Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
1.
Genesis ; 58(5): e23359, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32191380

RESUMO

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1-Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast-specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1-Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.


Assuntos
DNA Nucleotidiltransferases/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Animais , Células Cultivadas , DNA Nucleotidiltransferases/metabolismo , Fibroblastos/metabolismo , Gástrula/metabolismo , Marcação de Genes/métodos , Células HaCaT , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Zigoto/metabolismo
2.
Traffic ; 16(7): 712-26, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25753619

RESUMO

Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine-based signal (GYDQL) in its C-terminal tail and a non-classical motif in its fifth inter-TM loop. Using the yeast two-hybrid system, we showed that the GYDQL motif specifically interacted with the µ subunit of the adaptor protein complex 3 (AP-3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP-3-depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP-3 knockdown cells where it also accumulated in the trans-Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63-GYDQL chimeric protein were not increased when clathrin-mediated endocytosis was impaired, our data show that the tyrosine-based motif of cystinosin is a 'strong' AP-3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway.


Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Lisossomos/metabolismo , Sinais Direcionadores de Proteínas , Sistemas de Transporte de Aminoácidos Neutros/química , Endocitose , Células HeLa , Humanos , Transporte Proteico , Tetraspanina 30/metabolismo
3.
J Am Soc Nephrol ; 27(6): 1678-88, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26449607

RESUMO

Cystinosis is a rare autosomal recessive storage disorder characterized by defective lysosomal efflux of cystine due to mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin. Lysosomal cystine accumulation leads to crystal formation and functional impairment of multiple organs. Moreover, cystinosis is the most common inherited cause of renal Fanconi syndrome in children. Oral cysteamine therapy delays disease progression by reducing intracellular cystine levels. However, because cysteamine does not correct all complications of cystinosis, including Fanconi syndrome, we hypothesized that cystinosin could have novel roles in addition to transporting cystine out of the lysosome. By coimmunoprecipitation experiments and mass spectrometry, we found cystinosin interacts with almost all components of vacuolar H(+)-ATPase and the Ragulator complex and with the small GTPases Ras-related GTP-binding protein A (RagA) and RagC. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) pathway was downregulated in proximal tubular cell lines derived from Ctns(-/-) mice. Decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in these cells, suggesting that the downregulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine. Our results show a dual role for cystinosin as a cystine transporter and as a component of the mTORC1 pathway, and provide an explanation for the appearance of Fanconi syndrome in cystinosis. Furthermore, this study highlights the need to develop new treatments not dependent on lysosomal cystine depletion alone for this devastating disease.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/fisiologia , Cistinose/etiologia , Complexos Multiproteicos/fisiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos
4.
Pediatr Nephrol ; 29(4): 505-11, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23824180

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease, affecting approximately 1 in 1,000 people. The disease is characterized by the development of numerous large fluid-filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments which manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein.


Assuntos
Regulação da Expressão Gênica/fisiologia , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Humanos , Mutação , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Transcrição Gênica
5.
J Cachexia Sarcopenia Muscle ; 13(3): 1686-1703, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35277933

