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1.
Nat Commun ; 11(1): 1092, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107390

RESUMO

Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, however, relatively few specific miRNAs have been identified that control synapse formation. Using a conditional transgenic toolkit for competitive inhibition of miRNA function in Drosophila, we performed an unbiased screen for novel regulators of synapse morphogenesis at the larval neuromuscular junction (NMJ). From a set of ten new validated regulators of NMJ growth, we discovered that miR-34 mutants display synaptic phenotypes and cell type-specific functions suggesting distinct downstream mechanisms in the presynaptic and postsynaptic compartments. A search for conserved downstream targets for miR-34 identified the junctional receptor CNTNAP4/Neurexin-IV (Nrx-IV) and the membrane cytoskeletal effector Adducin/Hu-li tai shao (Hts) as proteins whose synaptic expression is restricted by miR-34. Manipulation of miR-34, Nrx-IV or Hts-M function in motor neurons or muscle supports a model where presynaptic miR-34 inhibits Nrx-IV to influence active zone formation, whereas, postsynaptic miR-34 inhibits Hts to regulate the initiation of bouton formation from presynaptic terminals.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Terminações Pré-Sinápticas/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a Calmodulina/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Larva/crescimento & desenvolvimento , Morfogênese/genética , Mutação , Junção Neuromuscular/citologia , Junção Neuromuscular/crescimento & desenvolvimento
2.
BMC Plant Biol ; 20(1): 38, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992205

RESUMO

BACKGROUD: Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) to communicate each other and to coordinate their collective behaviors. Recently, accumulating evidence shows that host plants are able to sense and respond to bacterial AHLs. Once primed, plants are in an altered state that enables plant cells to more quickly and/or strongly respond to subsequent pathogen infection or abiotic stress. RESULTS: In this study, we report that pretreatment with N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) confers resistance against the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (PstDC3000) in Arabidopsis. Pretreatment with 3OC8-HSL and subsequent pathogen invasion triggered an augmented burst of hydrogen peroxide, salicylic acid accumulation, and fortified expression of the pathogenesis-related genes PR1 and PR5. Upon PstDC3000 challenge, plants treated with 3OC8-HSL showed increased activities of defense-related enzymes including peroxidase, catalase, phenylalanine ammonialyase, and superoxide dismutase. In addition, the 3OC8-HSL-primed resistance to PstDC3000 in wild-type plants was impaired in plants expressing the bacterial NahG gene and in the npr1 mutant. Moreover, the expression levels of isochorismate synthases (ICS1), a critical salicylic acid biosynthesis enzyme, and two regulators of its expression, SARD1 and CBP60g, were potentiated by 3OC8-HSL pretreatment followed by pathogen inoculation. CONCLUSIONS: Our data indicate that 3OC8-HSL primes the Arabidopsis defense response upon hemibiotrophic bacterial infection and that 3OC8-HSL-primed resistance is dependent on the SA signaling pathway. These findings may help establish a novel strategy for the control of plant disease.


Assuntos
4-Butirolactona/análogos & derivados , Arabidopsis , Imunidade Vegetal/efeitos dos fármacos , Pseudomonas syringae/patogenicidade , Ácido Salicílico/metabolismo , 4-Butirolactona/farmacologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/efeitos dos fármacos , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Genes de Plantas , Transferases Intramoleculares/efeitos dos fármacos , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Percepção de Quorum/fisiologia , Transdução de Sinais/efeitos dos fármacos
3.
Cell Mol Life Sci ; 77(1): 195-212, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31177295

RESUMO

During ciliogenesis, the mother centriole transforms into a basal body competent to nucleate a cilium. The mother centriole and basal body possess sub-distal appendages (SDAs) and basal feet (BF), respectively. SDAs and BF are thought to be equivalent structures. In contrast to SDA assembly, little is known about the players involved in BF assembly and its assembly order. Furthermore, the contribution of BF to ciliogenesis is not understood. Here, we found that SDAs are distinguishable from BF and that the protein NPHP5 is a novel SDA and BF component. Remarkably, NPHP5 is specifically required for BF assembly in cells able to form basal bodies but is dispensable for SDA assembly. Determination of the hierarchical assembly reveals that NPHP5 cooperates with a subset of SDA/BF proteins to organize BF. The assembly pathway of BF is similar but not identical to that of SDA. Loss of NPHP5 or a BF protein simultaneously inhibits BF assembly and primary ciliogenesis, and these phenotypes could be rescued by manipulating the expression of certain components in the BF assembly pathway. These findings define a novel role for NPHP5 in specifically regulating BF assembly, a process which is tightly coupled to primary ciliogenesis.


