscopy (AFM) is a prominent imaging technology that observes large-scale structural dynamics of biomolecules near the physiological condition, but the AFM data are limited to the surface shape of specimens. Rigid-body fitting methods were developed to obtain molecular structures that fit to an AFM image, without accounting for conformational changes. Here we developed a method to fit flexibly a three-dimensional biomolecular structure into an AFM image. First, we describe a method to produce a pseudo-AFM image from a given three-dimensional structure in a differentiable form. Then, using a correlation function between the experimental AFM image and the computational pseudo-AFM image, we developed a flexible fitting molecular dynamics (MD) simulation method, by which we obtain protein structures that well fit to the given AFM image. We first test it with a twin-experiment; for a synthetic AFM image produced from a protein structure different from its native conformation, the flexible fitting MD simulations sampled those that fit well the AFM image. Then, parameter dependence in the protocol is discussed. Finally, we applied the method to a real experimental AFM image for a flagellar protein FlhA, demonstrating its applicability. We also test the rigid-body fitting of a fixed structure to the AFM image. Our method will be a general tool for structure modeling based on AFM images and is publicly available through CafeMol software.

biophysics↗

bioRxiv2019

Molecular dynamics simulation of proton-transfer coupled rotations in ATP synthase FO motor

The FO motor in FOF1 ATP synthase rotates its rotor driven by the proton motive force. While earlier studies elucidated basic mechanisms therein, recent advances in high-resolution cryo-electron microscopy enabled to investigate proton-transfer coupled FO rotary dynamics at structural details. Here, developing a hybrid Monte Carlo/molecular dynamics simulation method, we studied reversible dynamics of a yeast mitochondrial FO. We obtained the 36{degrees}-stepwise rotations of FO per one proton transfer in the ATP synthesis mode and the proton pumping in the ATP hydrolysis mode. In both modes, the most prominent path alternatively sampled states with two and three deprotonated glutamates in c-ring, by which the c-ring rotates one step. The free energy transduction efficiency in the model FO motor reaches ~ 90% in optimal conditions. Moreover, mutations in key glutamate and a highly conserved arginine increased proton leakage and markedly decreased the coupling, in harmony with previous experiments.

biophysics↗

bioRxiv2019

Diverse stepping motions of cytoplasmic dynein revealed by kinetic modeling

Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of microtubule (MT) using ATP hydrolysis free energy. By employing its two heads (motor domains), cytoplasmic dynein shows various bipedal stepping motions; the inchworm and hand-over-hand motions, as well as non-alternate steps of one head. However, the molecular basis to achieve such diverse stepping manners remains obscure. Here, we propose a kinetic model for bipedal motions of cytoplasmic dynein and performed Gillespie Monte Carlo simulations that reproduces most experimental data obtained to date. The model represents status of each motor domain as five states according to conformations, nucleotide- and MT-binding conditions of the domain. Also, the relative positions of the two domains were approximated by three discrete states. Accompanied by ATP hydrolysis cycles, the model dynein stochastically and processively moved forward in multiple steps via diverse pathways, including inchworm and hand-over-hand motions, same as experimental data. The model reproduced key experimental motility-related parameters including velocity and run-length as functions of ATP concentration and external force. Our model reveals that, in a typical inchworm motion, the leading domain moves via the ATP-dependent power-stroke of the linker coupled with a small change in the stalk angle, whereas the lagging domain moves via diffusion dragged by the leading domain. Moreover, the hand-over-hand motion in the model dynein clearly differs from that of kinesin by the usage of the power-stroke.\n\nAuthor SummaryCytoplasmic dynein is a two-headed molecular motor, which moves linearly and transports intra-cellar organelles along microtubules driven by ATP hydrolysis free energy. In contrast to other better-known molecular motors, such as kinesin, dynein is known to take various stepping motions including motions akin to human walking and inchworm-like motions. However, molecular mechanisms underpinning the diverse stepping motions are unclear. Here, based on recent high-resolution structure information and single-molecule motility assay data, we designed a kinetic model that explicitly include two heads, each of which makes ATP hydrolysis cycles and moves along the microtubules. Using the model, we performed Monte Carlo simulations. The simulation reproduced most of currently available experimental results. More importantly, the simulation suggested molecular mechanisms of various stepping motions. While stepping motions apparently resemble to those proposed before, once looking into details, we found the resulting mechanisms distinct from previously proposed ones in the usage of ATP and protein conformation changes coupled with stepping motions.

biophysics↗

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↗