RESUMO

BACKGROUND: Cancer patients at advanced stages experience a severe depletion of skeletal muscle compartment together with a decrease in muscle function, known as cancer cachexia. Cachexia contributes to reducing quality of life, treatment efficiency, and lifespan of cancer patients. However, the systemic nature of the syndrome is poorly documented. Here, we hypothesize that glucocorticoids would be important systemic mediators of cancer cachexia. METHODS: To explore the role of glucocorticoids during cancer cachexia, biomolecular analyses were performed on several tissues (adrenal glands, blood, hypothalamus, liver, and skeletal muscle) collected from ApcMin/+ male mice, a mouse model of intestine and colon cancer, aged of 13 and 23 weeks, and compared with wild type age-matched C57BL/6J littermates. RESULTS: Twenty-three-week-old Apc mice recapitulated important features of cancer cachexia including body weight loss (-16%, P < 0.0001), muscle atrophy (gastrocnemius muscle: -53%, P < 0.0001), and weakness (-50% in tibialis anterior muscle force, P < 0.0001), increased expression of atrogens (7-fold increase in MuRF1 transcript level, P < 0.0001) and down-regulation of Akt-mTOR pathway (3.3-fold increase in 4EBP1 protein content, P < 0.0001), together with a marked transcriptional rewiring of hepatic metabolism toward an increased expression of gluconeogenic genes (Pcx: +90%, Pck1: +85%), and decreased expression of glycolytic (Slc2a2: -40%, Gk: -30%, Pklr: -60%), ketogenic (Hmgcs2: -55%, Bdh1: -80%), lipolytic/fatty oxidation (Lipe: -50%, Mgll: -60%, Cpt2: -60%, Hadh: -30%), and lipogenic (Acly: -30%, Acacb: -70%, Fasn: -45%) genes. The hypothalamic pituitary-adrenal axis was activated, as evidenced by the increase in the transcript levels of genes encoding corticotropin-releasing hormone in the hypothalamus (2-fold increase, P < 0.01), adrenocorticotropic hormone receptor (3.4-fold increase, P < 0.001), and steroid biosynthesis enzymes (Cyp21a1, P < 0.0001, and Cyp11b1, P < 0.01) in the adrenal glands, as well as by the increase in corticosterone level in the serum (+73%, P < 0.05), skeletal muscle (+17%, P < 0.001), and liver (+24%, P < 0.05) of cachectic 23-week-old Apc mice. A comparative transcriptional analysis with dexamethasone-treated C57BL/6J mice indicated that the activation of the hypothalamic-pituitary-adrenal axis in 23-week-old ApcMin/+ mice was significantly associated with the transcription of glucocorticoid-responsive genes in skeletal muscle (P < 0.05) and liver (P < 0.001). The transcriptional regulation of glucocorticoid-responsive genes was also observed in the gastrocnemius muscle of Lewis lung carcinoma tumour-bearing mice and in KPC mice (tibialis anterior muscle and liver). CONCLUSIONS: These findings highlight the role of the hypothalamic-pituitary-adrenal-glucocorticoid pathway in the transcriptional regulation of skeletal muscle catabolism and hepatic metabolism during cancer cachexia. They also provide the paradigm for the design of new therapeutic strategies.


Assuntos
Carcinoma Pulmonar de Lewis , Sistema Hipófise-Suprarrenal , Idoso , Animais , Caquexia/genética , Caquexia/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Expressão Gênica , Glucocorticoides , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipotálamo-Hipofisário/patologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Sistema Hipófise-Suprarrenal/metabolismo , Sistema Hipófise-Suprarrenal/patologia , Qualidade de Vida
6.
Commun Biol ; 5(1): 1068, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207615

RESUMO

TGF-ß signaling is involved in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis, representing one of the four major pathways genetically altered in 100% of PDAC cases. TGF-ß exerts complex and pleiotropic effects in cancers, notably via the activation of SMAD pathways, predominantly SMAD2/3/4. Though SMAD2 and 3 are rarely mutated in cancers, SMAD4 is lost in about 50% of PDAC, and the role of SMAD2/3 in a SMAD4-null context remains understudied. We herein provide evidence of a SMAD2/3 oncogenic effect in response to TGF-ß1 in SMAD4-null human PDAC cancer cells. We report that inactivation of SMAD2/3 in SMAD4-negative PDAC cells compromises TGF-ß-driven collective migration mediated by FAK and Rho/Rac signaling. Moreover, RNA-sequencing analyses highlight a TGF-ß gene signature related to aggressiveness mediated by SMAD2/3 in the absence of SMAD4. Using a PDAC patient cohort, we reveal that SMAD4-negative tumors with high levels of phospho-SMAD2 are more aggressive and have a poorer prognosis. Thus, loss of SMAD4 tumor suppressive activity in PDAC leads to an oncogenic gain-of-function of SMAD2/3, and to the onset of associated deleterious effects.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteína Smad3/metabolismo , Carcinogênese/genética , Carcinoma Ductal Pancreático/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , RNA , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias Pancreáticas
7.
J Biol Chem ; 284(31): 21011-26, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19491093

RESUMO

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway.