Assuntos
Corpos Basais/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Cílios/metabolismo , Corpos Basais/ultraestrutura , Linhagem Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Cílios/ultraestrutura , Humanos , Mapas de Interação de Proteínas
4.
PLoS One ; 14(12): e0225621, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31821324

RESUMO

Markers in monocytes, precursors of macrophages, which are related to CAD, are largely unknown. Therefore, we aimed to identify genes in monocytes predictive of a new ischemic event in patients with CAD and/or discriminate between stable CAD and acute coronary syndrome. We included 66 patients with stable CAD, of which 24 developed a new ischemic event, and 19 patients with ACS. Circulating CD14+ monocytes were isolated with magnetic beads. RNA sequencing analysis in monocytes of patients with (n = 13) versus without (n = 11) ischemic event at follow-up and in patients with ACS (n = 12) was validated with qPCR (n = 85). MT-COI, STRN and COX10 predicted new ischemic events in CAD patients (power for separation at 1% error rate of 0.97, 0.90 and 0.77 respectively). Low MT-COI and high STRN were also related to shorter time between blood sampling and event. COX10 and ZNF484 together with MT-COI, STRN and WNK1 separated ACS completely from stable CAD patients. RNA expressions in monocytes of MT-COI, COX10, STRN, WNK1 and ZNF484 were independent of cholesterol lowering and antiplatelet treatment. They were independent of troponin T, a marker of myocardial injury. But, COX10 and ZNF484 in human plaques correlated to plaque markers of M1 macrophage polarization, reflecting vascular injury. Expression of MT-COI, COX10, STRN and WNK1, but not that of ZNF484, PBMCs paired with that in monocytes. The prospective study of relation of MT-COI, COX10, STRN, WNK1 and ZNF484 with unstable CAD is warranted.


Assuntos
Síndrome Coronariana Aguda/diagnóstico , Proteínas de Ligação a Calmodulina/metabolismo , Doença da Artéria Coronariana/diagnóstico , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Placa Aterosclerótica/patologia , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/patologia , Idoso , Alquil e Aril Transferases/sangue , Alquil e Aril Transferases/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Proteínas de Ligação a Calmodulina/sangue , Colesterol/sangue , Angiografia Coronária , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/patologia , Diagnóstico Diferencial , Complexo IV da Cadeia de Transporte de Elétrons/sangue , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Proteínas do Tecido Nervoso/sangue , Placa Aterosclerótica/sangue , Estudos Prospectivos , RNA-Seq , Proteína Quinase 1 Deficiente de Lisina WNK/sangue
5.
Endocrinol Metab (Seoul) ; 34(3): 291-301, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31565882

RESUMO

BACKGROUND: Striatin and caveolin-1 (cav-1) are scaffolding/regulating proteins that are associated with salt-sensitive high blood pressure and promote renal sodium and water reabsorption, respectively. The mineralocorticoid receptor (MR) interacts with striatin and cav-1, while aldosterone increases striatin and cav-1 levels. However, no in vivo data have been reported for the levels of these proteins in the kidney. METHODS: Male Wistar rats were intraperitoneally injected with normal saline solution, aldosterone alone (Aldo: 150 µg/kg body weight), or aldosterone after pretreatment with eplerenone, an MR blocker, 30 minutes before the aldosterone injection (eplerenone [Ep.]+Aldo). Thirty minutes after the aldosterone injection, the amount and localization of striatin and cav-1 were determined by Western blot analysis and immunohistochemistry, respectively. RESULTS: Aldosterone increased striatin levels by 150% (P<0.05), and cav-1 levels by 200% (P<0.001). Eplerenone had no significant effect on striatin levels, but partially blocked the aldosterone-induced increase in cav-1 levels. Aldosterone stimulated striatin and cav-1 immunoreactivity in both the cortex and medulla. Eplerenone reduced cav-1 immunostaining in both areas; however, striatin intensity was reduced in the cortex, but increased in the medulla. CONCLUSION: This is the first in vivo study demonstrating that aldosterone rapidly enhances renal levels of striatin and cav-1. Aldosterone increases striatin levels via an MR-independent pathway, whereas cav-1 is partially regulated through MR.


