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1.
Am J Pathol ; 194(1): 135-149, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37918800

RESUMO

Osteophytes in osteoarthritis (OA) joints contribute to restriction of joint movement, joint pain, and OA progression, but little is known about osteophyte regulators. Examination of gene expression related to cartilage extracellular matrix, endochondral ossification, and growth factor signaling in articular cartilage and osteophytes obtained from OA knee joints showed that several genes such as COL1A1, VCAN, BGLAP, BMP8B, RUNX2, and SOST were overexpressed in osteophytes compared with articular cartilage. Ratios of mesenchymal stem/progenitor cells, which were characterized by co-expression of CD105 and CD166, were significantly higher in osteophytic cells than articular cells. A three-dimensional culture method for cartilage and osteophyte cells was developed by modification of cultures of self-assembled spheroid cell organoids (spheroids). These spheroids cultured in the media for mesenchymal stem cells containing transforming growth factor-ß3 showed characteristic morphologies and gene expression profiles of articular cartilage and osteophytes, respectively. The effects of IL-1ß, tumor necrosis factor-α, and IL-6 on the spheroids of articular and osteophytic cells were studied. To the best of our knowledge, they provide the first evidence that IL-6 suppresses the spheroid size of osteophytic cells by inducing apoptosis and reducing extracellular matrix molecules. These data show that IL-6 is the suppressor of osteophyte growth and suggest that IL-6 expression and/or activity are implicated in the regulation of osteophyte formation in pathologic joints.


Assuntos
Cartilagem Articular , Osteoartrite do Joelho , Osteoartrite , Osteófito , Humanos , Cartilagem Articular/patologia , Condrócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-6/metabolismo , Articulação do Joelho/patologia , Osteoartrite/patologia , Osteoartrite do Joelho/metabolismo , Osteófito/genética , Osteófito/metabolismo , Osteófito/patologia
2.
Proc Natl Acad Sci U S A ; 117(51): 32739-32749, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33273113

RESUMO

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Complexos Multiproteicos/genética , Proteínas de Plantas/genética , Compartimento Celular , Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Complexos Multiproteicos/metabolismo , Proteínas de Plantas/metabolismo , Transporte Proteico , Homologia de Sequência de Aminoácidos
3.
Plant Cell ; 30(11): 2677-2703, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30309901

RESUMO

Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.


Assuntos
Proteínas de Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Trifosfato de Adenosina/metabolismo , Cloroplastos/metabolismo , Malato Desidrogenase/metabolismo , Transporte Proteico
4.
Proc Natl Acad Sci U S A ; 113(3): 775-80, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26739563

RESUMO

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Cistinúria/metabolismo , Túbulos Renais Proximais/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Animais , Anticorpos/metabolismo , Western Blotting , Transportador 3 de Aminoácido Excitatório/metabolismo , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Rim/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Proteolipídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética
5.
Proc Natl Acad Sci U S A ; 112(5): 1553-8, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25605899

RESUMO

Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Trifosfato de Adenosina/biossíntese , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/química , Transferência Ressonante de Energia de Fluorescência , Hipóxia/enzimologia , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mitocôndrias/enzimologia , Fosforilação Oxidativa , Conformação Proteica
6.
Proc Natl Acad Sci U S A ; 111(1): 273-8, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24344269

RESUMO

The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/metabolismo , Genes de Troca , Fosforilação Oxidativa , Animais , Técnicas Biossensoriais , Bovinos , Sobrevivência Celular , Fase G1 , Células HEK293 , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Oligomicinas/química , Análise de Sequência com Séries de Oligonucleotídeos , Consumo de Oxigênio , Fosforilação , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Fase de Repouso do Ciclo Celular , Fatores de Tempo
7.
J Atheroscler Thromb ; 2024 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-39085140

RESUMO

AIM: Postprandial hypertriglyceridemia (PHTG) is an independent risk factor for coronary heart diseases. PHTG exhibits accumulation of apoB-48 containing chylomicron remnants (CM-Rs) and apoB-100 containing VLDL remnants (VLDL-Rs), which are both known to be atherogenic. However, unlike VLDL-Rs, structural and functional characterization of CM-Rs remains to be elucidated due to challenges in separating CM-Rs from VLDL-Rs. Recently, we successfully isolated CM-Rs and VLDL-Rs utilizing anti-apoB-48 or apoB-100 specific antibodies. This study aimed to characterize the proteome of CM-Rs along with that of VLDL-Rs. METHODS: Eight healthy subjects were enrolled. Venous blood was drawn 3 hours after high-fat-containing meals. We isolated CM-Rs and VLDL-Rs from sera through combination of ultracentrifugation and immunoprecipitation using apoB-48 or apoB-100 specific antibodies, followed by shotgun proteomic analysis. RESULTS: We identified 42 CM-Rs or VLDL-Rs-associated proteins, including 11 potential newly identified proteins such as platelet basic protein (PPBP) and platelet factor 4, which are chemokines secreted from platelets. ApoA-I, apoA-IV, and clusterin, which are also known as HDL-associated proteins, were significantly more abundant in CM-Rs. Interestingly, apoC-I, which reduces the activity of lipoprotein lipase and eventually inhibits catabolism of remnant proteins, was also more abundant in CM-Rs. Moreover, we identified proteins involved in complement regulation such as complement C3 and vitronectin, and those involved in acute-phase response such as PPBP, serum amyloid A protein 2, and protein S100-A8, in both CM-Rs and VLDL-Rs. CONCLUSIONS: We have firstly characterized the proteome of CM-Rs. These findings may provide an explanation for the atherogenic properties of CM-Rs.

8.
J Hum Genet ; 58(11): 711-9, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24026176

RESUMO

α-synuclein (SNCA) is an established susceptibility gene for Parkinson's disease (PD), one of the most common human neurodegenerative disorders. Increased SNCA is considered to lead to PD and dementia with Lewy bodies. Four single-nucleotide polymorphisms (SNPs) in SNCA 3' region were prominently associated with PD among different ethnic groups. To examine how these SNPs influence disease susceptibility, we analyzed their potential effects on SNCA gene expression. We found that rs356219 showed allele-specific features. Gel shift assay using nuclear extracts from SH-SY5Y cells showed binding of one or more proteins to the protective allele, rs356219-A. We purified the rs356219-A-protein complex with DNA affinity beads and identified a bound protein using mass spectrometry. This protein, YY1 (Yin Yang 1), is an ubiquitous transcription factor with multiple functions. We next investigated SNCA expression change in SH-SY5Y cells by YY1 transfection. We also analyzed the expression of antisense noncoding RNA (ncRNA) RP11-115D19.1 in SNCA 3'-flanking region, because rs356219 is located in intron of RP11-115D19.1. Little change was observed in SNCA expression levels; however, RP11-115D19.1 expression was prominently stimulated by YY1. In autopsied cortices, positive correlation was observed among RP11-115D19.1, SNCA and YY1 expression levels, suggesting their functional interactions in vivo. Knockdown of RP11-115D19.1 increased SNCA expression significantly in SH-SY5Y cells, suggesting its repressive effect on SNCA expression. Our findings of the protective allele-specific YY1 and antisense ncRNA raised a novel possible mechanism to regulate SNCA expression.


Assuntos
Regiões 3' não Traduzidas , Proteínas Nucleares/metabolismo , RNA Antissenso/biossíntese , RNA não Traduzido/biossíntese , Fatores de Transcrição/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Idoso , Povo Asiático , Proteínas de Ciclo Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/química , Polimorfismo de Nucleotídeo Único , Ligação Proteica , RNA Antissenso/genética , RNA não Traduzido/genética , Fatores de Transcrição/química , População Branca , alfa-Sinucleína/genética
9.
J Clin Lipidol ; 13(2): 317-325, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30745272

RESUMO

BACKGROUND: We previously reported that the patients with cholesteryl ester transfer protein (CETP) deficiency (CETP-D) show marked changes in the size and lipid compositions of high-density lipoprotein (HDL) and that they are not protected from atherosclerotic cardiovascular diseases, despite increased serum HDL-cholesterol (HDL-C) levels. HDL particles carry a variety of proteins, some of which are known to have antiatherogenic functions. OBJECTIVE: This study aimed to investigate the protein composition of HDL particles in patients with CETP-D. METHODS: Eight patients with complete deficiency of CETP and 8 normolipidemic healthy subjects were enrolled. We performed shotgun proteomic analysis to investigate the proteome of ultracentrifugally isolated HDL. RESULTS: We identified 79 HDL-associated proteins involved in lipid metabolism, protease inhibition, complement regulation, and acute-phase response, including 5 potential newly identified HDL-associated proteins such as angiopoietin-like3 (ANGPTL3). Spectral counts of apolipoprotein (apo) E were increased in patients with CETP-D compared with controls (60.3 ± 6.9 vs 43.7 ± 2.5, P < .001), which is concordant with our previous report. Complement regulatory proteins such as C3, C4a, C4b, and C9 were also significantly enriched in HDL from patients with CETP-D. Furthermore, apoC-III and ANGPTL3, both of which are now known to associate with increased atherosclerotic cardiovascular diseases, were enriched in patients with CETP-D compared with normolipidemic subjects (35.9 ± 5.3 vs 27.1 ± 3.7, 2.3 ± 1.1 vs 0.4 ± 1.1, respectively; P < .01). CONCLUSION: We have characterized HDL-associated proteins in patients with CETP-D. We identified a significant increase in the amount of apoE, apoC-III, ANGPTL3, and complement regulatory proteins. These proteomic changes might be partly responsible for the enhanced atherogenicity of patients with CETP-D.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/deficiência , Erros Inatos do Metabolismo Lipídico/metabolismo , Lipoproteínas HDL/metabolismo , Proteômica , Reação de Fase Aguda/complicações , Proteínas de Transferência de Ésteres de Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Proteínas do Sistema Complemento/metabolismo , Feminino , Humanos , Erros Inatos do Metabolismo Lipídico/sangue , Erros Inatos do Metabolismo Lipídico/complicações , Erros Inatos do Metabolismo Lipídico/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico
10.
PLoS One ; 12(3): e0173908, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28291804