Assuntos
Canais de Cátion TRPP/química , Canais de Cátion TRPP/metabolismo , Substituição de Aminoácidos/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Células COS , Cálcio/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Genes Reporter , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Relação Estrutura-Atividade
8.
Hum Mol Genet ; 17(20): 3105-17, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18632682

RESUMO

Polycystin-1 (PC1), the product of the PKD1 gene mutated in the majority of autosomal dominant polycystic kidney disease (ADPKD) cases, undergoes a cleavage resulting in the intracellular release of its C-terminal tail (CTT). Here, we demonstrate that the PC1 CTT co-localizes with and binds to beta-catenin in the nucleus. This interaction requires a nuclear localization motif present in the PC1 CTT as well as the N-terminal portion of beta-catenin. The PC1 CTT inhibits the ability of both beta-catenin and Wnt ligands to activate T-cell factor (TCF)-dependent gene transcription, a major effector of the canonical Wnt signaling pathway. The PC1 CTT may produce this effect by reducing the apparent affinity of the interaction between beta-catenin and the TCF protein. DNA microarray analysis reveals that the canonical Wnt signaling pathway is activated in ADPKD patient cysts. Our results suggest a novel mechanism through which PC1 cleavage may impact upon Wnt-dependent signaling and thereby modulate both developmental processes and cystogenesis.


Assuntos
Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/química , Canais de Cátion TRPP/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Ligantes , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Rim Policístico Autossômico Dominante/etiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Biologia de Sistemas , Fatores de Transcrição TCF/genética , Transfecção
9.
Sci Rep ; 10(1): 3880, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127548

RESUMO

Transforming growth factor (TGFß) is a secreted factor, which accumulates in tissues during many physio- and pathological processes such as embryonic development, wound healing, fibrosis and cancer. In order to analyze the effects of increased microenvironmental TGFß concentration in vivo, we developed a conditional transgenic mouse model (Flpo/Frt system) expressing bioactive TGFß in fibroblasts, a cell population present in the microenvironment of almost all tissues. To achieve this, we created the genetically-engineered [Fsp1-Flpo; FSFTGFßCA] mouse model. The Fsp1-Flpo allele consists in the Flpo recombinase under the control of the Fsp1 (fibroblast-specific promoter 1) promoter. The FSFTGFßCA allele consists in a transgene encoding a constitutively active mutant form of TGFß (TGFßCA) under the control of a Frt-STOP-Frt (FSF) cassette. The FSFTGFßCA allele was created to generate this model, and functionally validated by in vitro, ex vivo and in vivo techniques. [Fsp1-Flpo; FSFTGFßCA] animals do not present any obvious phenotype despite the correct expression of TGFßCA transgene in fibroblasts. This [Fsp1-Flpo; FSFTGFßCA] model is highly pertinent for future studies on the effect of increased microenvironmental bioactive TGFß concentrations in mice bearing Cre-dependent genetic alterations in other compartments (epithelial or immune compartments for instance). These dual recombinase system (DRS) approaches will enable scientists to study uncoupled spatiotemporal regulation of different genetic alterations within the same mouse, thus better replicating the complexity of human diseases.


Assuntos
Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Expressão Gênica , Engenharia Genética , Células Hep G2 , Humanos , Camundongos , Camundongos Transgênicos , Modelos Animais
11.
Cell Death Dis ; 10(12): 886, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767842

