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1.
J Cell Sci ; 134(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635908

RESUMO

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise five major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with fluorescence recovery after photobleaching (FRAP) and fluorescence loss and localisation after photobleaching (FLAP), we show that desmosomes consist of two contrasting protein moieties or modules: a very stable moiety of desmosomal cadherins, desmoplakin and plakoglobin, and a highly mobile plakophilin (Pkp2a). As desmosomes mature from Ca2+ dependence to Ca2+-independent hyper-adhesion, their stability increases, but Pkp2a remains highly mobile. We show that desmosome downregulation during growth-factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable moiety and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in desmosomes. This article has an associated First Person interview with the first author of the paper.


Assuntos
Desmossomos , Placofilinas , Caderinas , Membrana Celular , Desmogleínas , Desmoplaquinas/genética , Humanos , Placofilinas/genética , gama Catenina
2.
Sci Rep ; 9(1): 2524, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792430

RESUMO

Superresolution microscopy offers the advantage of imaging biological structures within cells at the nano-scale. Here we apply two superresolution microscopy techniques, specifically 3D structured illumination microscopy (3D-SIM) and direct stochastic optical reconstruction microscopy (dSTORM), a type of single molecule localisation microscopy, to localise IRSp53 protein and its I-BAR domain in relation to F-actin within filopodia. IRSp53 generates dynamic (extending and retracting) filopodia 300 nm wide with a distinct gap between IRSp53 and F-actin. By contrast, protrusions induced by the I-BAR domain alone are non-dynamic measuring between 100-200 nm in width and exhibit a comparatively closer localisation of the I-BAR domain with the F-actin. The data suggest that IRSp53 membrane localisation is spatially segregated to the lateral edges of filopodia, in contrast to the I-BAR domain is uniformly distributed throughout the membranes of protrusions. Modeling of fluorescence recovery after photobleaching (FRAP) data suggests that a greater proportion of I-BAR domain is associated with membranes when compared to full length IRSp53. The significance of this new data relates to the role filopodia play in cell migration and its importance to cancer.


Assuntos
Actinas/genética , Membrana Celular/ultraestrutura , Proteínas do Tecido Nervoso/ultraestrutura , Imagem Individual de Molécula/métodos , Actinas/ultraestrutura , Animais , Membrana Celular/genética , Movimento Celular/genética , Recuperação de Fluorescência Após Fotodegradação/métodos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Proteínas do Tecido Nervoso/genética , Ligação Proteica/genética , Domínios Proteicos/genética
3.
PLoS One ; 11(9): e0153832, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27655137

RESUMO

The receptor for advanced glycation end-products (RAGE) is involved in the onset and progression of several inflammatory diseases. The RAGE primary transcript undergoes numerous alternative splicing (AS) events, some of which are species-specific. Here, we characterize the mouse-specific mRAGE_v4 splice variant, which is conserved in rodents and absent in primates. mRAGE_v4 derives from exon 9 skipping and encodes a receptor (M-RAGE) that lacks 9 amino acids between the transmembrane and the immunoglobulin (Ig) domains. RNA-Seq data confirm that in mouse lung mRAGE_v4 is the most abundant RAGE mRNA isoform after mRAGE, which codes for full-length RAGE (FL-RAGE), while in heart all RAGE variants are almost undetectable. The proteins M-RAGE and FL-RAGE are roughly equally abundant in mouse lung. Contrary to FL-RAGE, M-RAGE is extremely resistant to shedding because it lacks the peptide motif recognized by both ADAM10 and MMP9, and does not contribute significantly to soluble cRAGE formation. Thus, a cassette exon in RAGE corresponds to a specific function of the RAGE protein-the ability to be shed. Given the differences in RAGE AS variants between rodents and humans, caution is due in the interpretation of results obtained in mouse models of RAGE-dependent human pathologies.

4.
Cell ; 163(6): 1388-99, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26627736

RESUMO

Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance.


