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1.
Front Cell Dev Biol ; 11: 1270980, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38125876

RESUMO

Autosomal recessive polycystic kidney disease (ARPKD; MIM#263200) is a severe, hereditary, hepato-renal fibrocystic disorder that leads to early childhood morbidity and mortality. Typical forms of ARPKD are caused by pathogenic variants in the PKHD1 gene, which encodes the fibrocystin/polyductin (FPC) protein. MYC overexpression has been proposed as a driver of renal cystogenesis, but little is known about MYC expression in recessive PKD. In the current study, we provide the first evidence that MYC is overexpressed in kidneys from ARPKD patients and confirm that MYC is upregulated in cystic kidneys from cpk mutant mice. In contrast, renal MYC expression levels were not altered in several Pkhd1 mutant mice that lack a significant cystic kidney phenotype. We leveraged previous observations that the carboxy-terminus of mouse FPC (FPC-CTD) is proteolytically cleaved through Notch-like processing, translocates to the nucleus, and binds to double stranded DNA, to examine whether the FPC-CTD plays a role in regulating MYC/Myc transcription. Using immunofluorescence, reporter gene assays, and ChIP, we demonstrate that both human and mouse FPC-CTD can localize to the nucleus, bind to the MYC/Myc P1 promoter, and activate MYC/Myc expression. Interestingly, we observed species-specific differences in FPC-CTD intracellular trafficking. Furthermore, our informatic analyses revealed limited sequence identity of FPC-CTD across vertebrate phyla and database queries identified temporal differences in PKHD1/Pkhd1 and CYS1/Cys1 expression patterns in mouse and human kidneys. Given that cystin, the Cys1 gene product, is a negative regulator of Myc transcription, these temporal differences in gene expression could contribute to the relative renoprotection from cystogenesis in Pkhd1-deficient mice. Taken together, our findings provide new mechanistic insights into differential mFPC-CTD and hFPC-CTD regulation of MYC expression in renal epithelial cells, which may illuminate the basis for the phenotypic disparities between human patients with PKHD1 pathogenic variants and Pkhd1-mutant mice.

2.
Front Mol Biosci ; 9: 946344, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36710876

RESUMO

Transcription factor Ap2b (TFAP2B), an AP-2 family transcription factor, binds to the palindromic consensus DNA sequence, 5'-GCCN3-5GGC-3'. Mice lacking functional Tfap2b gene die in the perinatal or neonatal period with cystic dilatation of the kidney distal tubules and collecting ducts, a phenotype resembling autosomal recessive polycystic kidney disease (ARPKD). Human ARPKD is caused by mutations in PKHD1, DZIP1L, and CYS1, which are conserved in mammals. In this study, we examined the potential role of TFAP2B as a common regulator of Pkhd1 and Cys1. We determined the transcription start site (TSS) of Cys1 using 5' Rapid Amplification of cDNA Ends (5'RACE); the TSS of Pkhd1 has been previously established. Bioinformatic approaches identified cis-regulatory elements, including two TFAP2B consensus binding sites, in the upstream regulatory regions of both Pkhd1 and Cys1. Based on reporter gene assays performed in mouse renal collecting duct cells (mIMCD-3), TFAP2B activated the Pkhd1 and Cys1 promoters and electromobility shift assay (EMSA) confirmed TFAP2B binding to the in silico identified sites. These results suggest that Tfap2b participates in a renal epithelial cell gene regulatory network that includes Pkhd1 and Cys1. Disruption of this network impairs renal tubular differentiation, causing ductal dilatation that is the hallmark of recessive PKD.

3.
Kidney Int ; 98(5): 1225-1241, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32610050

RESUMO

Polycystin-1 (PC1) and -2 (PC2), products of the PKD1 and PKD2 genes, are mutated in autosomal dominant polycystic kidney disease (ADPKD). They localize to the primary cilia; however, their ciliary function is in dispute. Loss of either the primary cilia or PC1 or PC2 causes cyst formation. However, loss of both cilia and PC1 or PC2 inhibits cyst growth via an unknown pathway. To help define a pathway, we studied cilium length in human and mouse kidneys. We found cilia are elongated in kidneys from patients with ADPKD and from both Pkd1 and Pkd2 knockout mice. Cilia elongate following polycystin inactivation. The role of intraflagellar transport proteins in Pkd1-deficient mice is also unknown. We found that inactivation of Ift88 (a gene expressing a core component of intraflagellar transport) in Pkd1 knockout mice, as well as in a new Pkd2 knockout mouse, shortened the elongated cilia, impeded kidney and liver cystogenesis, and reduced cell proliferation. Multi-stage in vivo analysis of signaling pathways revealed ß-catenin activation as a prominent, early, and sustained event in disease onset and progression in Pkd2 single knockout but not in Pkd2.Ift88 double knockout mouse kidneys. Additionally, AMPK, mTOR and ERK pathways were altered in Pkd2 single knockout mice but only AMPK and mTOR pathway alteration were rescued in Pkd2.Ift88 double knockout mice. Thus, our findings advocate an essential role of polycystins in the structure and function of the primary cilia and implicate ß-catenin as a key inducer of cystogenesis downstream of the primary cilia. Our data suggest that modulating cilium length and/or its associated signaling events may offer novel therapeutic approaches for ADPKD.


