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1.
Semin Cell Dev Biol ; 133: 3-9, 2023 01 15.
Article in English | MEDLINE | ID: mdl-35227625

ABSTRACT

Axon growth and guidance in the developing nervous system rely on intracellular membrane dynamics that involve endosome maturation and transport, as well as its regulated tethering to the endoplasmic reticulum (ER). Recent studies have identified several key molecules, such as protrudin, which plays a dynamic role at membrane contact sites between the ER and endosomes/lysosomes, and myosin Va, which acts as a sensor for ER-derived Ca2+ that triggers peri-ER membrane export. These molecules form different types of multiprotein complexes at the interface of organelles and, in response to their surrounding microenvironments, such as Ca2+ concentrations and lipid contents, regulate the directional movement of endosomal vesicles in extending axons. Here, we review the molecular mechanisms underlying membrane dynamics and inter-organelle interactions during neuronal morphogenesis.


Subject(s)
Endoplasmic Reticulum , Endosomes , Lysosomes , Mitochondrial Membranes , Axons
2.
Cell Mol Life Sci ; 79(4): 220, 2022 Apr 04.
Article in English | MEDLINE | ID: mdl-35368213

ABSTRACT

During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.


Subject(s)
Neovascularization, Pathologic , Neovascularization, Physiologic , Animals , Cell Movement/genetics , Focal Adhesion Protein-Tyrosine Kinases/genetics , Human Umbilical Vein Endothelial Cells , Humans , Mice , Neovascularization, Pathologic/genetics , Neovascularization, Physiologic/genetics , Vesicular Transport Proteins
3.
Genes Cells ; 23(6): 418-434, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29644770

ABSTRACT

Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) responds to growth factors and nutrient availability. Amino acids induce the recruitment of mTORC1 to the lysosomal membrane and its consequent activation, but the molecular mechanism of such activation has remained unclear. We have now examined the role of TMEM55B, a lysosomal protein of unknown molecular function, in this process on the basis of the results of proteomics and immunofluorescence analyses showing that TMEM55B interacts with many proteins that participate in mTORC1 activation including components of the vacuolar-type proton ATPase (V-ATPase) and Ragulator complexes at the lysosomal membrane. The amino acid-induced phosphorylation of the mTORC1 substrates S6K and 4E-BP was attenuated in TMEM55B-depleted cells compared with control cells. Depletion of TMEM55B was also found to evoke lysosomal stress as showed by translocation of the transcription factor TFEB to the nucleus. Furthermore, recruitment of the V1 domain subcomplex of V-ATPase to lipid rafts was abrogated in TMEM55B-depleted cells. Collectively, our results suggest that TMEM55B contributes to assembly of the V-ATPase complex in lipid rafts of the lysosomal membrane and to subsequent activation of mTORC1.


Subject(s)
Amino Acids/metabolism , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Phosphoinositide Phosphatases/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Vesicular Transport Proteins/metabolism , Animals , Enzyme Activation , Female , HEK293 Cells , HeLa Cells , Homeostasis , Humans , Mice, Inbred C57BL , Mice, Knockout , Phosphoinositide Phosphatases/chemistry , Phosphorylation , Protein Interaction Domains and Motifs , Signal Transduction , Vesicular Transport Proteins/chemistry
4.
Genes Cells ; 23(7): 599-605, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29845697

ABSTRACT

Mammalian target of rapamycin complex 1 (mTORC1) kinase is a master regulator of the cellular response to nutrition-related signals such as insulin and amino acids. mTORC1 is activated on the lysosomal membrane and induces phosphorylation of a variety of downstream molecules. We previously showed that activated mTORC1 induces protein phosphatase 2A (PP2A)-mediated dephosphorylation of the transcription factor forkhead box K1 (FOXK1). The mechanism underlying the signal transduction from the cytoplasmic mTORC1 to the nuclear FOXK1 has remained unclear, however, we now show that a nuclear-cytoplasmic transport system is necessary for the mTORC1-FOXK1 signal transduction. This reaction is mediated by a shuttling protein B56, which is a regulatory subunit of PP2A and plays an essential role in the mTORC1-dependent dephosphorylation of FOXK1. These results suggest that PP2AB56 phosphatase contributes to the signaling for mTORC1-dependent transcriptional regulation.


