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1.
Nat Commun ; 12(1): 3803, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155197

RESUMO

The adenomatous polyposis coli (APC) is a frequently mutated tumour suppressor gene in cancers. However, whether APC is regulated at the epitranscriptomic level remains elusive. In this study, we analysed TCGA data and separated 200 paired oesophageal squamous cell carcinoma (ESCC) specimens and their adjacent normal tissues and demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in tumour tissues. m6A-RNA immunoprecipitation sequencing revealed that METTL3 upregulates the m6A modification of APC, which recruits YTHDF for APC mRNA degradation. Reduced APC expression increases the expression of ß-catenin and ß-catenin-mediated cyclin D1, c-Myc, and PKM2 expression, thereby leading to enhanced aerobic glycolysis, ESCC cell proliferation, and tumour formation in mice. In addition, downregulated APC expression correlates with upregulated METTL3 expression in human ESCC specimens and poor prognosis in ESCC patients. Our findings reveal a mechanism by which the Wnt/ß-catenin pathway is upregulated in ESCC via METTL3/YTHDF-coupled epitranscriptomal downregulation of APC.


Assuntos
Adenosina/análogos & derivados , Proteínas do Citoesqueleto/genética , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Animais , Carcinogênese , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Metiltransferases/genética , Camundongos , Prognóstico , RNA Mensageiro/metabolismo , Efeito Warburg em Oncologia , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
2.
Nat Commun ; 12(1): 3624, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131132

RESUMO

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Artrite/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Artrite/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Caderinas/metabolismo , Proteínas do Citoesqueleto/genética , Feminino , Proteínas de Homeodomínio , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos , beta Catenina/metabolismo
3.
World J Gastroenterol ; 27(15): 1595-1615, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33958846

RESUMO

BACKGROUND: Expression of the full-length isoform of Abelson interactor 1 (ABI1), ABI1-p65, is increased in colorectal carcinoma (CRC) and is thought to be involved in one or more steps leading to tumor progression or metastasis. The ABI1 splice isoform-L (ABI1-SiL) has conserved WAVE2-binding and SH3 domains, lacks the homeo-domain homologous region, and is missing the majority of PxxP- and Pro-rich domains found in full-length ABI1-p65. Thus, ABI1-SiL domain structure suggests that the protein may regulate CRC cell morphology, adhesion, migration, and metastasis via interactions with the WAVE2 complex pathway. AIM: To investigate the potential role and underlying mechanisms associated with ABI1-SiL-mediated regulation of CRC. METHODS: ABI1-SiL mRNA expression in CC tissue and cell lines was measured using both qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. A stably ABI1-SiL overexpressing SW480 cell model was constructed using Lipofectamine 2000, and cells selected with G418. Image J software, CCK8, and transwell assays were used to investigate SW480 cell surface area, proliferation, migration, and invasion. Immunoprecipitation, Western blot, and co-localization assays were performed to explore intermolecular interactions between ABI1-SiL, WAVE2, and ABI1-p65 proteins. RESULTS: ABI1-SiL was expressed in normal colon tissue and was significantly decreased in CRC cell lines and tissues. Overexpression of ABI1-SiL in SW480 cells significantly increased the cell surface area and inhibited the adhesive and migration properties of the cells, but did not alter their invasive capacity. Similar to ABI1-p65, ABI1-SiL still binds WAVE2, and the ABI1-p65 isoform in SW480 cells. Furthermore, co-localization assays confirmed these intermolecular interactions. CONCLUSION: These results support a model in which ABI1-SiL plays an anti-oncogenic role by competitively binding to WAVE2 and directly interacting with phosphorylated and non-phosphorylated ABI1-p65, functioning as a dominant-negative form of ABI1-p65.


Assuntos
Neoplasias Colorretais , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Isoformas de Proteínas
4.
Nat Commun ; 12(1): 2889, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001871

RESUMO

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Sítios de Ligação/genética , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Endocitose/genética , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
5.
Nat Commun ; 12(1): 3104, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035280

RESUMO

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.