Assuntos
Cistos , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Animais , Cílios , Cistos/genética , Humanos , Rim , Fígado , Camundongos , Camundongos Knockout , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética
4.
FASEB J ; 33(9): 9945-9958, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31157564

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder causing renal failure. Mutations of polycystic kidney disease 1 (PKD1) account for most ADPKD cases. Defective ciliary localization of polycystin-1 (PC1), a large integral membrane protein encoded by PKD1, underlies the pathogenesis of a subgroup of patients with ADPKD. However, the mechanisms by which PC1 and other ciliary proteins traffic to the primary cilium remain poorly understood. A ciliary targeting sequence (CTS) that resides in ciliary receptors is considered to function in the process. It has been reported that the VxP motif in the intracellular C-terminal tail of PC1 functions as a CTS in an ADP ribosylation factor 4 (Arf4)/ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1)-dependent manner. However, other recent studies have revealed that this motif is dispensable for PC1 trafficking to cilia. In this study, we identified a novel CTS consisting of 8 residues (RHKVRFEG) in the PC1 C tail. We found that this motif is sufficient to bind protein phosphatase 1 (PP1)α, a ubiquitously expressed phosphatase in the phosphoprotein phosphatase (PPP) family. Mutations in this CTS motif disrupt binding with PP1α and impair ciliary localization of PC1. Additionally, short hairpin RNA-mediated knockdown of PP1α results in reduced ciliary localization of PC1 and elongated cilia, suggesting a role for PP1α in the regulation of ciliary structure and function.-Luo, C., Wu, M., Su, X., Yu, F., Brautigan, D. L., Chen, J., Zhou, J. Protein phosphatase 1α interacts with a novel ciliary targeting sequence of polycystin-1 and regulates polycystin-1 trafficking.


Assuntos
Proteína Fosfatase 1/metabolismo , Canais de Cátion TRPP/metabolismo , Alanina , Sequência de Aminoácidos , Animais , Linhagem Celular , Camundongos , Camundongos Knockout , Mutagênese , Proteína Fosfatase 1/genética , Transporte Proteico , Canais de Cátion TRPP/genética
5.
PeerJ ; 6: e5178, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30013843

RESUMO

BACKGROUND: The importance of circulating antibodies as biomarkers of kidney disease has recently been recognized. However, no study has systematically described the methodology of sample preparation and storage regarding antibodies as biomarkers of kidney disease. It remains unknown whether repetitive freeze-thaw cycles, physical disturbances, storage at different temperatures or for different periods of time, or haemolytic or turbid serum samples affect antibody measurements. The aim of this study was to investigate the stabilities of antibodies associated with kidney disease in serum samples under various relevant clinical and research conditions. METHODS: We stored serum samples in the following different conditions: repetitive freeze-thaw cycles (1, 6 or 12 times), long-term storage (7 or 12 months at -80 °C), physical disturbance (1 or 8 h), and storage at 4 °C (1, 3 or 6 weeks) and room temperature (1 or 7 days). The stabilities of the anti-phospholipase A2 receptor (anti-PLA2R), anti-glomerular basement membrane, anti-myeloperoxidase and anti-proteinase 3 antibodies were evaluated with enzyme-linked immunosorbent assays (ELISA). RESULTS: We found that repetitive freeze-thaw cycles did not have a significant effect on the stabilities of the abovementioned antibodies in clear serum samples. The ELISA readings of haemolytic and turbid serum samples tended to increase and decrease, respectively. Neither long-term storage at -80 °C nor physical disturbance had a significant effect on anti-PLA2R antibody stability in sealed serum samples. The concentrations of most of these antibodies increased in unsealed serum samples that were stored at 4 °C for more than 6 weeks or at room temperature for more than 7 days. DISCUSSION: Our findings revealed that the abovementioned circulating antibodies that are used as biomarkers for kidney disease had stable physicochemical properties, structures and immunoreactivities such that they were not influenced by repetitive freeze-thaw cycles, physical disturbances or long-term storage at -80 °C. However, the ELISA readings tended to change for haemolytic, turbid and unsealed serum samples.