Assuntos
Aldosterona/administração & dosagem , Proteínas de Ligação a Calmodulina/metabolismo , Caveolina 1/metabolismo , Rim/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Animais , Rim/efeitos dos fármacos , Masculino , Ratos Wistar
6.
Am J Physiol Renal Physiol ; 317(6): F1503-F1512, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532245

RESUMO

We have defined a population of stem cell antigen (Sca)-1+/CD34+/lin- mesenchymal stem cells in the mouse urinary bladder. These cells are reduced after partial bladder outlet obstruction (PO). To test the role of Sca-1 expressed by these cells, we analyzed bladders from Sca-1 knockout (KO) mice in both uninjured male mice and male mice subjected to PO. We found that loss of Sca-1 alone had little effect on bladder development or function but reduced the total number of mesenchymal stem cells by 30%. After PO, bladders from Sca-1-null KO male mice were larger, with more collagen and less muscle, than obstructed wild-type mice. Steady-state levels of caldesmon were significantly reduced and levels of fibroblast-specific protein 1 were significantly increased in Sca-1 KO mice compared with wild-type mice after PO. In investigating the effects of PO on cell proliferation, we found that loss of Sca-1 changed the timing of cell division in CD34+/lin-, collagen-producing, and smooth muscle cells. PO in combination with loss of Sca-1 drastically reduced the ability of CD34+/lin- cells to form colonies in vitro. Our findings therefore support the hypothesis that Sca-1 protects the bladder from fibrotic remodeling after obstruction, in part by influencing the proliferation of cells responding to the injury.


Assuntos
Antígenos Ly/uso terapêutico , Proteínas de Membrana/uso terapêutico , Bexiga Urinária/patologia , Animais , Antígenos/imunologia , Antígenos/uso terapêutico , Antígenos CD34/metabolismo , Antígenos Ly/genética , Antígenos Ly/imunologia , Proteínas de Ligação a Calmodulina/metabolismo , Proliferação de Células , Fibrose , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Substâncias Protetoras , Células-Tronco , Obstrução do Colo da Bexiga Urinária/patologia
7.
Plant Physiol ; 181(3): 1314-1327, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31548265

RESUMO

Calmodulin (CaM) regulates plant disease responses through its downstream calmodulin-binding proteins (CaMBPs) often by affecting the biosynthesis or signaling of phytohormones, such as jasmonic acid (JA) and salicylic acid. However, how these CaMBPs mediate plant hormones and other stress resistance-related signaling remains largely unknown. In this study, we conducted analyses in Arabidopsis (Arabidopsis thaliana) on the functions of AtIQM1 (IQ-Motif Containing Protein1), a Ca2+-independent CaMBP, in JA biosynthesis and defense against the necrotrophic pathogen Botrytis cinerea using molecular, biochemical, and genetic analyses. IQM1 directly interacted with and promoted CATALASE2 (CAT2) expression and CAT2 enzyme activity and indirectly increased the activity of the JA biosynthetic enzymes ACX2 and ACX3 through CAT2, thereby positively regulating JA content and B. cinerea resistance. In addition, in vitro assays showed that in the presence of CaM5, IQM1 further enhanced the activity of CAT2, suggesting that CaM5 may affect the activity of CAT2 by combining with IQM1 in the absence of Ca2+ Our data indicate that IQM1 is a key regulatory factor in signaling of plant disease responses mediated by JA. The study also provides new insights that CaMBP may play a critical role in the cross talk of multiple signaling pathways in the context of plant defense processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Botrytis/fisiologia , Proteínas de Ligação a Calmodulina/metabolismo , Doenças das Plantas/imunologia , Reguladores de Crescimento de Planta/metabolismo , Motivos de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Sinalização do Cálcio , Proteínas de Ligação a Calmodulina/genética , Ciclopentanos/metabolismo , Resistência à Doença , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Estômatos de Plantas/enzimologia , Estômatos de Plantas/genética , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Ácido Salicílico/metabolismo
8.
Cell Struct Funct ; 44(2): 95-104, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31548446