Assuntos
Membrana Celular/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Integrinas/genética , Proteínas de Membrana/genética , Camundongos Knockout , Movimento (Física) , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Proteínas de Neoplasias/genética , Ligação Proteica
6.
Nat Commun ; 12(1): 2522, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947846

RESUMO

Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.


Assuntos
Autofagossomos/genética , Autofagia/genética , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Estabilidade Proteica , Receptores de Trombopoetina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Tirosina Quinase 3 Semelhante a fms/metabolismo
7.
Acta Biochim Biophys Sin (Shanghai) ; 53(5): 593-600, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33792654

RESUMO

CMYA1 (cardiomyopathy-associated protein 1, also termed Xin) localizes to the intercalated disks (ICDs) of the myocardium and functions to maintain ICD structural integrity and support signal transduction among cardiomyocytes. Our previous study showed that CMYA1 overexpression impairs the function of gap junction intercellular communication processes. Successful model generation was verified based on PCR, western blot analysis, immunohistochemistry, and immunofluorescence analysis. Myocardial CMYA1 expression was confirmed at both the mRNA and the protein levels in the CMYA1-OE transgenic mice. Masson's trichrome staining and electron microscopy revealed myocardial fibrosis and uneven bead width or the interruption of ICDs in the hearts of the CMYA1-OE transgenic mice. Furthermore, the Cx43 protein level was reduced in the CMYA1-OE mice, and co-immunoprecipitation assays of heart tissue protein extracts revealed a physical interaction between CMYA1 and Cx43. Electrocardiogram analysis enabled the detection of an obvious ventricular bigeminy for the CMYA1-OE mice. In summary, analysis of our mouse model indicates that elevated CMYA1 levels may induce myocardial fibrosis, impair ICDs, and downregulate the expression of Cx43. The observed ventricular bigeminy in the CMYA1-OE mice may be mediated by the reduced Cx43 protein level.


Assuntos
Proteínas do Citoesqueleto/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica , Miocárdio/metabolismo , Animais , Conexina 43/biossíntese , Conexina 43/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Feminino , Fibrose , Camundongos , Camundongos Transgênicos , Miocárdio/patologia
8.
Neuron ; 109(10): 1636-1656.e8, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-33831348

RESUMO

Ample evidence indicates that individuals with intellectual disability (ID) are at increased risk of developing stress-related behavioral problems and mood disorders, yet a mechanistic explanation for such a link remains largely elusive. Here, we focused on characterizing the syndromic ID gene oligophrenin-1 (OPHN1). We find that Ophn1 deficiency in mice markedly enhances helpless/depressive-like behavior in the face of repeated/uncontrollable stress. Strikingly, Ophn1 deletion exclusively in parvalbumin (PV) interneurons in the prelimbic medial prefrontal cortex (PL-mPFC) is sufficient to induce helplessness. This behavioral phenotype is mediated by a diminished excitatory drive onto Ophn1-deficient PL-mPFC PV interneurons, leading to hyperactivity in this region. Importantly, suppressing neuronal activity or RhoA/Rho-kinase signaling in the PL-mPFC reverses helpless behavior. Our results identify OPHN1 as a critical regulator of adaptive behavioral responses to stress and shed light onto the mechanistic links among OPHN1 genetic deficits, mPFC circuit dysfunction, and abnormalities in stress-related behaviors.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Interneurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Estresse Psicológico/metabolismo , Animais , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Desamparo Aprendido , Humanos , Interneurônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Parvalbuminas/genética , Parvalbuminas/metabolismo , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Estresse Psicológico/fisiopatologia , Transmissão Sináptica
9.
Nat Commun ; 12(1): 2448, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907196