6.
Mol Med Rep ; 15(1): 375-379, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27959435

RESUMO

Previous studies have demonstrated that oridonin, a tetracyclic diterpenoid compound extracted from Rabdosia rubescens, inhibits proliferation and induces apoptosis in several tumor cell lines. However, the mechanism by which oridonin inhibits the cell cycle remains poorly understood. In the present study, possible mechanisms by which oridonin affects cell cycle progression were explored in A549 lung cancer cells. Flow cytometry analysis indicated that oridonin inhibited the proliferation of A549 cells by inducing G2/M cell cycle arrest in a dose­dependent manner. Western blot analysis revealed that in oridonin treated cells, phosphorylated (p­)ATM serine/threonine kinase (S1981), p­checkpoint kinase 2 (CHK2) (T68), p­p53, and phosphorylated H2A histone family member X protein levels were visibly increased, indicating that oridonin promoted G2/M arrest in A549 cells through the ATM­p53­CHK2 pathway. This data suggests that oridonin promotes G2/M arrest in A549 cells by facilitating ATM activation, which is likely a common mechanism in other tumor cell types when using this drug for cancer treatment.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Diterpenos do Tipo Caurano/farmacologia , Ativação Enzimática/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Células A549 , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diterpenos do Tipo Caurano/química , Humanos , Isodon/química , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
7.
J Cell Sci ; 129(19): 3675-3684, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27505895

RESUMO

Mutation of PKD1, encoding the protein polycystin-1 (PC1), is the main cause of autosomal dominant polycystic kidney disease (ADPKD). The signaling pathways downstream of PC1 in ADPKD are still not fully understood. Here, we provide genetic evidence for the necessity of Gα12 (encoded by Gna12, hereafter Gα12) for renal cystogenesis induced by Pkd1 knockout. There was no phenotype in mice with deletion of Gα12 (Gα12-/-). Polyinosine-polycytosine (pI:pC)-induced deletion of Pkd1 (Mx1Cre+Pkd1f/fGα12+/+) in 1-week-old mice resulted in multiple kidney cysts by 9 weeks, but the mice with double knockout of Pkd1 and Gα12 (Mx1Cre+Pkd1f/fGα12-/-) had no structural and functional abnormalities in the kidneys. These mice could survive more than one year without kidney abnormalities except multiple hepatic cysts in some mice, which indicates that the effect of Gα12 on cystogenesis is kidney specific. Furthermore, Pkd1 knockout promoted Gα12 activation, which subsequently decreased cell-matrix and cell-cell adhesion by affecting the function of focal adhesion and E-cadherin, respectively. Our results demonstrate that Gα12 is required for the development of kidney cysts induced by Pkd1 mutation in mouse ADPKD.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Rim/metabolismo , Rim/patologia , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/metabolismo , Animais , Caderinas/metabolismo , Junções Célula-Matriz , Células Epiteliais/metabolismo , Deleção de Genes , Técnicas de Inativação de Genes , Fígado/metabolismo , Fígado/patologia , Camundongos , Modelos Biológicos , Fibras de Estresse/metabolismo
8.
Oncol Rep ; 35(5): 2707-14, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26986476

RESUMO

As an effective tumor suppressor, phosphatase and tensin homolog (PTEN) has attracted the increased attention of scientists. Recent studies have shown that PTEN plays unique roles in the DNA damage response (DDR) and can interact with the Chk1 pathway. However, little is known about how PTEN contributes to DDR through the ATM-Chk2 pathway. It is well-known that etoposide induces G2/M arrest in a variety of cell lines, including MCF-7 cells. The DNA damage-induced G2/M arrest results from the activation of protein kinase ataxia telangiectasia mutated (ATM), followed by the activation of Chk2 that subsequently inactivates CDC25C, resulting in G2/M arrest. In the present study, we assessed the contribution of PTEN to the etoposide-induced G2/M cell cycle arrest. PTEN was knocked down in MCF-7 cells by specific shRNA, and the effects of PTEN on the ATM-Chk2 pathway were investigated through various approaches. The results showed that knockdown of PTEN strongly antagonized ATM activation in response to etoposide treatment, and thereby reduced the phosphorylation level of ATM substrates, including H2AX, P53 and Chk2. Furthermore, depletion of PTEN reduced the etoposide-induced phosphorylation of CDC25C and strikingly compromised etoposide-induced G2/M arrest in the MCF-7 cells. Altogether, we demonstrated that PTEN plays a unique role in etoposide-induced G2/M arrest by facilitating the activation of the ATM pathway, and PTEN was required for the proper activation of checkpoints in response to DNA damage in MCF-7 cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Etoposídeo/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , PTEN Fosfo-Hidrolase/fisiologia , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Células MCF-7 , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
9.
Hum Mol Genet ; 25(3): 448-58, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26612203