RESUMO

KCBP is a microtubule (MT) minus-end-directed kinesin widely conserved in plants. It was shown in Arabidopsis that KCBP controls trichome cell shape by orchestrating MT and actin cytoskeletons using its tail and motor domains. In contrast, the KCBP knockout (KO) line in the moss Physcomitrella patens showed a defect in nuclear and organelle positioning in apical stem cells. Moss KCBP is postulated to transport the nucleus and chloroplast via direct binding to their membranes, since it binds to and transports liposomes composed of phospholipids in vitro. However, domains required for cargo transport in vivo have not been mapped. Here, we performed a structure-function analysis of moss KCBP. We found that the FERM domain in the tail region, which is known to bind to lipids as well as other proteins, is essential for both nuclear and chloroplast positioning, whereas the proximal MyTH4 domain plays a supporting role in chloroplast transport. After anaphase but prior to nuclear envelope re-formation, KCBP accumulates on the chromosomes, in particular at the centromeric region in a FERM-dependent manner. In the KCBP KO line, the rate of poleward chromosome movement in anaphase was reduced and lagging chromosomes occasionally appeared. These results suggest that KCBP binds to non-membranous naked chromosomes via an unidentified protein(s) for their transport. Finally, the liverwort orthologue of KCBP rescued the chromosome/chloroplast mis-positioning of the moss KCBP KO line, suggesting that the cargo transport function is conserved at least in bryophytes.Key words: kinesin, mitosis, chromosome segregation, kinetochore, dynein.


Assuntos
Anáfase , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Cromátides/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação a Calmodulina/deficiência , Proteínas de Ligação a Calmodulina/genética
9.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31167803

RESUMO

WT1 is a transcriptional activator that controls the boundary between multipotency and differentiation. The transcriptional cofactor BASP1 binds to WT1, forming a transcriptional repressor complex that drives differentiation in cultured cells; however, this proposed mechanism has not been demonstrated in vivo. We used the peripheral taste system as a model to determine how BASP1 regulates the function of WT1. During development, WT1 is highly expressed in the developing taste cells while BASP1 is absent. By the end of development, BASP1 and WT1 are co-expressed in taste cells, where they both occupy the promoter of WT1 target genes. Using a conditional BASP1 mouse, we demonstrate that BASP1 is critical to maintain the differentiated state of adult taste cells and that loss of BASP1 expression significantly alters the composition and function of these cells. This includes the de-repression of WT1-dependent target genes from the Wnt and Shh pathways that are normally only transcriptionally activated by WT1 in the undifferentiated taste cells. Our results uncover a central role for the WT1-BASP1 complex in maintaining cell differentiation in vivo.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Diferenciação Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Papilas Gustativas/citologia , Papilas Gustativas/metabolismo , Proteínas WT1/metabolismo , Animais , Biomarcadores , Proteínas de Ligação a Calmodulina/genética , Diferenciação Celular/genética , Proteínas do Citoesqueleto/genética , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Fenótipo , Ligação Proteica , Proteínas WT1/genética
10.
Artigo em Inglês | MEDLINE | ID: mdl-31202182

RESUMO

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Hiper-Homocisteinemia/metabolismo , Proteoma/metabolismo , Complexo Vitamínico B/análise , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Química Encefálica , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Cromatografia Líquida , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Hiper-Homocisteinemia/etiologia , Hiper-Homocisteinemia/genética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/química , Proteoma/genética , Complexo Vitamínico B/metabolismo
11.
Plant Mol Biol ; 101(1-2): 21-40, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31049793

RESUMO

KEY MESSAGE: Arabidopsis thaliana mlo3 mutant plants are not affected in pathogen infection phenotypes but-reminiscent of mlo2 mutant plants-exhibit spontaneous callose deposition and signs of early leaf senescence. The family of Mildew resistance Locus O (MLO) proteins is best known for its profound effect on the outcome of powdery mildew infections: when the appropriate MLO protein is absent, the plant is fully resistant to otherwise virulent powdery mildew fungi. However, most members of the MLO protein family remain functionally unexplored. Here, we investigate Arabidopsis thaliana MLO3, the closest relative of AtMLO2, AtMLO6 and AtMLO12, which are the Arabidopsis MLO genes implicated in the powdery mildew interaction. The co-expression network of AtMLO3 suggests association of the gene with plant defense-related processes such as salicylic acid homeostasis. Our extensive analysis shows that mlo3 mutants are unaffected regarding their infection phenotype upon challenge with the powdery mildew fungi Golovinomyces orontii and Erysiphe pisi, the oomycete Hyaloperonospora arabidopsidis, and the bacterial pathogen Pseudomonas syringae (the latter both in terms of basal and systemic acquired resistance), indicating that the protein does not play a major role in the response to any of these pathogens. However, mlo3 genotypes display spontaneous callose deposition as well as signs of early senescence in 6- or 7-week-old rosette leaves in the absence of any pathogen challenge, a phenotype that is reminiscent of mlo2 mutant plants. We hypothesize that de-regulated callose deposition in mlo3 genotypes might be the result of a subtle transient aberration of salicylic acid-jasmonic acid homeostasis during development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a Calmodulina/metabolismo , Resistência à Doença/genética , Glucanos/metabolismo , Doenças das Plantas/imunologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ascomicetos/fisiologia , Proteínas de Ligação a Calmodulina/genética , Ciclopentanos/metabolismo , Genótipo , Homeostase , Mutação , Oomicetos/fisiologia , Oxilipinas/metabolismo , Fenótipo , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Pseudomonas syringae/fisiologia , Ácido Salicílico/metabolismo
12.
Technol Cancer Res Treat ; 18: 1533033819841433, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30947633

RESUMO

Colorectal cancer is one of the most prevalent malignancies worldwide. ENKUR is a transient receptor potential canonical-binding protein that acts as a potential regulator or effector of transient receptor potential canonical channels. It also directly interacts with the p85 regulatory subunit of phosphoinositide 3-kinase. However, the role of ENKUR in colorectal cancer remains unclear. In the present study, the expression profiles of ENKUR in the The Cancer Genome Atlas and ONCOMINE databases were analyzed. Significant downregulation of ENKUR was observed in clinical tumor samples of various cancer types, including colorectal cancer. Decreased ENKUR messenger RNA expression and ENKUR protein level were detected in 6 human colorectal cancer cell lines. Silencing of ENKUR in colorectal cancer cells led to enhanced cell proliferation, migration, and invasion, while the opposite effects were achieved in ENKUR-overexpressing cells. Furthermore, ENKUR-underexpressing cells exhibited increased activity of phosphoinositide 3-kinase /Akt signaling pathway. Downregulation of the epithelial marker, E-cadherin, and upregulation of the mesenchymal markers, vimentin and N-cadherin, were detected in ENKUR-underexpressing cells, suggesting the induction of epithelial-mesenchymal transition. In conclusion, the present study demonstrates that ENKUR may be responsible for alterations in the proliferative, migratory, and invasive potential of colorectal cancer cells through possible involvement in the phosphoinositide 3-kinase /Akt signaling pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/patologia , Proteínas de Ligação a Calmodulina/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Proteínas de Ligação a Calmodulina/genética , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Células Tumorais Cultivadas
13.
Medicine (Baltimore) ; 98(2): e13847, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30633156

RESUMO

BACKGROUND: Bladder cancer (BC) is one of the most common malignant neoplasms in the genitourinary tract. We employed the GSE13507 data set from the Gene Expression Omnibus (GEO) database in order to identify key genes related to tumorigenesis, progression, and prognosis in BC patients. METHODS: The data set used in this study included 10 normal bladder mucosae tissue samples and 165 primary BC tissue samples. Differentially expressed genes (DEGs) in the 2 types of samples were identified by GEO2R. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the online website DAVID. The online website STRING was used to construct a protein-protein interaction network. Moreover, the plugins in MCODE and cytoHubba in Cytoscape were employed to find the hub genes and modules in these DEGs. RESULTS: We identified 154 DEGs comprising 135 downregulated genes and 19 upregulated genes. The GO enrichment results were mainly related to the contractile fiber part, extracellular region part, actin cytoskeleton, and extracellular region. The KEGG pathway enrichment results mainly comprised type I diabetes mellitus, asthma, systemic lupus erythematosus, and allograft rejection. A module was identified from the protein-protein interaction network. In total, 15 hub genes were selected and 3 of them comprising CALD1, CNN1, and TAGLN were associated with both overall survival and disease-free survival. CONCLUSION: CALD1, CNN1, and TAGLN may be potential biomarkers for diagnosis as well as therapeutic targets in BC patients.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Biomarcadores Tumorais/metabolismo , Biologia Computacional , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Mapas de Interação de Proteínas , Neoplasias da Bexiga Urinária/mortalidade
14.
J Biomed Sci ; 26(1): 12, 2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678675

RESUMO

BACKGROUND: Osteoclasts (OCs) are motile multinucleated cells derived from differentiation and fusion of hematopoietic progenitors of the monocyte-macrophage lineage that undergo a multistep process called osteoclastogenesis. The biological function of OCs is to resorb bone matrix for controlling bone strength and integrity, which is essential for bone development. The bone resorption function is based on the remodelling of the actin cytoskeleton into an F-actin-rich structure known as the sealing zone for bone anchoring and matrix degradation. Non-muscle caldesmon (l-CaD) is known to participate in the regulation of actin cytoskeletal remodeling, but its function in osteoclastogenesis remains unclear. METHODS/RESULTS: In this study, gain and loss of the l-CaD level in RAW264.7 murine macrophages followed by RANKL induction was used as an experimental approach to examine the involvement of l-CaD in the control of cell fusion into multinucleated OCs in osteoclastogenesis. In comparison with controls, l-CaD overexpression significantly increased TRAP activity, actin ring structure and mineral substrate resorption in RANKL-induced cells. In contrast, gene silencing against l-CaD decreased the potential for RANKL-induced osteoclastogenesis and mineral substrate resorption. In addition, OC precursor cells with l-CaD overexpression and gene silencing followed by RANKL induction caused 13% increase and 24% decrease, respectively, in cell fusion index. To further understand the mechanistic action of l-CaD in the modulation of OC fusion, atomic force microscopy was used to resolve the mechanical changes of cell spreading and adhesion force in RANKL-induced cells with and without l-CaD overexpression or gene silencing. CONCLUSIONS: l-CaD plays a key role in the regulation of actin cytoskeletal remodeling for the formation of actin ring structure at the cell periphery, which may in turn alter the mechanical property of cell-spreading and cell surface adhesion force, thereby facilitating cell-cell fusion into multinucleated OCs during osteoclastogenesis.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Diferenciação Celular , Osteoclastos/metabolismo , Osteogênese , Animais , Macrófagos/metabolismo , Camundongos , Células RAW 264.7
15.
Eur J Pharmacol ; 842: 167-176, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30391746

RESUMO

The goals of this study were to examine the cellular signaling pathways associated with the phosphorylation of caldesmon, the phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17), and the 20-kDa regulatory light chain of myosin (MLC20) induced by levobupivacaine in isolated rat aortas. The effects of genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, 1-butanol, and ML-7 HCl on levobupivacaine-induced contraction were assessed. The effect of genistein on the simultaneous calcium-tension curves induced by levobupivacaine was examined. The effects of GF109203X, genistein, PD98059 and extracellular signal-regulated kinase (ERK) siRNA on levobupivacaine-induced caldesmon phosphorylation were investigated. The effect of genistein on the ERK and tyrosine phosphorylation induced by levobupivacaine was examined. The effect of GF109203X, PD98059, Y-27632, SP600125, and ML-7 HCl on the levobupivacaine-induced phosphorylation of CPI-17 and MLC20 were investigated. Genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, ML-7 HCl, and 1-butanol attenuated levobupivacaine-induced contraction. Genistein caused a right downward shift of the calcium-tension curves induced by levobupivacaine. Genistein attenuated levobupivacaine-induced phosphorylation of protein tyrosine, ERK and caldesmon. PD98059, ERK siRNA and GF109203X attenuated levobupivacaine-induced caldesmon phosphorylation. GF109203X, Y-27632, SP600125, ML-7 HCl and PD98059 attenuated CPI-17 phosphorylation and MLC20 phosphorylation induced by levobupivacaine. These results suggest that levobupivacaine-induced caldesmon phosphorylation contributing to levobupivacaine-induced contraction is mediated by a pathway involving ERK, which is activated by tyrosine kinase or protein kinase C (PKC). The phosphorylation of CPI-17 and MLC20 induced by levobupivacaine is mediated by cellular signaling pathways involving PKC, Rho-kinase, and c-Jun NH2-terminal kinase or PKC, Rho-kinase, ERK, and myosin light chain kinase.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Levobupivacaína/farmacologia , Proteínas Tirosina Quinases/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Relação Dose-Resposta a Droga , Masculino , Músculo Liso Vascular/citologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
16.
New Phytol ; 222(1): 335-348, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30372534