Assuntos
Evolução Biológica , Genes Essenciais , Saccharomyces cerevisiae/genética , Deleção de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/metabolismo
5.
Am J Hum Genet ; 97(3): 483-92, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26320891

RESUMO

Progeroid disorders overlapping with De Barsy syndrome (DBS) are collectively denoted as autosomal-recessive cutis laxa type 3 (ARCL3). They are caused by biallelic mutations in PYCR1 or ALDH18A1, encoding pyrroline-5-carboxylate reductase 1 and pyrroline-5-carboxylate synthase (P5CS), respectively, which both operate in the mitochondrial proline cycle. We report here on eight unrelated individuals born to non-consanguineous families clinically diagnosed with DBS or wrinkly skin syndrome. We found three heterozygous mutations in ALDH18A1 leading to amino acid substitutions of the same highly conserved residue, Arg138 in P5CS. A de novo origin was confirmed in all six probands for whom parental DNA was available. Using fibroblasts from affected individuals and heterologous overexpression, we found that the P5CS-p.Arg138Trp protein was stable and able to interact with wild-type P5CS but showed an altered sub-mitochondrial distribution. A reduced size upon native gel electrophoresis indicated an alteration of the structure or composition of P5CS mutant complex. Furthermore, we found that the mutant cells had a reduced P5CS enzymatic activity leading to a delayed proline accumulation. In summary, recurrent de novo mutations, affecting the highly conserved residue Arg138 of P5CS, cause an autosomal-dominant form of cutis laxa with progeroid features. Our data provide insights into the etiology of cutis laxa diseases and will have immediate impact on diagnostics and genetic counseling.


Assuntos
Opacidade da Córnea/genética , Opacidade da Córnea/patologia , Cútis Laxa/genética , Cútis Laxa/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Mutação de Sentido Incorreto/genética , Ornitina-Oxo-Ácido Transaminase/genética , Sequência de Aminoácidos , Sequência de Bases , Genes Dominantes/genética , Humanos , Dados de Sequência Molecular , Linhagem , Prolina/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Pele/patologia , Especificidade da Espécie
6.
Proc Natl Acad Sci U S A ; 112(35): E4864-73, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26283369

RESUMO

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.


Assuntos
Adesões Focais/metabolismo , Nanoestruturas , Talina/fisiologia , Humanos , Microscopia de Fluorescência
7.
Antioxid Redox Signal ; 21(12): 1726-40, 2014 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-24094148

RESUMO

AIMS: Using primary cultures of mouse hippocampal neurons, we studied the molecular and functional interactions between high mobility group box-1 (HMGB1) and the N-methyl-d-aspartate receptor (NMDAR), two proteins playing a key role in neuronal hyperexcitability. By measuring NMDA-induced calcium (Ca(2+)) increase in neuronal somata and neurotoxicity as functional read-out parameters, we explored the role of the redox state of HMGB1, the receptor involved, and the molecular signaling underlying its interactions with postsynaptic NMDAR. We also investigated whether HMGB1 redox state affects its proconvulsive effects in mice. RESULTS: Nonoxidizable HMGB1 with a triple cysteine-to-serine replacement (3S-HMGB1) was ineffective on NMDA response. Conversely, the disulfide-containing form of HMGB1 dose dependently enhanced NMDA-induced Ca(2+) increase in neuronal cell bodies. This effect was prevented by BoxA, a competitive HMGB1 antagonist, and by Rhodobacter sphaeroides lipopolysaccharide (LPS-RS), a toll-like receptor 4 (TLR4) selective antagonist, and it was abrogated in neurons lacking TLR4 while persisting in the absence of receptor for advanced glycation end products (RAGE). TLR4 and NMDAR subunit 1 (NR1) and 2B (NR2B) were colocalized in neurons. Disulfide HMGB1 effect on NMDA-induced Ca(2+) influx was prevented by 3-O-methylsphingomyelin (3-O-MS) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine, (PP2) selective inhibitors of neutral sphingomyelinase and Src-family Tyr kinases, respectively. Disulfide HMGB1, but not 3S-HMGB1, increased Tyr(1472) phosphorylation of the NR2B subunit of the NMDAR, which is known to increase Ca(2+) channel permeability. Similarly, disulfide HMGB1 increased NMDA-induced neuronal cell death in vitro and enhanced kainate-induced seizures in vivo. INNOVATION AND CONCLUSION: We describe a novel molecular neuronal pathway activated by HMGB1 that could be targeted in vivo to prevent neurodegeneration and seizures mediated by excessive NMDARs stimulation.


Assuntos
Dissulfetos/metabolismo , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução
8.
PLoS One ; 8(12): e82590, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24358210

RESUMO

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/genética , Proteínas dos Microfilamentos/fisiologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Células Cultivadas , Quimiotaxia de Leucócito/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Esfingosina-1-Fosfato
9.
J Exp Med ; 209(9): 1519-28, 2012 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-22869893

RESUMO

Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S-HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states.


Assuntos
Citocinas/metabolismo , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Leucócitos/citologia , Animais , Anticorpos Monoclonais/farmacologia , Movimento Celular/efeitos dos fármacos , Fatores Quimiotáticos/metabolismo , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/imunologia , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/lesões , Mutação , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Nat Med ; 16(4): 413-9, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20348922

RESUMO

Brain inflammation is a major factor in epilepsy, but the impact of specific inflammatory mediators on neuronal excitability is incompletely understood. Using models of acute and chronic seizures in C57BL/6 mice, we discovered a proconvulsant pathway involving high-mobility group box-1 (HMGB1) release from neurons and glia and its interaction with Toll-like receptor 4 (TLR4), a key receptor of innate immunity. Antagonists of HMGB1 and TLR4 retard seizure precipitation and decrease acute and chronic seizure recurrence. TLR4-defective C3H/HeJ mice are resistant to kainate-induced seizures. The proconvulsant effects of HMGB1, like those of interleukin-1beta (IL-1beta), are partly mediated by ifenprodil-sensitive N-methyl-d-aspartate (NMDA) receptors. Increased expression of HMGB1 and TLR4 in human epileptogenic tissue, like that observed in the mouse model of chronic seizures, suggests a role for the HMGB1-TLR4 axis in human epilepsy. Thus, HMGB1-TLR4 signaling may contribute to generating and perpetuating seizures in humans and might be targeted to attain anticonvulsant effects in epilepsies that are currently resistant to drugs.


Assuntos
Proteína HMGB1/fisiologia , Convulsões/fisiopatologia , Receptor 4 Toll-Like/fisiologia , Animais , Anticonvulsivantes/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epilepsia/fisiopatologia , Proteína HMGB1/antagonistas & inibidores , Hipocampo/fisiologia , Humanos , Interleucina-1beta/fisiologia , Ácido Caínico/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/fisiologia , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/antagonistas & inibidores
11.
J Biotechnol ; 139(2): 152-5, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19041912

RESUMO

Positive transcription elongation factor b (P-TEFb) is an important transcriptional regulator which controls 70-80% of RNA polymerase II transcription. It has been reported that the human I-mfa (inhibitor of MyoD family a) domain-containing protein (HIC) interacts with P-TEFb and that expression of HIC cDNA stimulates P-TEFb-dependent transcription. Interestingly, our recent study shows that transcriptional stimulation by HIC is predominately due to the 3' untranslated region (3'UTR) of HIC mRNA rather than its coding region. In this report, we investigate the effects of HIC 3'UTR on recombinant protein expression in mammalian cells. In transient transfections, overexpression of HIC 3'UTR stimulates transgene expression in several mammalian cell lines and significantly increases the production of human erythropoietin and interferon-gamma in Chinese hamster ovary (CHO) cells. This is the first report that demonstrates the improvement of expression of biopharmaceutical proteins by overexpressing a non-coding 3'UTR in CHO cells.


Assuntos
Regiões 3' não Traduzidas , Interferon gama/biossíntese , Fatores de Regulação Miogênica/genética , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Eritropoetina/genética , Eritropoetina/metabolismo , Expressão Gênica , Humanos , Interferon gama/genética , Modelos Biológicos , Fatores de Regulação Miogênica/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Transgenes
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