RESUMO

Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.


Assuntos
Proteínas de Membrana/metabolismo , Proteômica , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Carpas
11.
Sci Rep ; 6: 36946, 2016 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-27833131

RESUMO

The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, Nε-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest.


Assuntos
Adenoviridae/genética , Aminoácidos/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Células A549 , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Archaea/genética , Proteínas Arqueais/metabolismo , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Código Genético , Vetores Genéticos , Células HEK293 , Células HT29 , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Lisina/análogos & derivados , Lisina/química
12.
Nat Commun ; 6: 6137, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25635515

RESUMO

Augmented AMP-activated protein kinase (AMPK) activity inhibits cell migration, possibly contributing to the clinical benefits of chemical AMPK activators in preventing atherosclerosis, vascular remodelling and cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we identify PDZ and LIM domain 5 (Pdlim5) as a novel AMPK substrate and show that it plays a critical role in the inhibition of cell migration. AMPK directly phosphorylates Pdlim5 at Ser177. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation. Consistent with this observation, S177D-Pdlim5 suppresses Rac1 activity at the cell periphery and displaces the Arp2/3 complex from the leading edge. Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery. Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Linhagem Celular , Movimento Celular/fisiologia , Camundongos , Fosforilação , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo
13.
Neurochem Int ; 61(2): 240-50, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22609377

RESUMO

The CNS synapse is an adhesive junction differentiated for chemical neurotransmission and is equipped with presynaptic vesicles and postsynaptic neurotransmitter receptors. Cell adhesion molecule cadherins not only maintain connections between pre- and postsynaptic membranes but also modulate the efficacy of synaptic transmission. Although the components of the cadherin-mediated adhesive apparatus have been studied extensively in various cell systems, the complete picture of these components, particularly at the synaptic junction, remains elusive. Here, we describe the proteomic assortment of the N-cadherin-mediated synaptic adhesion apparatus in cultured hippocampal neurons. N-cadherin immunoprecipitated from Triton X-100-solubilized neuronal extract contained equal amounts of ß- and α-catenins, as well as F-actin-related membrane anchor proteins such as integrins bridged with α-actinin-4, and Na(+)/K(+)-ATPase bridged with spectrins. A close relative of ß-catenin, plakoglobin, and its binding partner, desmoplakin, were also found, suggesting that a subset of the N-cadherin-mediated adhesive apparatus also anchors intermediate filaments. Moreover, dynein heavy chain and LEK1/CENPF/mitosin were found. This suggests that internalized pools of N-cadherin in trafficking vesicles are conveyed by dynein motors on microtubules. In addition, ARVCF and NPRAP/neurojungin/δ2-catenin, but not p120ctn/δ1-catenin or plakophilins-1, -2, -3, -4 (p0071), were found, suggesting other possible bridges to microtubules. Finally, synaptic stimulation by membrane depolarization resulted in an increased 93-kDa band, which corresponded to proteolytically truncated ß-catenin. The integration of three different classes of cytoskeletal systems found in the synaptic N-cadherin complex may imply a dynamic switching of adhesive scaffolds in response to synaptic activity.


Assuntos
Caderinas/metabolismo , Citoesqueleto/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteômica/métodos , Actinas/metabolismo , Animais , Western Blotting , Cateninas/metabolismo , Cromatografia Líquida , Feminino , Hipocampo/citologia , Imunoprecipitação , Proteínas de Filamentos Intermediários/metabolismo , Espectrometria de Massas , Camundongos , Microtúbulos/metabolismo , Plasticidade Neuronal/fisiologia , Gravidez , Ratos , Sinapses/metabolismo , Sinapses/fisiologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Tripsina/química , delta Catenina
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