RESUMO

Plants have evolved an array of responses that provide them with protection from attack by microorganisms and other predators. Many of these mechanisms depend upon interactions between the plant hormones jasmonate (JA) and ethylene (ET). However, the molecular basis of these interactions is insufficiently understood. Gene expression and physiological assays with mutants were performed to investigate the role of Arabidopsis BIG gene in stress responses. BIG transcription is downregulated by methyl JA (MeJA), necrotrophic infection or mechanical injury. BIG deficiency promotes JA-dependent gene induction, increases JA production but restricts the accumulation of both ET and salicylic acid. JA-induced anthocyanin accumulation and chlorophyll degradation are enhanced and stomatal immunity is impaired by BIG disruption. Bacteria- and lipopolysaccaride (LPS)-induced stomatal closure is reduced in BIG gene mutants, which are hyper-susceptible to microbial pathogens with different lifestyles, but these mutants are less attractive to phytophagous insects. Our results indicate that BIG negatively and positively regulate the MYC2 and ERF1 arms of the JA signalling pathway. BIG warrants recognition as a new and distinct regulator that regulates JA responses, the synergistic interactions of JA and ET, and other hormonal interactions that reconcile the growth and defense dilemma in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Imunidade Vegetal , Estômatos de Plantas/imunologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a Calmodulina/genética , Regulação para Baixo/genética , Etilenos , Regulação da Expressão Gênica de Plantas , Mutação/genética , Ácido Salicílico/metabolismo
17.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 395-408, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30290240

RESUMO

Cell migration is a critical mechanism controlling tissue morphogenesis, epithelial wound healing and tumor metastasis. Migrating cells depend on orchestrated remodeling of the plasma membrane and the underlying actin cytoskeleton, which is regulated by the spectrin-adducin-based membrane skeleton. Expression of adducins is altered during tumorigenesis, however, their involvement in metastatic dissemination of tumor cells remains poorly characterized. This study investigated the roles of α-adducin (ADD1) and γ-adducin (ADD3) in regulating migration and invasion of non-small cell lung cancer (NSCLC) cells. ADD1 was mislocalized, whereas ADD3 was markedly downregulated in NSCLC cells with the invasive mesenchymal phenotype. CRISPR/Cas9-mediated knockout of ADD1 and ADD3 in epithelial-type NSCLC and normal bronchial epithelial cells promoted their Boyden chamber migration and Matrigel invasion. Furthermore, overexpression of ADD1, but not ADD3, in mesenchymal-type NSCLC cells decreased cell migration and invasion. ADD1-overexpressing NSCLC cells demonstrated increased adhesion to the extracellular matrix (ECM), accompanied by enhanced assembly of focal adhesions and hyperphosphorylation of Src and paxillin. The increased adhesiveness and decreased motility of ADD1-overexpressing cells were reversed by siRNA-mediated knockdown of Src. By contrast, the accelerated migration of ADD1 and ADD3-depleted NSCLC cells was ECM adhesion-independent and was driven by the upregulated expression of pro-motile cadherin-11. Overall, our findings reveal a novel function of adducins as negative regulators of NSCLC cell migration and invasion, which could be essential for limiting lung cancer progression and metastasis.


Assuntos
Caderinas/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Junções Célula-Matriz/metabolismo , Neoplasias Pulmonares/metabolismo , Caderinas/biossíntese , Caderinas/genética , Proteínas de Ligação a Calmodulina/biossíntese , Proteínas de Ligação a Calmodulina/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Junções Célula-Matriz/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Células Epiteliais/metabolismo , Adesões Focais/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
18.
J Matern Fetal Neonatal Med ; 32(6): 916-921, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29046106

RESUMO

PURPOSE: The decreased placental perfusion is the underlying reason for intrauterine growth restriction that in turn leads to reduced placental perfusion and ischemia. However, there are several issues to be understood in the pathophysiology of intrauterine growth restriction. We aimed to study whether any compensatory response in placental vascular bed occur in pregnancies complicated with intrauterine growth restriction by the immunohistochemical staining of von Willebrand factor and caldesmon in placental tissues. MATERIALS AND METHODS: A total of 103 pregnant women was enrolled in the study including 50 patients who were complicated with IUGR and 50 uncomplicated control patients. The study was designed in a prospective manner. All placentas were also stained with von Willebrand factor and caldesmon monoclonal kits. RESULTS: The immunohistochemical staining of von Willebrand factor and caldesmon expressions in placental tissues were different between normal and intrauterine growth restriction group. The percentages of 2+ and 3+ von Willebrand factor expression were higher in the intrauterine growth restriction group comparing with the normal group, although the difference was not statistically significant. The intensity of caldesmon expression was significantly lower in the intrauterine growth restriction group in comparison with the normal group (p < .001). CONCLUSION: Angiogenesis occurs as a placental response to intrauterine growth restriction which is a hypoxic condition. But newly formed vessels are immature and not strong enough. Our study is important to clarify the pathophysiology and placental compensatory responses in intrauterine growth restriction.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Retardo do Crescimento Fetal/etiologia , Placenta/irrigação sanguínea , Fator de von Willebrand/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Retardo do Crescimento Fetal/metabolismo , Idade Gestacional , Humanos , Placenta/metabolismo , Gravidez , Estudos Prospectivos , Adulto Jovem
19.
J Exp Bot ; 70(2): 387-396, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30590729

RESUMO

Plant growth and development are a genetically predetermined series of events but can change dramatically in response to environmental stimuli, involving perpetual pattern formation and reprogramming of development. The rate of growth is determined by cell division and subsequent cell expansion, which are restricted and controlled by the cell wall-plasma membrane-cytoskeleton continuum, and are coordinated by intricate networks that facilitate intra- and intercellular communication. An essential role in cellular signaling is played by calcium ions, which act as universal second messengers that transduce, integrate, and multiply incoming signals during numerous plant growth processes, in part by regulation of the microtubule cytoskeleton. In this review, we highlight recent advances in the understanding of calcium-mediated regulation of microtubule-associated proteins, their function at the microtubule cytoskeleton, and their potential role as hubs in crosstalk with other signaling pathways.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Plantas/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo
20.
FASEB J ; 33(4): 4729-4740, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30592649

RESUMO

The adherens junctions (AJs) and tight junctions (TJs) provide critical adhesive contacts between neighboring epithelial cells and are crucial for epithelial adhesion, integrity, and barrier functions in a wide variety of tissues and organisms. The striatin protein family, which are part of the striatin interaction phosphatases and kinases complex, are multidomain scaffolding proteins that play important biologic roles. We have previously shown that striatin colocalizes with the tumor suppressor protein adenomatous polyposis coli in the TJs of epithelial cells. Here we show that striatin affects junction integrity and cell migration, probably through a mechanism that involves the adhesion molecule E-cadherin. Cells engaged in cell-cell adhesion expressed a high MW-modified form of striatin that forms stable associations with detergent-insoluble, membrane-bound cellular fractions. In addition, striatin has recently been suggested to be a target of the poly (ADP-ribose) polymerases Tankyrase 1, and we have found that striatin interacts with Tankyrase 1 and is subsequently poly-ADP-ribosylated. Taken together, our results suggest that striatin is a novel cell-cell junctional protein that functions to maintain correct cell adhesion and may have a role in establishing the relationship between AJs and TJs that is fundamental for epithelial cell-cell adhesion.-Lahav-Ariel, L., Caspi, M., Nadar-Ponniah, P. T., Zelikson, N., Hofmann, I., Hanson, K. K., Franke, W. W., Sklan, E. H., Avraham, K. B., Rosin-Arbesfeld, R. Striatin is a novel modulator of cell adhesion.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Adesão Celular/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Junções Aderentes/metabolismo , Animais , Western Blotting , Células COS , Células CACO-2 , Caderinas/genética , Caderinas/metabolismo , Proteínas de Ligação a Calmodulina/genética , Adesão Celular/genética , Chlorocebus aethiops , Cães , Células Hep G2 , Humanos , Imunoprecipitação , Células MCF-7 , Células Madin Darby de Rim Canino , Proteínas de Membrana/genética , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Tanquirases/metabolismo , Junções Íntimas/metabolismo
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