RESUMO

Epilepsy or seizure disorder is among the least understood chronic medical conditions affecting over 65 million people worldwide. Here, we show that disruption of the polycystic kidney disease 2-like 1 (Pkd2l1 or Pkdl), encoding polycystin-L (PCL), a non-selective cation channel, increases neuronal excitability and the susceptibility to pentylenetetrazol-induced seizure in mice. PCL interacts with ß2-adrenergic receptor (ß2AR) and co-localizes with ß2AR on the primary cilia of neurons in the brain. Pkdl deficiency leads to the loss of ß2AR on neuronal cilia, which is accompanied with a remarkable reduction in cAMP levels in the central nervous system (CNS). The reduction of cAMP levels is associated with a reduction in the activation of cAMP response element-binding protein, but not the activation of Ca(2+)/calmodulin-dependent protein kinase II, Akt or mitogen-activated protein kinases. Our data, thus, indicate for the first time that a ciliary protein complex is required for the control of neuronal excitability in the CNS.


Assuntos
Canais de Cálcio/genética , Córtex Cerebral/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Epilepsia/genética , Hipocampo/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores de Superfície Celular/genética , Tálamo/metabolismo , Animais , Canais de Cálcio/deficiência , Córtex Cerebral/patologia , Cílios/metabolismo , Cílios/patologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Epilepsia/patologia , Potenciais Pós-Sinápticos Excitadores , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/patologia , Humanos , Transporte de Íons , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Pentilenotetrazol , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Superfície Celular/deficiência , Transdução de Sinais , Tálamo/patologia
10.
J Cell Sci ; 128(22): 4063-73, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26430213

RESUMO

Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD.


Assuntos
Cílios/metabolismo , Túbulos Renais Coletores/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Células HEK293 , Humanos , Túbulos Renais Coletores/citologia , Camundongos , Canais de Cátion TRPP/genética
11.
Hum Mol Genet ; 23(20): 5441-51, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24939912

RESUMO

Bardet-Biedl syndrome (BBS) and autosomal dominant polycystic kidney disease (ADPKD) are two genetically distinct ciliopathies but share common phenotypes such as renal cysts. Seven BBS proteins form a complex called the BBSome which is localized at the basal body or ciliary axoneme and regulates the ciliary entry or flagellar exit of several signaling molecules. Here, we demonstrate that, unlike the seven-span somatostatin receptor 3 or the leptin receptor that interacts with all subunits of the BBSome, the ADPKD protein polycystin-1 (PC1) interacts with BBS1, BBS4, BBS5 and BBS8, four of the seven components of the BBSome. Only depletion or mutation of BBS1, but not depletion of BBS5 and BBS8, or knockout of BBS4, impairs ciliary trafficking of PC1 in kidney epithelial cells. Depletion of these BBS proteins affects neither the ciliary length nor the plasma membrane targeting of PC1. Expression of a pathogenic BBS3/Arl6 mutant (T31R) that locks Arl6 in the GDP form leads to stunted cilia and inhibition of PC1 on primary cilia. We propose that the 11-span membrane protein PC1 is a BBSome cargo and that the components of the BBSome may possess subunit-specific functions. Moreover, physical interactions between the BBS and ADPKD proteins may underline the overlapping renal phenotypes in these two diseases.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Rim/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Canais de Cátion TRPP/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Linhagem Celular , Cílios/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Transporte Proteico , Canais de Cátion TRPP/genética
12.
PLoS One ; 9(5): e95630, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24851866

RESUMO

FPC (fibrocystin or polyductin) is a single transmembrane receptor-like protein, responsible for the human autosomal recessive polycystic kidney disease (ARPKD). It was recently proposed that FPC undergoes a Notch-like cleavage and subsequently the cleaved carboxy(C)-terminal fragment translocates to the nucleus. To study the functions of the isolated C-tail, we expressed the intracellular domain of human FPC (hICD) in renal epithelial cells. By 3-dimensional (3D) tubulogenesis assay, we found that in contrast to tubule-like structures formed from control cells, hICD-expressing cells exclusively formed cyst-like structures. By western blotting, we showed that the Akt/mTOR pathway, indicated by increased phosphorylation of Akt at serine 473 and S6 kinase 1 at threonine 389, was constitutively activated in hICD-expressing cells, similar to that in FPC knockdown cells and ARPKD kidneys. Moreover, application of mTOR inhibitor rapamycin reduced the size of the cyst-like structures formed by hICD-expressing cells. Application of either LY294002 or wortmannin inhibited the activation of both S6K1 and Akt. Expression of full-length FPC inhibited the activation of S6 and S6 kinase whereas co-expression of hICD with full-length FPC antagonized the inhibitory effect of full-length FPC on mTOR. Taken together, we propose that FPC modulates the PI3K/Akt/mTOR pathway and the cleaved C-tail regulates the function of the full-length protein.


Assuntos
Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Células Cultivadas , Pré-Escolar , Ativação Enzimática , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lactente , Recém-Nascido , Rim/citologia , Rim/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/genética , Proteínas Quinases S6 Ribossômicas/metabolismo
13.
PLoS One ; 8(12): e83062, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24349431

RESUMO

Cystin is a novel cilia-associated protein that is disrupted in the cpk mouse, a well-characterized mouse model of autosomal recessive polycystic kidney disease (ARPKD). Interestingly, overexpression of the Myc gene is evident in animal models of ARPKD and is thought to contribute to the renal cystic phenotype. Using a yeast two-hybrid approach, the growth suppressor protein necdin, known to modulate Myc expression, was found as an interacting partner of cystin. Deletion mapping demonstrated that the C-terminus of cystin and both termini of necdin are required for their mutual interaction. Speculating that these two proteins may function to regulate gene expression, we developed a luciferase reporter assay and observed that necdin strongly activated the Myc P1 promoter, and cystin did so more modestly. Interestingly, the necdin effect was significantly abrogated when cystin was co-transfected. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed a physical interaction with both necdin and cystin and the Myc P1 promoter, as well as between these proteins. The data suggest that these proteins likely function in a regulatory complex. Thus, we speculate that Myc overexpression in the cpk kidney results from the dysregulation of the cystin-necdin regulatory complex and c-Myc, in turn, contributes to cystogenesis in the cpk mouse.


Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Rim Policístico Autossômico Recessivo/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Animais , Proteínas de Membrana/genética , Camundongos , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Rim Policístico Autossômico Recessivo/genética , Rim Policístico Autossômico Recessivo/patologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética
14.
J Am Soc Nephrol ; 24(10): 1571-86, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24009235

RESUMO

Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.


Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Recessivo/genética , Canais de Cátion TRPP/genética , Adulto , Estudos de Casos e Controles , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/metabolismo
15.
Hum Mol Genet ; 19(17): 3306-19, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20554582

RESUMO

Autosomal recessive polycystic kidney disease (ARPKD) is a significant hereditary renal disease occurring in infancy and childhood, which presents with greatly enlarged echogenic kidneys, ultimately leading to renal insufficiency and end-stage renal disease. ARPKD is caused by mutations in a single gene PKHD1, which encodes fibrocystin/polyductin (FPC), a large single transmembrane protein generally known to be on the primary cilium, basal body and plasma membrane. Here, using our newly generated antibody raised against the entire C-terminal intracellular cytoplasmic domain (ICD) of FPC, as well as our previously well-characterized antibody against a peptide of ICD, we report for the first time that at least one isoform of FPC is localized to the centrosome and mitotic spindle of dividing cells in multiple cell lines, including MDCK, mIMCD3, LLC-PK1, HEK293, RCTEC and HFCT cells. Using short-hairpin-mediated RNA interference, we show that the inhibition of FPC function in MDCK and mIMCD3 cells leads to centrosome amplification, chromosome lagging and multipolar spindle formation. Consistent with our in vitro findings, we also observed centrosome amplification in the kidneys from human ARPKD patients. These findings demonstrate a novel function of FPC in centrosome duplication and mitotic spindle assembly during cell division. We propose that mitotic defects due to FPC dysfunction contribute to cystogenesis in ARPKD.