Subject(s)
Forkhead Transcription Factors/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Protein Phosphatase 2/metabolism , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Cytoplasm/metabolism , Humans , Nuclear Proteins/metabolism , Phosphorylation , Signal Transduction , TOR Serine-Threonine Kinases/metabolism
5.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(7): 312-320, 2019.
Article in English | MEDLINE | ID: mdl-31406056

ABSTRACT

Intracellular organelles were long viewed as isolated compartments floating in the cytosol. However, this view has been radically changed within the last decade by the discovery that most organelles communicate with the endoplasmic reticulum (ER) network via membrane contact sites (MCSs) that are essential for intracellular homeostasis. Protrudin is an ER resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking. More recently, however, protrudin has been found to serve as a tethering factor at MCSs. The roles performed by protrudin at MCSs are mediated by its various domains, including inactivation of the small GTPase Rab11, bending of the ER membrane, and functional interactions with other molecules such as the motor protein KIF5 and the ER protein VAP. Mutations in the protrudin gene (ZFYVE27) are associated with hereditary spastic paraplegia, an axonopathy that results from defective ER structure. This review, examines the pleiotropic molecular functions of protrudin and its role in interorganellar communication.


Subject(s)
Intracellular Membranes/metabolism , Organelles/metabolism , Vesicular Transport Proteins/metabolism , Animals , Base Sequence , Humans , Mutation , Protein Domains , Protein Transport , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/genetics
6.
J Biol Chem ; 289(19): 12946-61, 2014 May 09.
Article in English | MEDLINE | ID: mdl-24668814

ABSTRACT

Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.


Subject(s)
Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/pathology , Genetic Diseases, Inborn/metabolism , Genetic Diseases, Inborn/pathology , Paraplegia/metabolism , Paraplegia/pathology , Amino Acid Substitution , Animals , Carrier Proteins/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum Stress/genetics , Genetic Diseases, Inborn/genetics , Kinesins/genetics , Kinesins/metabolism , Mice , Mice, Transgenic , Mutation, Missense , Paraplegia/genetics , Proteomics , Spastic Paraplegia, Hereditary/genetics , Spastic Paraplegia, Hereditary/metabolism , Vesicular Transport Proteins
7.
J Biol Chem ; 289(37): 25639-54, 2014 Sep 12.
Article in English | MEDLINE | ID: mdl-25077969

ABSTRACT

Sonic hedgehog (Shh) is a secreted morphogen that controls the patterning and growth of various tissues in the developing vertebrate embryo, including the central nervous system. Ablation of the FK506-binding protein 38 (FKBP38) gene results in activation of the Shh signaling pathway in mouse embryos, but the molecular mechanism by which FKBP38 suppresses Shh signaling has remained unclear. With the use of a proteomics approach, we have now identified ANKMY2, a protein with three ankyrin repeats and a MYND (myeloid, Nervy, and DEAF-1)-type Zn(2+) finger domain, as a molecule that interacts with FKBP38. Co-immunoprecipitation analysis confirmed that endogenous FKBP38 and ANKMY2 interact in the mouse brain. Depletion or overexpression of ANKMY2 resulted in down- and up-regulation of Shh signaling, respectively, in mouse embryonic fibroblasts. Furthermore, combined depletion of both FKBP38 and ANKMY2 attenuated Shh signaling in these cells, suggesting that ANKMY2 acts downstream of FKBP38 to activate the Shh signaling pathway. Targeting of the zebrafish ortholog of mouse Ankmy2 (ankmy2a) in fish embryos with an antisense morpholino oligonucleotide conferred a phenotype reflecting loss of function of the Shh pathway, suggesting that the regulation of Shh signaling by ANKMY2 is conserved between mammals and fish. Our findings thus indicate that the FKBP38-ANKMY2 axis plays a key role in regulation of Shh signaling in vivo.


Subject(s)
Carrier Proteins/genetics , Hedgehog Proteins/metabolism , Signal Transduction/genetics , Tacrolimus Binding Proteins/metabolism , Animals , Gene Expression Regulation, Developmental , Hedgehog Proteins/genetics , Mice , Tacrolimus Binding Proteins/genetics , Trans-Activators/metabolism , Zebrafish/genetics
8.
Genes Cells ; 19(2): 97-111, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24251978

ABSTRACT

Protrudin is a membrane protein that regulates polarized vesicular transport. Now, we have identified a novel isoform of protrudin (protrudin-L) that contains an additional seven amino acids between the FFAT motif and the coiled-coil domain compared with the conventional isoform (protrudin-S) as a result of alternative splicing of a microexon (exon L). Protrudin-L mRNA was found to be mostly restricted to the central nervous system in mice, whereas protrudin-S mRNA was detected in all tissues examined. With the use of a splicing reporter minigene that produces two distinct fluorescent proteins in a manner dependent on the splicing pattern of protrudin transcripts, we found that most neurons express protrudin-L, whereas astrocytes express both protrudin isoforms and oligodendrocytes express only protrudin-S. Protrudin-L associated to a greater extent with vesicle-associated membrane protein-associated protein (VAP) than protrudin-S. Expression of protrudin-L in hippocampal neurons of protrudin-deficient mice also promoted neurite outgrowth more efficiently than protrudin-S. Our results suggest that protrudin-L is a neuron-specific protrudin isoform that promotes axonal elongation and contributes to the establishment of neuronal polarity.


Subject(s)
Neurons/metabolism , R-SNARE Proteins/metabolism , Vesicular Transport Proteins/metabolism , Alternative Splicing , Animals , Cell Line, Tumor , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Organ Specificity , Protein Isoforms/genetics , Protein Isoforms/metabolism , R-SNARE Proteins/genetics , Vesicular Transport Proteins/genetics
9.
Mol Brain ; 16(1): 11, 2023 01 19.
Article in English | MEDLINE | ID: mdl-36658656

ABSTRACT

Although dyslipidemia in the brain has been implicated in neurodegenerative disorders, the molecular mechanisms underlying its pathogenesis have been largely unclear. PDZD8 is a lipid transfer protein and mice deficient in PDZD8 (PDZD8-KO mice) manifest abnormal accumulation of cholesteryl esters (CEs) in the brain due to impaired lipophagy, the degradation system of lipid droplets. Here we show the detailed mechanism of PDZD8-dependent lipophagy. PDZD8 transports cholesterol to lipid droplets (LDs), and eventually promotes fusion of LDs and lysosomes. In addition, PDZD8-KO mice exhibit growth retardation, hyperactivity, reduced anxiety and fear, increased sensorimotor gating, and impaired cued fear conditioned memory and working memory. These results indicate that abnormal CE accumulation in the brain caused by PDZD8 deficiency affects emotion, cognition and adaptive behavior, and that PDZD8 plays an important role in the maintenance of brain function through lipid metabolism.


Subject(s)
Adaptor Proteins, Signal Transducing , Brain , Dyslipidemias , Animals , Mice , Brain/physiopathology , Cognition , Dyslipidemias/complications , Fear , Lipid Metabolism , Adaptor Proteins, Signal Transducing/genetics
10.
iScience ; 25(12): 105612, 2022 Dec 22.
Article in English | MEDLINE | ID: mdl-36465123

ABSTRACT

Dyslipidemia including the accumulation of cholesteryl esters (CEs) in the brain is associated with neurological disorders, although the underlying mechanism has been unclear. PDZD8, a Rab7 effector protein, transfers lipids between endoplasmic reticulum (ER) and Rab7-positive organelles and thereby promotes endolysosome maturation and contributes to the maintenance of neuronal integrity. Here we show that CEs accumulate in the brain of PDZD8-deficient mice as a result of impaired lipophagy. This CE accumulation was not affected by diet, implicating a defect in intracellular lipid metabolism. Whereas cholesterol synthesis appeared normal, degradation of lipid droplets (LDs) was defective, in the brain of PDZD8-deficient mice. PDZD8 may mediate the exchange of cholesterol and phosphatidylserine between ER and Rab7-positive organelles to promote the fusion of CE-containing LDs with lysosomes for their degradation. Our results thus suggest that PDZD8 promotes clearance of CEs from the brain by lipophagy, with this role of PDZD8 likely contributing to brain function.