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the solid tumors with the poorest prognosis. The stroma of this tumor is abundant and composed of extracellular matrix and stromal cells (including cancer-associated fibroblasts and immune cells). Nerve fibers invading this stroma represent a hallmark of PDAC, involved in neural remodeling, which participates in neuropathic pain, cancer cell dissemination and tumor relapse after surgery. Pancreatic cancer-associated neural remodeling is regulated through functional interplays mediated by physical and molecular interactions between cancer cells, nerve cells and surrounding Schwann cells, and other stromal cells. In the present study, we show that Schwann cells (glial cells supporting peripheral neurons) can enhance aggressiveness (migration, invasion, tumorigenicity) of pancreatic cancer cells in a transforming growth factor beta (TGFß)-dependent manner. Indeed, we reveal that conditioned medium from Schwann cells contains high amounts of TGFß able to activate the TGFß-SMAD signaling pathway in cancer cells. We also observed in human PDAC samples that high levels of TGFß signaling activation were positively correlated with perineural invasion. Secretome analyses by mass spectrometry of Schwann cells and pancreatic cancer cells cultured alone or in combination highlighted the central role of TGFß in neuro-epithelial interactions, as illustrated by proteomic signatures related to cell adhesion and motility. Altogether, these results demonstrate that Schwann cells are a meaningful source of TGFß in PDAC, which plays a crucial role in the acquisition of aggressive properties by pancreatic cancer cells.

12.
Mol Biol Cell ; 16(11): 5087-93, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16107561

RESUMO

Polycystin-1 (PC-1) is the product of the PKD1 gene, which is mutated in autosomal dominant polycystic kidney disease. We show that the Na,K-ATPase alpha-subunit interacts in vitro and in vivo with the final 200 amino acids of the polycystin-1 protein, which constitute its cytoplasmic C-terminal tail. Functional studies suggest that this association may play a role in the regulation of the Na,K-ATPase activity. Chinese hamster ovary cells stably expressing the entire PC-1 protein exhibit a dramatic increase in Na,K-ATPase activity, although the kinetic properties of the enzyme remain unchanged. These data indicate that polycystin-1 may contribute to the regulation of Na,K-ATPase activity in kidneys in situ, thus modulating renal tubular fluid and electrolyte transport.


Assuntos
Ouabaína/farmacologia , Proteínas , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Sódio/farmacologia , Animais , Células CHO/ultraestrutura , Linhagem Celular , Cricetinae , Cricetulus , Cães , Inibidores Enzimáticos , Escherichia , Mutação , Doenças Renais Policísticas , Proteínas Recombinantes , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Canais de Cátion TRPP , Transfecção
13.
J Clin Invest ; 114(10): 1433-43, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15545994

RESUMO

Polycystin-1, which is encoded by a gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), is involved in cell-matrix interactions as well as in ciliary signaling. The precise mechanisms by which it functions, however, remain unclear. Here we find that polycystin-1 undergoes a proteolytic cleavage that releases its C-terminal tail (CTT), which enters the nucleus and initiates signaling processes. The cleavage occurs in vivo in association with alterations in mechanical stimuli. Polycystin-2, the product of the second gene mutated in ADPKD, modulates the signaling properties of the polycystin-1 CTT. These data reveal a novel pathway by which polycystin-1 transmits messages directly to the nucleus.


Assuntos
Núcleo Celular/metabolismo , Proteínas/química , Proteínas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Cães , Embrião de Mamíferos , Células Epiteliais/citologia , Túbulos Renais/citologia , Túbulos Renais/embriologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Proteínas/genética , Deleção de Sequência , Estresse Mecânico , Canais de Cátion TRPP , Fator de Transcrição AP-1/metabolismo
14.
Stem Cell Reports ; 9(1): 264-278, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28648900

RESUMO

Understanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A.


Assuntos
Diferenciação Celular , Metilação de DNA , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/citologia , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/citologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Linhagem Celular , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/metabolismo , Humanos , Regiões Promotoras Genéticas , Células-Tronco/metabolismo
15.
Ann N Y Acad Sci ; 986: 360-8, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12763851

RESUMO

The sorting and regulation of the Na,K and H,K-ATPases requires that the pump proteins must associate, at least transiently, with kinases, phosphatases, scaffolding molecules, and components of the cellular trafficking machinery. The identities of these interacting proteins and the nature of their associations with the pump polypeptides have yet to be elucidated. We have begun a series of yeast two-hybrid screens employing structurally defined segments of pump polypeptides as baits in order to gain insight into the nature and function of these interacting proteins.