RESUMO

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Divisão Celular , Parede Celular/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Expressão Gênica , Hidrólise , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Transporte Proteico
10.
Nat Med ; 27(5): 785-789, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33795869

RESUMO

Leber congenital amaurosis due to CEP290 ciliopathy is being explored by treatment with the antisense oligonucleotide (AON) sepofarsen. One patient who was part of a larger cohort (ClinicalTrials.gov NCT03140969 ) was studied for 15 months after a single intravitreal sepofarsen injection. Concordant measures of visual function and retinal structure reached a substantial efficacy peak near 3 months after injection. At 15 months, there was sustained efficacy, even though there was evidence of reduction from peak response. Efficacy kinetics can be explained by the balance of AON-driven new CEP290 protein synthesis and a slow natural rate of CEP290 protein degradation in human foveal cone photoreceptors.


Assuntos
Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Ciliopatias/terapia , Proteínas do Citoesqueleto/genética , Terapia Genética/métodos , Amaurose Congênita de Leber/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciliopatias/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Células Fotorreceptoras/metabolismo , Visão Ocular/fisiologia , Campos Visuais/fisiologia
11.
Neuron ; 109(7): 1067-1069, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33831359

RESUMO

Axonal degeneration is controlled by the TIR domain NADase SARM1. In this issue of Neuron, Figley et al. (2021) reveal a key regulatory mechanism that controls SARM1's enzymatic activity, providing insight into how NAD+ biosynthesis by the NMNAT2 enzyme protects axons, and a new therapeutic path to tune SARM1 activity.


Assuntos
Proteínas do Domínio Armadillo , NAD , Proteínas do Domínio Armadillo/genética , Axônios , Proteínas do Citoesqueleto/genética , NAD+ Nucleosidase
12.
PLoS Negl Trop Dis ; 15(3): e0008352, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33760809

RESUMO

Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E paralogs, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlaps. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions.


Assuntos
Adaptação Fisiológica/genética , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4G em Eucariotos/metabolismo , Leishmania/genética , Sequência de Aminoácidos/genética , Animais , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica/genética , Humanos , Macrófagos/parasitologia , Psychodidae/parasitologia , Alinhamento de Sequência
13.
Genes (Basel) ; 12(2)2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670832

RESUMO

Leber congenital amaurosis (LCA) encompasses the earliest and most severe retinal dystrophies and can occur as a non-syndromic or a syndromic disease. Molecular diagnosis in LCA is of particular importance in clinical decision-making and patient care since it can provide ocular and extraocular prognostics and identify patients eligible to develop gene-specific therapies. Routine high-throughput molecular testing in LCA yields 70%-80% of genetic diagnosis. In this study, we aimed to investigate the non-coding regions of one non-syndromic LCA gene, RPGRIP1, in a series of six families displaying one single disease allele after a gene-panel screening of 722 LCA families which identified 26 biallelic RPGRIP1 families. Using trio-based high-throughput whole locus sequencing (WLS) for second disease alleles, we identified a founder deep intronic mutation (NM_020366.3:c.1468-128T>G) in 3/6 families. We employed Sanger sequencing to search for the pathologic variant in unresolved LCA cases (106/722) and identified three additional families (two homozygous and one compound heterozygous with the NM_020366.3:c.930+77A>G deep intronic change). This makes the c.1468-128T>G the most frequent RPGRIP1 disease allele (8/60, 13%) in our cohort. Studying patient lymphoblasts, we show that the pathologic variant creates a donor splice-site and leads to the insertion of the pseudo-exon in the mRNA, which we were able to hamper using splice-switching antisense oligonucleotides (AONs), paving the way to therapies.


Assuntos
Proteínas do Citoesqueleto/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Amaurose Congênita de Leber/genética , Distrofias Retinianas/genética , Adolescente , Adulto , Alelos , Criança , Pré-Escolar , Análise Mutacional de DNA , Éxons , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Íntrons/genética , Amaurose Congênita de Leber/patologia , Masculino , Mutação/genética , Patologia Molecular , Linhagem , Distrofias Retinianas/patologia , Adulto Jovem
14.
Cancer Sci ; 112(5): 1785-1797, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33788346

RESUMO

The pathogenesis and cisplatin chemoresistance of ovarian cancer (OC) are still unclear. Vacuolar protein sorting-associated 33B (VPS33B) has not been reported in OC to date. In this study, immunohistochemistry was used to detect VPS33B protein expression between OC and ovarian tissues. MTT, EdU, colony formation, cell cycle, in vivo tumorigenesis, western blot, ChIP, EMSA, co-immunoprecipitation (CoIP), qRT-PCR, and microconfocal microscopy were used to explore the function and molecular mechanisms of VPS33B in OC cells. The results of the present study demonstrated that VPS33B protein expression was obviously reduced in OC compared with that in ovarian tissues. Overexpressed VPS33B suppressed cell cycle transition, cell growth, and chemoresistance to cisplatin in vitro and in vivo. Analysis of the mechanism indicated that overexpressed VPS33B regulated the epidermal growth factor receptor (EGFR)/PI3K/AKT/c-Myc/p53/miR-133a-3p feedback loop and reduced the expression of the cell cycle factor CDK4. Nasopharyngeal epithelium-specific protein 1 (NESG1) as a tumor suppressor not only interacted with VPS33B, but was also induced by VPS33B by the attenuation of PI3K/AKT/c-Jun-mediated transcription inhibition. Overexpressed NESG1 further suppressed cell growth by mediating VPS33B-modulated signals in VPS33B-overexpressing OC cells. Finally, NESG1 induced VPS33B expression by reducing the inhibition of PI3K/AKT/c-Jun-mediated transcription. Our study is the first to demonstrate that VPS33B serves as a tumor suppressor, and VPS33B can interact with NESG1 to suppress cell growth and promote cisplatin sensitivity by regulating the EGFR/PI3K/AKT/c-Myc/p53/miR-133a-3p feedback loop in OC cells.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Antineoplásicos/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cisplatino/farmacologia , Quinase 4 Dependente de Ciclina/metabolismo , Proteínas do Citoesqueleto/genética , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Transporte Vesicular/genética
15.
Neuron ; 109(7): 1118-1136.e11, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33657413

RESUMO

Axon degeneration is a central pathological feature of many neurodegenerative diseases. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+)-cleaving enzyme whose activation triggers axon destruction. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD+, activates SARM1 via an unknown mechanism. Using structural, biochemical, biophysical, and cellular assays, we demonstrate that SARM1 is activated by an increase in the ratio of NMN to NAD+ and show that both metabolites compete for binding to the auto-inhibitory N-terminal armadillo repeat (ARM) domain of SARM1. We report structures of the SARM1 ARM domain bound to NMN and of the homo-octameric SARM1 complex in the absence of ligands. We show that NMN influences the structure of SARM1 and demonstrate via mutagenesis that NMN binding is required for injury-induced SARM1 activation and axon destruction. Hence, SARM1 is a metabolic sensor responding to an increased NMN/NAD+ ratio by cleaving residual NAD+, thereby inducing feedforward metabolic catastrophe and axonal demise.


Assuntos
Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Axônios/patologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , NAD/metabolismo , Degeneração Neural/genética , Degeneração Neural/patologia , Mononucleotídeo de Nicotinamida/metabolismo , Animais , Ativação Enzimática , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Conformação Proteica
16.
Cell Mol Life Sci ; 78(8): 4003-4018, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33783564

RESUMO

Kindlin3 (K3), a FERM domain containing protein expressed in hematopoietic cells controls integrin activation and thus hemostatic and inflammatory responses. However, its role in the mechanics of plasma membrane remains unclear. Here, we show that genetic knockout of K3 in microglia and macrophages resulted in defective plasma membrane tension and membrane blebbing. Atomic force microscopy (AFM) of K3-deficient cells revealed a significant loss in membrane-to-cortex attachment (MCA), and consequently reduced membrane tension. This loss in MCA is amplified by the mislocalization of the cell cortex proteins-ezrin, radixin, and moesin (ERM)-to the plasma membrane of microglia and macrophages. Re-expression of K3 in K3-deficient macrophages rescued the defects and localization of ERMs implying a key role for K3 in MCA. Analysis of two K3 mutants, K3int affecting integrin binding and activation, and K3pxn/act disrupting binding to paxillin and actin but not integrin functions, demonstrated that the role of K3 in membrane mechanics is separate from integrin activation. The K3pxn/act mutant substantially diminished both membrane tension and Yes-associated protein (YAP) translocation to the nucleus, while preserving integrin activation, cell spreading, and migration. Together, our results show that K3 coordinates membrane mechanics, ERM protein recruitment to the membrane, and YAP translocation by linking integrin at the membrane to paxillin and actin of the cytoskeleton. This novel function of K3 is distinct from its role in integrin activation.


Assuntos
Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Proteínas de Neoplasias/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Membrana Celular/genética , Proteínas do Citoesqueleto/genética , Técnicas de Inativação de Genes , Humanos , Integrinas/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/genética , Células RAW 264.7
17.
Hum Genet ; 140(7): 1047-1060, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33721060

RESUMO

Mutations in proteins involved in cell division and chromosome segregation, such as microtubule-regulating, centrosomal and kinetochore proteins, are associated with microcephaly and/or short stature. In particular, the kinetochore plays an essential role in mitosis and cell division by mediating connections between chromosomal DNA and spindle microtubules. To date, only a few genes encoding proteins of the kinetochore complex have been identified as causes of syndromes that include microcephaly. We report a male patient with a rare de novo missense variant in NUF2, after trio whole-exome sequencing analysis. The patient presented with microcephaly and short stature, with additional features, such as bilateral vocal cord paralysis, micrognathia and atrial septal defect. NUF2 encodes a subunit of the NDC80 complex in the outer kinetochore, important for correct microtubule binding and spindle assembly checkpoint. The mutated residue is buried at the calponin homology (CH) domain at the N-terminus of NUF2, which interacts with the N-terminus of NDC80. The variant caused the loss of hydrophobic interactions in the core of the CH domain of NUF2, thereby impairing the stability of NDC80-NUF2. Analysis using a patient-derived lymphoblastoid cell line revealed markedly reduced protein levels of both NUF2 and NDC80, aneuploidy, increased micronuclei formation and spindle abnormality. Our findings suggest that NUF2 may be the first member of the NDC80 complex to be associated with a human disorder.


Assuntos
Anormalidades Múltiplas/genética , Aneuploidia , Proteínas de Ciclo Celular/genética , Transtornos Cromossômicos/genética , Segregação de Cromossomos , Mutação de Sentido Incorreto , Anormalidades Múltiplas/patologia , Adolescente , Estatura/genética , Linhagem Celular , Transtornos Cromossômicos/patologia , Proteínas do Citoesqueleto/genética , Regulação para Baixo , Transtornos do Crescimento/genética , Humanos , Masculino , Microcefalia/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fuso Acromático/patologia , Ubiquitina/metabolismo , Sequenciamento Completo do Genoma
18.
Hum Genet ; 140(7): 1031-1043, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33689014