11.
Nat Cell Biol ; 5(1): 28-37, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12510191

ABSTRACT

The mitochondrial localization of the membrane proteins Bcl-2 and Bcl-x(L) is essential for their anti-apoptotic function. Here we show that mitochondrial FK506-binding protein 38 (FKBP38), unlike FKBP12, binds to and inhibits calcineurin in the absence of the immunosuppressant FK506, suggesting that FKBP38 is an inherent inhibitor of this phosphatase. FKBP38 is associated with Bcl-2 and Bcl-x(L) in immunoprecipitation assays and colocalizes with these proteins in mitochondria; in addition, the expression of FKBP38 mutant proteins induces a marked redistribution of Bcl-2 and Bcl-x(L). Overexpression of FKBP38 blocks apoptosis, whereas functional inhibition of this protein by a dominant-negative mutant or by RNA interference promotes apoptosis. Thus, FKBP38 might function to inhibit apoptosis by anchoring Bcl-2 and Bcl-x(L) to mitochondria.


Subject(s)
Apoptosis/physiology , Calcineurin Inhibitors , Mitochondria/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Saccharomyces cerevisiae/physiology , Tacrolimus Binding Proteins/metabolism , Cell Line , HeLa Cells , Humans , Microscopy, Fluorescence , Protein Binding , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/metabolism , Transfection , bcl-X Protein
12.
Front Cell Dev Biol ; 8: 615600, 2020.
Article in English | MEDLINE | ID: mdl-33385000

ABSTRACT

Endosome maturation refers to the conversion of early endosomes (EEs) to late endosomes (LEs) for subsequent fusion with lysosomes. It is an incremental process that involves a combination of endosome fusion and fission and which occurs at contact sites between endosomes and the endoplasmic reticulum (ER), with knowledge of the underlying mechanisms having increased greatly in recent years. Protrudin is an ER-resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking, whereas PDZD8 is a mammalian paralog of a subunit of the yeast ERMES (ER-mitochondrial encounter structure) complex that possesses lipid transfer activity. A complex of protrudin and PDZD8 was recently found to promote endosome maturation by mediating lipid transfer at ER-endosome membrane contact sites. This review focuses on the roles of the protrudin-PDZD8 complex in tethering of endosomes to the ER, in mediating lipid transfer at such contact sites, and in regulating endosome dynamics, especially in neuronal cells. It also addresses the physiological contribution of endosome maturation mediated by this complex to neuronal polarity and integrity.

13.
Mol Brain ; 13(1): 146, 2020 11 10.
Article in English | MEDLINE | ID: mdl-33172474

ABSTRACT

Protrudin is a protein that resides in the membrane of the endoplasmic reticulum and is highly expressed in the nervous system. Although mutations in the human protrudin gene (ZFYVE27, also known as SPG33) give rise to hereditary spastic paraplegia (HSP), the physiological role of the encoded protein has been largely unclear. We therefore generated mice deficient in protrudin and subjected them to a battery of behavioral tests designed to examine their intermediate phenotypes. The protrudin-deficient mice were found to have a reduced body size and to manifest pleiotropic behavioral abnormalities, including hyperactivity, depression-like behavior, and deficits in attention and fear-conditioning memory. They exhibited no signs of HSP, however, consistent with the notion that HSP-associated mutations of protrudin may elicit neural degeneration, not as a result of a loss of function, but rather as a result of a gain of toxic function. Overall, our results suggest that protrudin might play an indispensable role in normal neuronal development and behavior.