Assuntos
Proteínas de Transporte/metabolismo , Bombas de Íon/química , Bombas de Íon/metabolismo , Animais , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte/química , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo
17.
J Biol Chem ; 278(38): 36786-93, 2003 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-12840011

RESUMO

Mutations in PKD1 and PKD2, the genes that encode polycystin-1 and polycystin-2 respectively, account for almost all cases of autosomal dominant polycystic kidney disease. Although the polycystins are believed to interact in vivo, the two proteins often display dissimilar patterns and gradients of expression during development. In an effort to understand this apparent discrepancy, we investigated how changes in polycystin-2 expression can affect the subcellular localization of polycystin-1. We show that, when polycystin-1 is expressed alone in a PKD2 null cell line, it localizes to the cell surface, as well as to the endoplasmic reticulum. When co-expressed with polycystin-2, however, polycystin-1 is not seen at the cell surface and co-localizes completely with polycystin-2 in the endoplasmic reticulum. The localization of a polycystin-1 fusion protein was similarly affected by changes in its level of expression relative to that of polycystin-2. This phenomenon was observed in populations as well as in individual COS-7 cells. Our data suggest that the localization of polycystin-1 can be regulated via the relative expression level of polycystin-2 in mammalian cells. This mechanism may help to explain the divergent patterns and levels of expression observed for the polycystins, and may provide clues as to how the function of these two proteins are regulated during development.


Assuntos
Proteínas de Membrana/biossíntese , Biossíntese de Proteínas , Proteínas , Animais , Western Blotting , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , DNA Complementar/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Testes de Precipitina , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Canais de Cátion TRPP , Transfecção
18.
Am J Pathol ; 160(3): 973-83, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11891195

RESUMO

Autosomal-dominant polycystic kidney disease, one of the most frequent human genetic disorders, is genetically heterogeneous. Most cases result from mutations of PKD1 or PKD2 encoding polycystin-1 or polycystin-2, respectively. Polycystin-1 is a large transmembrane protein containing several domains involved in cell-cell and/or cell-matrix interactions. Polycystin-2 is transmembrane glycoprotein sharing homology with some families of cation channels. Despite a large number of reports, the tissue distribution of these two proteins, especially of polycystin-1, is still debated. We investigated the expression pattern of PKD1 and PKD2 transcripts and proteins during human embryogenesis and kidney development, using Northern blot analysis, in situ hybridization, and immunohistochemical methods. For each gene, the expression pattern of transcripts and protein was concordant. In human 5- to 6-week-old embryos, both genes are widely expressed, mainly in neural tissue, cardiomyocytes, endodermal derivatives, and mesonephros. At this age, PKD2 but not PKD1 expression is observed in the ureteric bud and the uninduced metanephros. Thereafter, PKD2 is diffusely expressed at all stages of nephron development, whereas high PKD1 expression first appears in differentiated proximal tubules. Proximal tubule expression of both genes decreases from weeks 20 to 24 onwards. PKD1 transcripts, later restricted to distal tubules in fetal nephrogenesis, are no longer detected in adult kidneys, which nevertheless maintain a faint expression of polycystin-1, whereas persistent expression of PKD2 transcripts and protein is observed throughout nephrogenesis. Overall, contrary to previous observations, we found profound differences in the spatiotemporal expression of PKD1 and PKD2 during nephrogenesis, PKD2 being expressed earlier and more diffusely than PKD1. These data suggest that polycystins could interact with different partners, at least during kidney development.


Assuntos
Embrião de Mamíferos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Rim/fisiologia , Proteínas de Membrana/genética , Proteínas/genética , Adulto , Feminino , Humanos , Proteínas de Membrana/biossíntese , Gravidez , Biossíntese de Proteínas , Canais de Cátion TRPP , Transcrição Gênica
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa