Publications by authors named "Masaki Inagaki"

103 Publications

Primary cilia-dependent lipid raft/caveolin dynamics regulate adipogenesis.

Cell Rep 2021 Mar;34(10):108817

Department of Physiology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. Electronic address:

Primary cilia play a pivotal role in signal transduction and development and are known to serve as signaling hubs. Recent studies have shown that primary cilium dysfunction influences adipogenesis, but the mechanisms are unclear. Here, we show that mesenchymal progenitors C3H10T1/2 depleted of trichoplein, a key regulator of cilium formation, have significantly longer cilia than control cells and fail to differentiate into adipocytes. Mechanistically, the elongated cilia prevent caveolin-1- and/or GM3-positive lipid rafts from being assembled around the ciliary base where insulin receptor proteins accumulate, thereby inhibiting the insulin-Akt signaling. We further generate trichoplein knockout mice, in which adipogenic progenitors display elongated cilia and impair the lipid raft dynamics. The knockout mice on an extended high-fat diet exhibit reduced body fat and smaller adipocytes than wild-type (WT) mice. Overall, our results suggest a role for primary cilia in regulating adipogenic signal transduction via control of the lipid raft dynamics around cilia.
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http://dx.doi.org/10.1016/j.celrep.2021.108817DOI Listing
March 2021

[Targeting the ubiquitin system for treatment of cilia-related diseases].

Nihon Yakurigaku Zasshi 2021 ;156(1):4-8

Glocal Center for Advanced Medical Research, Mie University.

The ubiquitin system regulates a wide variety of cellular functions. Not surprisingly, dysregulation of the ubiquitin system is associated with various disorders. Therefore, drugs that can modulate the functions of the ubiquitin system have been actively developed to treat these disorders. Chemical knockdown of pathogenic proteins using the ubiquitin-proteasome system is also a promising approach. The ubiquitin system regulates the assemble and disassemble of primary cilia through balanced control over the ubiquitination and deubiquitination of ciliary proteins. Primary cilia are antenna-like structures present in many vertebrate cells that sense and transduce extracellular cues to control cellular processes such as proliferation and differentiation. Impairment of primary cilia is associated with many diseases, including cancer and ciliopathy, a group of multisystem developmental disorders. In this review, we focus on the role of the ubiquitin system on cilia-related disorders and discuss the possibility of the ubiquitin system as therapeutic targets for these diseases through regulation of primary cilia formation.
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http://dx.doi.org/10.1254/fpj.20072DOI Listing
January 2021

Targeting E3 Ubiquitin Ligases and Deubiquitinases in Ciliopathy and Cancer.

Int J Mol Sci 2020 Aug 19;21(17). Epub 2020 Aug 19.

Department of Integrative Pharmacology, Graduate School of Medicine, Mie University, Tsu, Mie 514-8507, Japan.

Cilia are antenna-like structures present in many vertebrate cells. These organelles detect extracellular cues, transduce signals into the cell, and play an essential role in ensuring correct cell proliferation, migration, and differentiation in a spatiotemporal manner. Not surprisingly, dysregulation of cilia can cause various diseases, including cancer and ciliopathies, which are complex disorders caused by mutations in genes regulating ciliary function. The structure and function of cilia are dynamically regulated through various mechanisms, among which E3 ubiquitin ligases and deubiquitinases play crucial roles. These enzymes regulate the degradation and stabilization of ciliary proteins through the ubiquitin-proteasome system. In this review, we briefly highlight the role of cilia in ciliopathy and cancer; describe the roles of E3 ubiquitin ligases and deubiquitinases in ciliogenesis, ciliopathy, and cancer; and highlight some of the E3 ubiquitin ligases and deubiquitinases that are potential therapeutic targets for these disorders.
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http://dx.doi.org/10.3390/ijms21175962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504095PMC
August 2020

A novel CDK-independent function of p27 in preciliary vesicle trafficking during ciliogenesis.

Biochem Biophys Res Commun 2020 06 16;527(3):716-722. Epub 2020 May 16.