Assuntos
Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/metabolismo , Fuso Acromático/metabolismo , Adolescente , Animais , Linhagem Celular Tumoral , Centrossomo/metabolismo , Criança , Pré-Escolar , Modelos Animais de Doenças , Cães , Feminino , Humanos , Lactente , Masculino , Camundongos , Rim Policístico Autossômico Recessivo/genética , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Fuso Acromático/genética
16.
Int J Mol Med ; 17(5): 749-54, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16596256

RESUMO

As an atypical member of the Rab family, Rab24 has several attributes distinguishing this protein from the other members. Based on the yeast two-hybrid system, interaction between human RAB24 and two proteins, cyclophilin A (CyP-A) and gamma-aminobutyric acid type A receptor-associated protein (GABARAP), was detected and identified in COS-7 cells. Interestingly, RAB24 is predominantly localized in the nuclei of COS-7 cells, which is different from previous reports using other cell lines. RAB24 (D123I) can trigger the accumulation of intracellular inclusions, with small quantities of intranuclear inclusions in some cells. The GTPase activity of RAB24 and its two mutants was detected.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Ciclofilina A/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose , Células COS , Chlorocebus aethiops , Ciclofilina A/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/genética , Mutação , Plasmídeos/genética , Ligação Proteica , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/genética
17.
Sheng Wu Gong Cheng Xue Bao ; 22(2): 338-43, 2006 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-16607966

RESUMO

Nowadays genetic tests are available in a growing number of countries, for an expanding set of conditions. Nonetheless, many Chinese people are still not familiar with the principle, testing types, technologies used in this process, application and benefits to society, and national or international administration of genetic testing. It is essential that this increased use of genetic testing should be accompanied by appropriate oversight. This article has roughly reviewed the proceeding of genetic testing these years, which will help us learn more about the new coming era of human genetics and molecular medical revolutions.


Assuntos
Técnicas Genéticas/tendências , Testes Genéticos/tendências , Predisposição Genética para Doença/prevenção & controle , Humanos
18.
Int J Mol Med ; 16(4): 701-7, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16142408

RESUMO

Phox homology (PX) domains specifically bind to phosphoinositides, and this interaction is crucial for their cellular function. A full-length cDNA of human PX domain containing serine/threonine kinase gene (PXK) that we termed PXK_v1 had previously been cloned. PXK_v1 consists of a S_TKc domain (serine/threonine kinases, catalytic domain), but lacks several residues that are indispensable for intrinsic catalytic activity. Evidence obtained in the present study demonstrated the existence of four other splice isoforms of human PXK in fetal brain, designated as PXK_v2, PXK_v3, PXK_v4 and PXK_v5. The results of RT-PCR indicated that human PXK_v1, PXK_v2 and PXK_v4 transcripts were widely expressed in human adult tissue, except heart tissue. In contrast, PXK_v3 transcripts were only expressed in peripheral blood leukocytes at a low level and PXK_v5 transcripts were not detectable in any of the tissue analyzed. Subcellular localization analysis of EGFP-PXK fusion proteins in COS7 cells indicated that EGFP-PXK_v3 had a different subcellular localization compared to other EGFP-PXK fusion proteins. Mutation analysis of EGFP-PXK_v1 showed that PXK_v1-Tyr56 and Arg92 are essential for subcellular localization of the protein in the cytoplasm.


Assuntos
Processamento Alternativo/genética , Perfilação da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/química , DNA Complementar/genética , Feminino , Biblioteca Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas do Tecido Nervoso , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transfecção
19.
DNA Seq ; 16(2): 137-42, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16147865

RESUMO

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.


Assuntos
Processamento Alternativo , Rim/metabolismo , Leucócitos/metabolismo , Fígado/metabolismo , Oxo-Ácido-Liases/biossíntese , Oxo-Ácido-Liases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/metabolismo , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilação , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina/química , Suínos , Treonina/química , Distribuição Tecidual
20.
Biochem Genet ; 43(3-4): 165-73, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15932064

RESUMO

By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human cDNA (C4orf13). This cDNA is 2706 bp in length, encoding a 340-amino-acid polypeptide that contains a typical SBF (sodium bile acid cotransporter family) domain and ten possible transmembrane segments. The putative protein C4orf13 shows high similarity with its orthologs in Mus musculus and Xenopus laevis. Human C4orf13 is mapped to chromosome 4q31.2 and contains 12 exons. RT-PCR analysis shows that human C4orf13 is widely expressed in human tissues, and the expression levels in liver and lung are relatively high, expression levels in placenta, kidney, spleen, and thymus are moderate, low levels of expression are detected in heart, prostate, and testis.


Assuntos
Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Simportadores/química , Xenopus laevis
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