Subject(s)
Attention/physiology , Behavior, Animal , Conditioning, Classical , Cues , Depression/physiopathology , Fear/physiology , Vesicular Transport Proteins/deficiency , Animals , Anxiety/complications , Anxiety/physiopathology , Depression/complications , Electrophysiological Phenomena , Gene Targeting , Hippocampus/physiopathology , Memory, Short-Term , Mice, Inbred C57BL , Mice, Knockout , Neuronal Plasticity , Phenotype , Reflex, Startle , Social Interaction , Vesicular Transport Proteins/metabolism
14.
Nat Commun ; 11(1): 4576, 2020 09 11.
Article in English | MEDLINE | ID: mdl-32917905

ABSTRACT

Endosome maturation depends on membrane contact sites (MCSs) formed between endoplasmic reticulum (ER) and endolysosomes (LyLEs). The mechanism underlying lipid supply for this process and its pathophysiological relevance remains unclear, however. Here, we identify PDZD8-the mammalian ortholog of a yeast ERMES subunit-as a protein that interacts with protrudin, which is located at ER-LyLE MCSs. Protrudin and PDZD8 promote the formation of ER-LyLE MCSs, and PDZD8 shows the ability to extract various lipids from the ER. Overexpression of both protrudin and PDZD8 in HeLa cells, as well as their depletion in mouse primary neurons, impairs endosomal homeostasis by inducing the formation of abnormal large vacuoles reminiscent of those apparent in spastin- or REEP1-deficient neurons. The protrudin-PDZD8 system is also essential for the establishment of neuronal polarity. Our results suggest that protrudin and PDZD8 cooperatively promote endosome maturation by mediating ER-LyLE tethering and lipid extraction at MCSs, thereby maintaining neuronal polarity and integrity.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Endosomes/physiology , Lipid Metabolism , Neurons/metabolism , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Disease Models, Animal , Endoplasmic Reticulum/metabolism , Female , HEK293 Cells , HeLa Cells , Humans , Lipids , Liposomes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Mitochondria , Protein Domains , Proteomics , Recombinant Proteins , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/genetics
15.
Genes Cells ; 13(6): 635-51, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18459960

ABSTRACT

FKBP38 (also known as FKBP8) is a transmembrane chaperone protein that inhibits apoptosis by recruiting the anti-apoptotic proteins Bcl-2 and Bcl-x(L) to mitochondria. We have now generated mice harboring a loss-of-function mutation in Fkbp38. The Fkbp38(-/-) mice die soon after birth manifesting defects in neural tube closure in the thoraco-lumbar-sacral region (spina bifida) as well as skeletal defects including scoliosis, rib deformities, club foot and curled tail. The neuroepithelium is disorganized and that formation of dorsal root ganglia is defective in Fkbp38(-/-) embryos, likely as a result of an increased frequency of apoptosis and aberrant migration of neuronal cells. Furthermore, the extension of nerve fibers in the spinal cord is abnormal in the mutant embryos. To explore the mechanisms underlying these characteristics, we screened for proteins that interact with FKBP38 in the yeast two-hybrid system and thereby identified protrudin, a protein that promotes process formation by regulating membrane trafficking. Protrudin was found to be hyperphosphorylated in the brain of Fkbp38(-/-) mice, suggesting that FKBP38 regulates protrudin-dependent membrane recycling and neurite outgrowth. Together, our findings suggest that FKBP38 is required for neuroectodermal organization during neural tube formation as a result of its anti-apoptotic activity and regulation of neurite extension.


Subject(s)
Apoptosis , Neurites/metabolism , Tacrolimus Binding Proteins/metabolism , Animals , Body Patterning , Brain/cytology , Brain/metabolism , Carrier Proteins/metabolism , Embryo, Mammalian/metabolism , Mice , Neural Tube Defects/metabolism , Neurons/metabolism , Osteogenesis , Phosphorylation , Tacrolimus Binding Proteins/genetics , Vesicular Transport Proteins
18.
Cell Rep ; 23(7): 1988-2000, 2018 05 15.
Article in English | MEDLINE | ID: mdl-29768199

ABSTRACT

The gene encoding the chromatin remodeler CHD8 is the most frequently mutated gene in individuals with autism spectrum disorder (ASD). Heterozygous mutations in CHD8 give rise to ASD that is often accompanied by macrocephaly, gastrointestinal complaints, and slender habitus. Whereas most phenotypes of CHD8 haploinsufficiency likely result from delayed neurodevelopment, the mechanism underlying slender habitus has remained unknown. Here, we show that CHD8 interacts with CCAAT/enhancer-binding protein ß (C/EBPß) and promotes its transactivation activity during adipocyte differentiation. Adipogenesis was impaired in Chd8-deleted preadipocytes, with the upregulation of C/EBPα and peroxisome-proliferator-activated receptor γ (PPARγ), two master regulators of this process, being attenuated in mutant cells. Furthermore, mice with CHD8 ablation in white preadipocytes had a markedly reduced white adipose tissue mass. Our findings reveal a mode of C/EBPß regulation by CHD8 during adipogenesis, with CHD8 deficiency resulting in a defect in the development of white adipose tissue.


Subject(s)
Adipogenesis , Autistic Disorder/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , DNA-Binding Proteins/metabolism , 3T3-L1 Cells , Adipocytes/metabolism , Adipocytes/pathology , Adipogenesis/genetics , Adipose Tissue, White/pathology , Animals , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Gene Expression Regulation , Genome , HEK293 Cells , Humans , Hypertrophy , Mice , Mice, Inbred C57BL , PPAR gamma/metabolism , Protein Binding
19.
Sci Rep ; 7: 41130, 2017 01 20.
Article in English | MEDLINE | ID: mdl-28106138

ABSTRACT

Alternative splicing gives rise to diversity of the proteome, and it is especially prevalent in the mammalian nervous system. Indeed, many factors that control the splicing process govern nervous system development. Among such factors, SRRM4 is an important regulator of aspects of neural differentiation including neurite outgrowth. The mechanism by which SRRM4 regulates neurite outgrowth has remained poorly understood, however. We now show that SRRM4 regulates the splicing of protrudin gene (Zfyve27) transcripts in neuronal cells. SRRM4 was found to promote splicing of protrudin pre-mRNA so as to include a microexon (exon L) encoding seven amino acids in a neuron-specific manner. The resulting protein (protrudin-L) promotes neurite outgrowth during neurogenesis. Depletion of SRRM4 in Neuro2A cells impaired inclusion of exon L in protrudin mRNA, resulting in the generation of a shorter protein isoform (protrudin-S) that is less effective at promoting neurite extension. SRRM4 was found to recognize a UGC motif that is located immediately upstream of exon L and is necessary for inclusion of exon L in the mature transcript. Deletion of exon L in Neuro2A or embryonic stem cells inhibited neurite outgrowth. Our results suggest that SRRM4 controls neurite outgrowth through regulation of alternative splicing of protrudin transcripts.


Subject(s)
Alternative Splicing , Nerve Tissue Proteins/genetics , Neuronal Outgrowth , Vesicular Transport Proteins/genetics , Animals , Cell Line, Tumor , Exons , Mice , Neurogenesis , RNA, Messenger/genetics
20.
Nihon Rinsho ; 62(2): 405-12, 2004 Feb.
Article in Japanese | MEDLINE | ID: mdl-14968553

ABSTRACT

Various apoptotic stimuli induce mitochondrial dysfunction. Bcl-2 and Bcl-xL antagonize apoptosis by blocking the release of caspase activators such as cytochrome c from mitochondria. We demonstrated that FKBP38, a member of the immunophilin family, interacts and targets these anti-apoptotic proteins Bcl-2 and Bcl-xL, thereby assisting them in their pro-survival role. FKBP38 is specifically localized on mitochondria, at which FKBP38 is colocalized with Bcl-2 and Bcl-xL. Expression of exogenous FKBP38 promotes mitochondrial targeting of Bcl-2 and Bcl-xL, while dominant-negative FKBP38 or siRNA of FKBP38 disturbs their localization. On the other hand, unlike FKBP12, FKBP38 inhibits serine/threonine phosphatase calcineurin in an FK506-independent manner. Overexpression of FKBP38 inhibits apoptosis, while expression of dominant-negative FKBP38 or depletion of endogenous FKBP38 increases the sensitivity for apoptosis. Thus, FKBP38 has unique features among members of the immunophilin family.


Subject(s)
Apoptosis/genetics , Calcineurin Inhibitors , Mitochondria/genetics , Tacrolimus Binding Proteins/physiology , Animals , Apoptosis/drug effects , Cytochromes c/metabolism , Depression, Chemical , Humans , Mitochondria/enzymology , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , Proto-Oncogene Proteins c-bcl-2/physiology , Tacrolimus Binding Proteins/pharmacology , bcl-X Protein
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