RESUMO

Cilia and flagella are formed around an evolutionary conserved microtubule-based axoneme and are required for fluid and mucus clearance, tissue homeostasis, cell differentiation and movement. The formation and maintenance of cilia and flagella require bidirectional transit of proteins along the axonemal microtubules, a process called intraflagellar transport (IFT). In humans, IFT defects contribute to a large group of systemic diseases, called ciliopathies, which often display overlapping phenotypes. By performing exome sequencing of a cohort of 167 non-syndromic infertile men displaying multiple morphological abnormalities of the sperm flagellum (MMAF) we identified two unrelated patients carrying a homozygous missense variant adjacent to a splice donor consensus site of IFT74 (c.256G > A;p.Gly86Ser). IFT74 encodes for a core component of the IFT machinery that is essential for the anterograde transport of tubulin. We demonstrate that this missense variant affects IFT74 mRNA splicing and induces the production of at least two distinct mutant proteins with abnormal subcellular localization along the sperm flagellum. Importantly, while IFT74 deficiency was previously implicated in two cases of Bardet-Biedl syndrome, a pleiotropic ciliopathy with variable expressivity, our data indicate that this missense mutation only results in primary male infertility due to MMAF, with no other clinical features. Taken together, our data indicate that the nature of the mutation adds a level of complexity to the clinical manifestations of ciliary dysfunction, thus contributing to the expanding phenotypical spectrum of ciliopathies.


Assuntos
Astenozoospermia/genética , Síndrome de Bardet-Biedl/genética , Proteínas do Citoesqueleto/genética , Flagelos/genética , Infertilidade Masculina/genética , Mutação de Sentido Incorreto/genética , Tubulina (Proteína)/genética , Animais , Axonema/genética , Cílios/genética , Homozigoto , Humanos , Masculino , Transporte Proteico/genética , Sítios de Splice de RNA/genética , Cauda do Espermatozoide/fisiologia , Sequenciamento Completo do Exoma/métodos
19.
Biomed Environ Sci ; 34(2): 139-151, 2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33685573

RESUMO

Objective: The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms. Methods: We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells. Results: The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial-mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation. Conclusion: Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Neoplasias Ovarianas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas do Citoesqueleto/genética , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fibras de Estresse/genética , Fibras de Estresse/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Cell Prolif ; 54(5): e13023, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33759281

RESUMO

BACKGROUND: Vascular smooth muscle cells (VSMC) switch to macrophage-like cells after cholesterol loading, and this change may play an important role in atherogenesis. Muscleblind-like splicing regulator 1 (MBNL1) is a well-known splicing factor that has been implicated in many cellular processes. However, the role of MBNL1 in VSMC macrophage-like transdifferentiation is largely unknown. In this study, we aim to characterize the role of MBNL1-induced gene splicing during atherogenesis. METHODS: The expression of MBNL1 and Abelson interactor 1 (Abi1) splice variants (Abi1-e10 and Abi1-Δe10) was compared between artery tissues from healthy donors and atherosclerosis patients. Regulatory mechanisms of MBNL1-induced Abi1 gene splicing were studied, and the signal pathways mediated by Abi1 splice variants were investigated in VSMC. RESULTS: Loss of MBNL1 was found in the macrophage-like VSMC (VSMC-M) in artery wall from atherosclerosis patients. In vitro and in vivo evidence confirmed that Abi1 is one of the MBNL1 target genes. Loss of MBNL1 significantly induces the Abi1-Δe10 isoform expression. Compared to the known actin organization activities of the Abi1 gene, we discovered a novel action of Abi1-Δe10, whereby Abi1-Δe10 activates Rac1 independent of upstream stimulation and triggers the Rac1-NOX1-ROS pathway, which results in increased expression of transcription factor Kruppel-like factor 4 (KLF4). While Abi1-Δe10 inhibits contractile VSMC biomarkers expression and cell contraction, it stimulates VSMC proliferation, migration and macrophage-like transdifferentiation. CONCLUSION: Loss-of-function of MBNL1 activates VSMC-M transdifferentiation to promote atherogenesis through regulating Abi1 RNA splicing.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Músculo Liso Vascular/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Desdiferenciação Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Músculo Liso Vascular/citologia , NADPH Oxidase 1/antagonistas & inibidores , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , Fenótipo , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo
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