Department of Physiology, Mie University Graduate School of Medicine, Tsu, Mie, 514-8507, Japan. Electronic address:

p27, a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27 is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27 is associated with ciliogenesis. Here, we have generated p27-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27 and, surprisingly, its 86-140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27 abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehd1-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27 in primary cilia formation.
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http://dx.doi.org/10.1016/j.bbrc.2020.05.048DOI Listing
June 2020

Cdc7 kinase stimulates Aurora B kinase in M-phase.

Sci Rep 2019 12 9;9(1):18622. Epub 2019 Dec 9.

Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, 156-8506, Japan.

The conserved serine-threonine kinase, Cdc7, plays a crucial role in initiation of DNA replication by facilitating the assembly of an initiation complex. Cdc7 is expressed at a high level and exhibits significant kinase activity not only during S-phase but also during G2/M-phases. A conserved mitotic kinase, Aurora B, is activated during M-phase by association with INCENP, forming the chromosome passenger complex with Borealin and Survivin. We show that Cdc7 phosphorylates and stimulates Aurora B kinase activity in vitro. We identified threonine-236 as a critical phosphorylation site on Aurora B that could be a target of Cdc7 or could be an autophosphorylation site stimulated by Cdc7-mediated phosphorylation elsewhere. We found that threonines at both 232 (that has been identified as an autophosphorylation site) and 236 are essential for the kinase activity of Aurora B. Cdc7 down regulation or inhibition reduced Aurora B activity in vivo and led to retarded M-phase progression. SAC imposed by paclitaxel was dramatically reversed by Cdc7 inhibition, similar to the effect of Aurora B inhibition under the similar situation. Our data show that Cdc7 contributes to M-phase progression and to spindle assembly checkpoint most likely through Aurora B activation.
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http://dx.doi.org/10.1038/s41598-019-54738-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901529PMC
December 2019

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Elife 2019 11 4;8. Epub 2019 Nov 4.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.
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http://dx.doi.org/10.7554/eLife.47789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927689PMC
November 2019

Intermediate filaments and IF-associated proteins: from cell architecture to cell proliferation.

Proc Jpn Acad Ser B Phys Biol Sci 2019 ;95(8):479-493

Department of Physiology, Mie University Graduate School of Medicine.

Intermediate filaments (IFs), in coordination with microfilaments and microtubules, form the structural framework of the cytoskeleton and nucleus, thereby providing mechanical support against cellular stresses and anchoring intracellular organelles in place. The assembly and disassembly of IFs are mainly regulated by the phosphorylation of IF proteins. These phosphorylation states can be tracked using antibodies raised against phosphopeptides in the target proteins. IFs exert their functions through interactions with not only structural proteins, but also non-structural proteins involved in cell signaling, such as stress responses, apoptosis, and cell proliferation. This review highlights findings related to how IFs regulate cell division through phosphorylation cascades and how trichoplein, a centriolar protein originally identified as a keratin-associated protein, regulates the cell cycle through primary cilium formation.
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http://dx.doi.org/10.2183/pjab.95.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819152PMC
February 2020

Vimentin Phosphorylation Is Required for Normal Cell Division of Immature Astrocytes.

Cells 2019 09 1;8(9). Epub 2019 Sep 1.

Laboratory of Astrocyte Biology and CNS Regeneration, Center for Brain Repair, Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, 40530 Gothenburg, Sweden.

Vimentin (VIM) is an intermediate filament (nanofilament) protein expressed in multiple cell types, including astrocytes. Mice with mutations of serine sites phosphorylated during mitosis (VIM) show cytokinetic failure in fibroblasts and lens epithelial cells, chromosomal instability, facilitated cell senescence, and increased neuronal differentiation of neural progenitor cells. Here we report that in vitro immature VIM astrocytes exhibit cytokinetic failure and contain vimentin accumulations that co-localize with mitochondria. This phenotype is transient and disappears with VIM astrocyte maturation and expression of glial fibrillary acidic protein (GFAP); it is also alleviated by the inhibition of cell proliferation. To test the hypothesis that GFAP compensates for the effect of VIM in astrocytes, we crossed the VIM and GFAP mice. Surprisingly, the fraction of VIM immature astrocytes with abundant vimentin accumulations was reduced when on GFAP background. This indicates that the disappearance of vimentin accumulations and cytokinetic failure in mature astrocyte cultures are independent of GFAP expression. Both VIM and VIMGFAP astrocytes showed normal mitochondrial membrane potential and vulnerability to HO, oxygen/glucose deprivation, and chemical ischemia. Thus, mutation of mitotic phosphorylation sites in vimentin triggers formation of vimentin accumulations and cytokinetic failure in immature astrocytes without altering their vulnerability to oxidative stress.
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http://dx.doi.org/10.3390/cells8091016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769829PMC
September 2019

Primary Cilia as Signaling Hubs in Health and Disease.

Adv Sci (Weinh) 2019 Jan 16;6(1):1801138. Epub 2018 Nov 16.

Department of Physiology Mie University Graduate School of Medicine Tsu Mie 514-8507 Japan.

Primary cilia detect extracellular cues and transduce these signals into cells to regulate proliferation, migration, and differentiation. Here, the function of primary cilia as signaling hubs of growth factors and morphogens is in focus. First, the molecular mechanisms regulating the assembly and disassembly of primary cilia are described. Then, the role of primary cilia in mediating growth factor and morphogen signaling to maintain human health and the potential mechanisms by which defects in these pathways contribute to human diseases, such as ciliopathy, obesity, and cancer are described. Furthermore, a novel signaling pathway by which certain growth factors stimulate cell proliferation through suppression of ciliogenesis is also described, suggesting novel therapeutic targets in cancer.
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http://dx.doi.org/10.1002/advs.201801138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325590PMC
January 2019

Chk1-mediated Cdc25A degradation as a critical mechanism for normal cell cycle progression.

J Cell Sci 2019 01 25;132(2). Epub 2019 Jan 25.

Department of Neural Regeneration and Cell Communication, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.

Chk1 (encoded by in mammals) is an evolutionarily conserved protein kinase that transduces checkpoint signals from ATR to Cdc25A during the DNA damage response (DDR). In mammals, Chk1 also controls cellular proliferation even in the absence of exogenous DNA damage. However, little is known about how Chk1 regulates unperturbed cell cycle progression, and how this effect under physiological conditions differs from its regulatory role in DDR. Here, we have established near-diploid HCT116 cell lines containing endogenous Chk1 protein tagged with a minimum auxin-inducible degron (mAID) through CRISPR/Cas9-based gene editing. Establishment of these cells enabled us to induce specific and rapid depletion of the endogenous Chk1 protein, which resulted in aberrant accumulation of DNA damage factors that induced cell cycle arrest at S or G2 phase. Cdc25A was stabilized upon Chk1 depletion before the accumulation of DNA damage factors. Simultaneous depletion of Chk1 and Cdc25A partially suppressed the defects caused by Chk1 single depletion. These results indicate that, similar to its function in DDR, Chk1 controls normal cell cycle progression mainly by inducing Cdc25A degradation.
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http://dx.doi.org/10.1242/jcs.223123DOI Listing
January 2019

Albatross/FBF1 contributes to both centriole duplication and centrosome separation.

Genes Cells 2018 Dec 13;23(12):1023-1042. Epub 2018 Nov 13.

Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Japan.

The centrosome is a small but important organelle that participates in centriole duplication, spindle formation, and ciliogenesis. Each event is regulated by key enzymatic reactions, but how these processes are integrated remains unknown. Recent studies have reported that ciliogenesis is controlled by distal appendage proteins such as FBF1, also known as Albatross. However, the precise role of Albatross in the centrosome cycle, including centriole duplication and centrosome separation, remains to be determined. Here, we report a novel function for Albatross at the proximal ends of centrioles. Using Albatross monospecific antibodies, full-length constructs, and siRNAs for rescue experiments, we found that Albatross mediates centriole duplication by recruiting HsSAS-6, a cartwheel protein of centrioles. Moreover, Albatross participates in centrosome separation during mitosis by recruiting Plk1 to residue S348 of Albatross after its phosphorylation. Taken together, our results show that Albatross is a novel protein that spatiotemporally integrates different aspects of centrosome function, namely ciliogenesis, centriole duplication, and centrosome separation.
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http://dx.doi.org/10.1111/gtc.12648DOI Listing
December 2018

Erbin Suppresses KSR1-Mediated RAS/RAF Signaling and Tumorigenesis in Colorectal Cancer.

Cancer Res 2018 09 6;78(17):4839-4852. Epub 2018 Jul 6.

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky.

Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that plays important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colorectal cancer. Analysis of Erbin expression in colorectal cancer patient specimens revealed that Erbin was downregulated at both mRNA and protein levels in tumor tissues. Knockdown of Erbin disrupted epithelial cell polarity and increased cell proliferation in 3D culture. In addition, silencing Erbin resulted in increased amplitude and duration of signaling through Akt and RAS/RAF pathways. Erbin loss induced epithelial-mesenchymal transition, which coincided with a significant increase in cell migration and invasion. Erbin interacted with kinase suppressor of Ras 1 (KSR1) and displaced it from the RAF/MEK/ERK complex to prevent signal propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promoted tumorigenesis and significantly reduced survival. Tumor organoids derived from Erbin/Apc double knockout mice displayed increased tumor initiation potential and activation of Wnt signaling. Results from gene set enrichment analysis revealed that Erbin expression associated positively with the E-cadherin adherens junction pathway and negatively with Wnt signaling in human colorectal cancer. Taken together, our study identifies Erbin as a negative regulator of tumor initiation and progression by suppressing Akt and RAS/RAF signaling These findings establish the scaffold protein Erbin as a negative regulator of EMT and tumorigenesis in colorectal cancer through direct suppression of Akt and RAS/RAF signaling. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510240PMC
September 2018

Tetraploidy in cancer and its possible link to aging.

Cancer Sci 2018 Sep 26;109(9):2632-2640. Epub 2018 Jul 26.

Department of Physiology, Mie University Graduate School of Medicine, Tsu, Japan.

Tetraploidy, a condition in which a cell has four homologous sets of chromosomes, is often seen as a natural physiological condition but is also frequently seen in pathophysiological conditions such as cancer. Tetraploidy facilitates chromosomal instability (CIN), which is an elevated level of chromosomal loss and gain that can cause production of a wide variety of aneuploid cells that carry structural and numerical aberrations of chromosomes. The resultant genomic heterogeneity supposedly expedites karyotypic evolution that confers oncogenic potential in spite of the reduced cellular fitness caused by aneuploidy. Recent studies suggest that tetraploidy might also be associated with aging; mice with mutations in an intermediate filament protein have revealed that these tetraploidy-prone mice exhibit tissue disorders associated with aging. Cellular senescence and its accompanying senescence-associated secretory phenotype have now emerged as critical factors that link tetraploidy and tetraploidy-induced CIN with cancer, and possibly with aging. Here, we review recent findings about how tetraploidy is related to cancer and possibly to aging, and discuss underlying mechanisms of the relationship, as well as how we can exploit the properties of cells exhibiting tetraploidy-induced CIN to control these pathological conditions.
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http://dx.doi.org/10.1111/cas.13717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125447PMC
September 2018

Regulation of keratin 5/14 intermediate filaments by CDK1, Aurora-B, and Rho-kinase.

Biochem Biophys Res Commun 2018 04 6;498(3):544-550. Epub 2018 Mar 6.

Department of Physiology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. Electronic address:

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.
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http://dx.doi.org/10.1016/j.bbrc.2018.03.016DOI Listing
April 2018

EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase.

Nat Commun 2018 02 22;9(1):758. Epub 2018 Feb 22.

Department of Physiology, Mie University Graduate School of Medicine, Tsu, Mie, 14101, Japan.

Ciliogenesis is generally inhibited in dividing cells, however, it has been unclear which signaling cascades regulate the phenomenon. Here, we report that epidermal growth factor receptor (EGFR) kinase suppresses ciliogenesis by directly phosphorylating the deubiquitinase USP8 on Tyr-717 and Tyr-810 in RPE1 cells. These phosphorylations elevate the deubiquitinase activity, which then stabilizes the trichoplein-Aurora A pathway, an inhibitory mechanism of ciliogenesis. EGFR knockdown and serum starvation result in ciliogenesis through downregulation of the USP8-trichoplein-Aurora A signal. Moreover, primary cilia abrogation, which is induced upon IFT20 or Cep164 depletion, ameliorates the cell cycle arrest of EGFR knockdown cells. The present data reveal that the EGFR-USP8-trichoplein-Aurora A axis is a critical signaling cascade that restricts ciliogenesis in dividing cells, and functions to facilitate cell proliferation. We further show that usp8 knockout zebrafish develops ciliopathy-related phenotypes including cystic kidney, suggesting that USP8 is a regulator of ciliogenesis in vertebrates.
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http://dx.doi.org/10.1038/s41467-018-03117-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823934PMC
February 2018

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice.

Mol Neurobiol 2018 Jul 27;55(7):5478-5489. Epub 2017 Sep 27.

Laboratory of Astrocyte Biology and CNS Regeneration, Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 440, 40530, Gothenburg, Sweden.

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIM ) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIM astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIM astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIM neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.
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http://dx.doi.org/10.1007/s12035-017-0759-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994207PMC
July 2018

Sphingosine 1-phosphate signaling through its receptor S1P promotes chromosome segregation and mitotic progression.

Sci Signal 2017 Mar 28;10(472). Epub 2017 Mar 28.

CNRS, Institut de Pharmacologie et de Biologie Structurale, 31400 Toulouse, France.

Sphingosine kinase 1 (SphK1) promotes cell proliferation and survival, and its abundance is often increased in tumors. SphK1 produces the signaling lipid sphingosine 1-phosphate (S1P), which activates signaling cascades downstream five G protein-coupled receptors (S1P) to modulate vascular and immune system function and promote proliferation. We identified a new function of the SphK1-S1P pathway specifically in the control of mitosis. SphK1 depletion in HeLa cells caused prometaphase arrest, whereas its overexpression or activation accelerated mitosis. Increasing the abundance of S1P promoted mitotic progression, overrode the spindle assembly checkpoint (SAC), and led to chromosome segregation defects. S1P was secreted through the transporter SPNS2 and stimulated mitosis by binding to and activating S1P on the extracellular side, which then activated the intracellular phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Knockdown of S1P prevented the S1P-induced spindle defect phenotype. RNA interference assays revealed that the mitotic kinase Polo-like kinase 1 (PLK1) was an important effector of S1P-S1P signaling-induced mitosis in HeLa cells. Our findings identify an extracellular signal and the downstream pathway that promotes mitotic progression and may indicate potential therapeutic targets to inhibit the proliferation of cancer cells.
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http://dx.doi.org/10.1126/scisignal.aah4007DOI Listing
March 2017

Mechanisms of ciliogenesis suppression in dividing cells.

Cell Mol Life Sci 2017 03 26;74(5):881-890. Epub 2016 Sep 26.

Department of Physiology, Mie University School of Medicine, Tsu, Mie, Japan.

The primary cilium is a non-motile and microtubule-enriched protrusion ensheathed by plasma membrane. Primary cilia function as mechano/chemosensors and signaling hubs and their disorders predispose to a wide spectrum of human diseases. Most types of cells assemble their primary cilia in response to cellular quiescence, whereas they start to retract the primary cilia upon cell-cycle reentry. The retardation of ciliary resorption process has been shown to delay cell-cycle progression to the S or M phase after cell-cycle reentry. Apart from this conventional concept of ciliary disassembly linked to cell-cycle reentry, recent studies have led to a novel concept, suggesting that cells can suppress primary cilia assembly during cell proliferation. Accumulating evidence has also demonstrated the importance of Aurora-A (a protein originally identified as one of mitotic kinases) not only in ciliary resorption after cell-cycle reentry but also in the suppression of ciliogenesis in proliferating cells, whereas Aurora-A activators are clearly distinct in both phenomena. Here, we summarize the current knowledge of how cycling cells suppress ciliogenesis and compare it with mechanisms underlying ciliary resorption after cell-cycle reentry. We also discuss a reciprocal relationship between primary cilia and cell proliferation.
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http://dx.doi.org/10.1007/s00018-016-2369-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5306231PMC
March 2017

Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues.

Biochem Biophys Res Commun 2016 09 23;478(3):1323-9. Epub 2016 Aug 23.

Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, 464-8681, Japan; Department of Physiology, Mie University School of Medicine, Tsu, Mie, 514-8507, Japan. Electronic address:

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.
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http://dx.doi.org/10.1016/j.bbrc.2016.08.122DOI Listing
September 2016

Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein-Aurora A pathway.

J Cell Biol 2016 Feb;212(4):409-23

Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan Department of Cellular Oncology, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly.
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http://dx.doi.org/10.1083/jcb.201507046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754717PMC
February 2016

Phospho-Specific Antibody Probes of Intermediate Filament Proteins.

Methods Enzymol 2016 24;568:85-111. Epub 2015 Oct 24.

Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan; Department of Cellular Oncology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan. Electronic address:

Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.
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http://dx.doi.org/10.1016/bs.mie.2015.07.010DOI Listing
October 2016

Current topics of functional links between primary cilia and cell cycle.

Cilia 2015 29;4:12. Epub 2015 Dec 29.

Division of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, 464-8681 Japan ; Department of Cellular Oncology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550 Japan.

Primary cilia, microtubule-based sensory structures, orchestrate various critical signals during development and tissue homeostasis. In view of the rising interest into the reciprocal link between ciliogenesis and cell cycle, we discuss here several recent advances to understand the molecular link between the individual step of ciliogenesis and cell cycle control. At the onset of ciliogenesis (the transition from centrosome to basal body), distal appendage proteins have been established as components indispensable for the docking of vesicles at the mother centriole. In the initial step of axonemal extension, CP110, Ofd1, and trichoplein, key negative regulators of ciliogenesis, are found to be removed by a kinase-dependent mechanism, autophagy, and ubiquitin-proteasome system, respectively. Of note, their disposal functions as a restriction point to decide that the axonemal nucleation and extension begin. In the elongation step, Nde1, a negative regulator of ciliary length, is revealed to be ubiquitylated and degraded by CDK5-SCF(Fbw7) in a cell cycle-dependent manner. With regard to ciliary length control, it has been uncovered in flagellar shortening of Chlamydomonas that cilia itself transmit a ciliary length signal to cytoplasm. At the ciliary resorption step upon cell cycle re-entry, cilia are found to be disassembled not only by Aurora A-HDAC6 pathway but also by Nek2-Kif24 and Plk1-Kif2A pathways through their microtubule-depolymerizing activity. On the other hand, it is becoming evident that the presence of primary cilia itself functions as a structural checkpoint for cell cycle re-entry. These data suggest that ciliogenesis and cell cycle intimately link each other, and further elucidation of these mechanisms will contribute to understanding the pathology of cilia-related disease including cancer and discovering targets of therapeutic interventions.
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http://dx.doi.org/10.1186/s13630-015-0021-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696186PMC
December 2015

Vimentin Phosphorylation Underlies Myofibroblast Sensitivity to Withaferin A In Vitro and during Corneal Fibrosis.

PLoS One 2015 17;10(7):e0133399. Epub 2015 Jul 17.

From the Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

Vimentin is a newly recognized target for corneal fibrosis. Using primary rabbit corneal fibroblasts and myofibroblasts we show that myofibroblasts, unlike fibroblasts, display impaired cell spreading and cell polarization, which is associated with increased levels of soluble serine-38 phosphorylated vimentin (pSer38Vim). This pSer38Vim isoform is inefficiently incorporated into growing vimentin intermediate filaments (IFs) of myofibroblasts during cell spreading, and as a result, myofibroblasts maintain higher soluble pSer38Vim levels compared to fibroblasts. Moreover, the soluble vimentin-targeting small molecule and fibrotic inhibitor withaferin A (WFA) causes a potent blockade of cell spreading selectively in myofibroblasts by targeting soluble pSer38Vim for hyperphosphorylation. WFA treatment does not induce vimentin hyperphosphorylation in fibroblasts. This hyperphosphorylated pSer38Vim species in WFA-treated myofibroblasts becomes complexed with adaptor protein filamin A (FlnA), and these complexes appear as short squiggles when displaced from focal adhesions. The extracellular-signal regulated kinase (ERK) is also phosphorylated (pERK) in response to WFA, but surprisingly, pERK does not enter the nucleus but remains bound to pSer38Vim in cytoplasmic complexes. Using a model of corneal alkali injury, we show that fibrotic corneas of wild type mice possess high levels of pERK, whereas injured corneas of vimentin-deficient (Vim KO) mice that heal with reduced fibrosis have highly reduced pERK expression. Finally, WFA treatment causes a decrease in pERK and pSer38Vim expression in healing corneas of wild type mice. Taken together, these findings identify a hereto-unappreciated role for pSer38Vim as an important determinant of myofibroblast sensitivity to WFA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133399PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506086PMC
May 2016

Sphingolipids inhibit vimentin-dependent cell migration.

J Cell Sci 2015 Jun 23;128(11):2057-69. Epub 2015 Apr 23.

Department of Biosciences, Åbo Akademi University, Tykistökatu 6A, FI-20520, Turku, Finland Minerva Foundation Institute for Medical Research, Biomedicum Helsinki, Tukholmankatu 8, 00290 Helsinki, Finland

The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.
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http://dx.doi.org/10.1242/jcs.160341DOI Listing
June 2015

Cytokinetic Failure-induced Tetraploidy Develops into Aneuploidy, Triggering Skin Aging in Phosphovimentin-deficient Mice.

J Biol Chem 2015 May 6;290(21):12984-98. Epub 2015 Apr 6.

From the Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464-8681, the Departments of Cellular Oncology and

Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ∼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIM(SA/SA)) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIM(SA/SA)-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIM(SA/SA) mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging.
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http://dx.doi.org/10.1074/jbc.M114.633891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505553PMC
May 2015

Novel insights into Chk1 regulation by phosphorylation.

Cell Struct Funct 2015 25;40(1):43-50. Epub 2014 Dec 25.

Division of Biochemistry, Aichi Cancer Center Research Institute; Department of Cellular Oncology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan.

Checkpoint kinase 1 (Chk1) is a conserved protein kinase central to the cell-cycle checkpoint during DNA damage response (DDR). Until recently, ATR, a protein kinase activated in response to DNA damage or stalled replication, has been considered as the sole regulator of Chk1. Recent progress, however, has led to the identification of additional protein kinases involved in Chk1 phosphorylation, affecting the subcellular localization and binding partners of Chk1. In fact, spatio-temporal regulation of Chk1 is of critical importance not only in the DDR but also in normal cell-cycle progression. In due course, many potent inhibitors targeted to Chk1 have been developed as anticancer agents and some of these inhibitors are currently in clinical trials. In this review, we summarize the current knowledge of Chk1 regulation by phosphorylation.
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http://dx.doi.org/10.1247/csf.14017DOI Listing
November 2015

Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle.

Cell Cycle 2014 ;13(20):3302-11

a The University of Queensland Diamantina Institute; Translational Research Institute ; Brisbane , Queensland , Australia.

Whereas many components regulating the progression from S phase through G2 phase into mitosis have been identified, the mechanism by which these components control this critical cell cycle progression is still not fully elucidated. Cyclin A/Cdk2 has been shown to regulate the timing of Cyclin B/Cdk1 activation and progression into mitosis although the mechanism by which this occurs is only poorly understood. Here we show that depletion of Cyclin A or inhibition of Cdk2 during late S/early G2 phase maintains the G2 phase arrest by reducing Cdh1 transcript and protein levels, thereby stabilizing Claspin and maintaining elevated levels of activated Chk1 which contributes to the G2 phase observed. Interestingly, the Cyclin A/Cdk2 regulated APC/C(Cdh1) activity is selective for only a subset of Cdh1 targets including Claspin. Thus, a normal role for Cyclin A/Cdk2 during early G2 phase is to increase the level of Cdh1 which destabilises Claspin which in turn down regulates Chk1 activation to allow progression into mitosis. This mechanism links S phase exit with G2 phase transit into mitosis, provides a novel insight into the roles of Cyclin A/Cdk2 in G2 phase progression, and identifies a novel role for APC/C(Cdh1) in late S/G2 phase cell cycle progression.
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http://dx.doi.org/10.4161/15384101.2014.949111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615124PMC
September 2015

Direct interaction of Plk4 with STIL ensures formation of a single procentriole per parental centriole.

Nat Commun 2014 Oct 24;5:5267. Epub 2014 Oct 24.

Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.

Formation of one procentriole next to each pre-existing centriole is essential for centrosome duplication, robust bipolar spindle assembly and maintenance of genome integrity. However, the mechanisms maintaining strict control over centriole copy number are incompletely understood. Here we show that Plk4 and STIL, the key regulators of centriole formation, form a protein complex that provides a scaffold for recruiting HsSAS-6, a major component of the centriolar cartwheel, at the onset of procentriole formation. Furthermore, we demonstrate that phosphorylation of STIL by Plk4 facilitates the STIL/HsSAS-6 interaction and centriolar loading of HsSAS-6. We also provide evidence that negative feedback by centriolar STIL regulates bimodal centriolar distribution of Plk4 and seemingly restricts occurrence of procentriole formation to one site on each parental centriole. Overall, these findings suggest a mechanism whereby coordinated action of three critical factors ensures formation of a single procentriole per parental centriole.
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http://dx.doi.org/10.1038/ncomms6267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220463PMC
October 2014

Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension.

Nat Commun 2014 Oct 1;5:5081. Epub 2014 Oct 1.

1] Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan [2] Department of Cellular Oncology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan.

Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.
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http://dx.doi.org/10.1038/ncomms6081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4205846PMC
October 2014

New insights into roles of intermediate filament phosphorylation and progeria pathogenesis.

IUBMB Life 2014 Mar 23;66(3):195-200. Epub 2014 Mar 23.

Division of Biochemistry, Aichi Cancer Center Research Institute, Kanokoden, Chikusa-Ku, Nagoya, Japan.

Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Because of their insoluble nature, cellular IFs had been considered to be stable for a long time. The discovery that a purified protein kinase phosphorylated a purified IF protein and in turn induced the disassembly of IF structure in vitro led to the novel concept of dynamic IF regulation. Since then, a variety of protein kinases have been identified to phosphorylate IF proteins such as vimentin in a spatiotemporal regulated manner. A series of studies using cultured cells have demonstrated that preventing IF phosphorylation during mitosis inhibits cytokinesis by the retention of an IF bridge-like structure (IF-bridge) connecting the two daughter cells. Knock-in mice expressing phosphodeficient vimentin variants developed binucleation/aneuploidy in lens epithelial cells, which promoted microophthalmia and lens cataract. Therefore, mitotic phosphorylation of vimentin is of great importance in the completion of cytokinesis, the impairment of which promotes chromosomal instability and premature aging. © 2014 IUBMB Life, 66(3):195-200, 2014.
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http://dx.doi.org/10.1002/iub.1260DOI Listing
March 2014