Assuntos
Aldosterona/urina , Cromatografia Líquida de Alta Pressão , Imunoensaio/normas , Medições Luminescentes/normas , Espectrometria de Massas em Tandem , Urinálise/métodos , Urinálise/normas , Automação , Humanos , Imunoensaio/instrumentação , Medições Luminescentes/instrumentação , Radioimunoensaio , Análise de RegressãoRESUMO
The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.
Assuntos
Polaridade Celular/fisiologia , Proteínas de Drosophila/metabolismo , Células Eucarióticas/enzimologia , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos/fisiologia , Animais , Encéfalo/enzimologia , Células COS , Proteínas de Drosophila/genética , Humanos , Substâncias Macromoleculares , Camundongos , Complexos Multiproteicos , Fosforilação , Ligação Proteica/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/genética , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas/genética , Células Tumorais Cultivadas , Cicatrização/fisiologiaRESUMO
The p75 neurotrophin receptor (p75NTR) is required for the activity of growth cone collapsing factors such as Nogo, MAG, OMgP, and ephrin A. Specifically, p75NTR associates with the Nogo receptor and GPI-linked ephrin A, and unliganded p75NTR mediates the biological effects of those proteins. Here we assess the requirement for p75NTR for the growth cone collapsing responses of semaphorins (Sema) 3A and 3F and ephrin B2 in sympathetic neurons. We show that the ability of Sema 3s or ephrin B2 to collapse growth cones is suppressed in p75NTR-/- sympathetic neurons. Ectopic expression of p75NTR restores the collapsing activity of Sema 3 in p75NTR-/- neurons. Moreover, p75NTR must be bound to its neurotrophin ligands to participate in Sema 3-mediated collapse. Ligand-bound p75NTR participates in Sema 3 and ephrin B2-mediated collapse via the Rho signaling pathway, since inhibition of Rho signaling is sufficient to suppress the effects of Sema 3s and ephrin B2 in p75NTR+/+ but not p75NTR-/- neurons. Our data suggest that in addition to its role as a co-receptor, p75NTR may provide an obligate parallel neurotrophin-activated inhibitory pathway that broadly sensitizes neurons to inhibitory cues.
Assuntos
Cones de Crescimento/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Efrina-B2/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Semaforinas/metabolismoRESUMO
The neurotrophin receptor TrkA plays critical roles in the nervous system by recruiting signaling molecules that activate pathways required for the growth and survival of neurons. Here, we report APPL1 as a TrkA-associated protein. APPL1 and TrkA co-immunoprecipitated in sympathetic neurons. We have identified two routes through which this association can occur. APPL1 was isolated as a binding partner for the TrkA-interacting protein GIPC1 from rat brain lysate by mass spectrometry. The PDZ domain of GIPC1 directly engaged the C-terminal sequence of APPL1. This interaction provides a means through which APPL1 may be recruited to TrkA. In addition, the APPL1 PTB domain bound to TrkA, indicating that APPL1 may associate with TrkA independently of GIPC1. Isolation of endosomal fractions by high-resolution centrifugation determined that APPL1, GIPC1, and phosphorylated TrkA are enriched in the same fractions. Reduction of APPL1 or GIPC1 protein levels suppressed nerve growth factor (NGF)-dependent MEK, extracellular signal-regulated kinase, and Akt activation and neurite outgrowth in PC12 cells. Together, these results indicate that GIPC1 and APPL1 play a role in TrkA function and suggest that a population of endosomes bearing a complex of APPL1, GIPC1, and activated TrkA may transmit NGF signals.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Fator de Crescimento Neural/metabolismo , Neuropeptídeos/metabolismo , Receptor trkA/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Células COS , Proteínas de Transporte/química , Células Cultivadas , Chlorocebus aethiops , Células Clonais , Técnica Direta de Fluorescência para Anticorpo , Glutationa Transferase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neuropeptídeos/química , Células PC12 , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptor trkA/genética , Proteínas Recombinantes de Fusão/metabolismo , Gânglio Cervical Superior/citologiaRESUMO
Protein electrophoresis is commonly used as an aid in the diagnosis of monoclonal gammopathies and is performed in many laboratories in Canada and throughout the world. However, unlike many other diagnostic tests, there is limited guidance for standardization and neither guidance nor specific recommendations for clinical reporting of serum (SPE) or urine (UPE) protein electrophoresis and immunotyping available in the literature. Therefore, a Canadian effort was undertaken to recommend standards that cover all aspects of clinical reporting with an ultimate goal towards reporting standardization. The Canadian Society of Clinical Chemists (CSCC) Monoclonal Gammopathy Interest Group (MGIG), which is composed of CSCC members with an interest in protein electrophoresis, has formed a Monoclonal Gammopathy Working Group (MGWG) to take initial steps towards standardization of SPE, UPE and immunotyping. Candidate standardization recommendations were developed, discussed and voted upon by the MGWG. Candidate recommendations that achieved 90% agreement are presented as consensus recommendations. Recommendations that did not achieve 90% consensus remain candidate recommendations and are presented with accompanying MGWG discussion. Eleven consensus recommendations along with candidate recommendations for nomenclature, protein fraction reporting, test utilization, interference handling and interpretive reporting options are presented.
Assuntos
Eletroforese das Proteínas Sanguíneas/métodos , Guias como Assunto , Paraproteinemias/sangue , Sociedades Médicas , Canadá , HumanosRESUMO
BACKGROUND: Testing for 25-hydroxyvitamin D (25(OH)D) has increased dramatically over the past decade and several automated immunoassays exist to measure serum 25(OH)D. Here we assess the performance of the recently released automated Bio-Rad BioPlex® 2200 25-OH vitamin D immunoassay, claimed to equally detect 25(OH)D2 and 25(OH)D3, and compare its results against a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and the well-established DiaSorin LIAISON® 25-OH vitamin D total immunoassay. METHODS: Imprecision was determined using third party controls over 20days. Linearity over the claimed measuring range was assessed using admixtures of a high and a low patient pool. Correlation between the BioPlex and LC-MS/MS (n=137) or the LIAISON (n=56) was assessed using patient samples with varying amounts of 25(OH)D3 and/or 25(OH)D2. RESULTS: The total imprecision was 9.4%, 6.9% and 4.5% at concentrations of 39.4nmol/L, 70.6nmol/L and 242.8nmol/L, respectively. The assay was linear from 33.1-375.0nmol/L with a R(2) of 0.993. Method comparison revealed a strong correlation between the BioPlex assay and LC-MS/MS for samples containing 25(OH)D2 alone (n=5; R(2)=0.999), 25(OH)D3 alone (n=119; R(2)=0.935) and both (n=13; R(2)=0.919). In samples tested by all three methods (n=56), the correlation between the BioPlex and the LIAISON (R(2)=0.853) was poorer than that of the BioPlex and LC-MS/MS (R(2)=0.942). CONCLUSION: The BioPlex assay is suitable for the measurement of total serum 25(OH)D. The strong correlation between the BioPlex assay and LC-MS/MS in detecting 25(OH)D2 and 25(OH)D3 provides evidence that the BioPlex assay is capable of the equivalent detection of both forms.
Assuntos
25-Hidroxivitamina D 2/sangue , Biomarcadores/sangue , Calcifediol/sangue , Cromatografia Líquida/métodos , Imunoensaio/métodos , Polímeros/química , Espectrometria de Massas em Tandem/métodos , Humanos , Reprodutibilidade dos TestesRESUMO
The mechanisms that regulate symmetric, proliferative divisions versus asymmetric, neurogenic divisions of mammalian neural precursors are still not well understood. We found that Lfc (Arhgef2), a Rho-specific guanine nucleotide exchange factor that interacts with spindle microtubules, and its negative regulator Tctex-1 (Dynlt1) determine the genesis of neurons from precursors in the embryonic murine cortex. Specifically, genetic knockdown of Arhgef2 in cortical precursors either in culture or in vivo inhibited neurogenesis and maintained cells as cycling radial precursors. Conversely, genetic knockdown of Dynlt1 in radial precursors promoted neurogenesis and depleted cycling cortical precursors. Coincident silencing of these two genes indicated that Tctex-1 normally inhibits the genesis of neurons from radial precursors by antagonizing the proneurogenic actions of Lfc. Moreover, Lfc and Tctex-1 were required to determine the orientation of mitotic precursor cell divisions in vivo. Thus, Lfc and Tctex-1 interact to regulate cortical neurogenesis, potentially by regulating mitotic spindle orientation.
Assuntos
Córtex Cerebral/embriologia , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Associadas aos Microtúbulos/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Células-Tronco/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Polaridade Celular/genética , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Regulação para Baixo/genética , Dineínas , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Mitose/genética , Neurônios/citologia , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/genética , Fuso Acromático/genética , Células-Tronco/citologia , Proteína rhoA de Ligação ao GTP/genética , Região do Complexo-t do GenomaRESUMO
Mutations in p73 are rare in cancer. Emerging evidence suggests that the relative expression of various p73 isoforms may contribute to tumorigenesis. Alternative promoters and N-terminal splicing result in the transcription and processing of either full-length (TA) or N-terminally truncated (deltaN) p73 isoforms. TAp73 possesses pro-apoptotic functions, while deltaNp73 has anti-apoptotic properties via functional inhibition of TAp73 and p53. Here, we report that TAp73, but not deltaNp73, is covalently modified by NEDD8 under physiologic conditions in an Mdm2-dependent manner. Co-expression of NEDP1, a cysteine protease that specifically cleaves NEDD8 conjugates, was shown to deneddylate TAp73. In addition, blockage of the endogenous NEDD8 pathway increased TAp73-mediated transactivation of p53- and p73-responsive promoter-driven reporter activity, and in conjunction, neddylated TAp73 species were found preferentially in the cytoplasm. These results suggest that Mdm2 attenuates TAp73 transactivation function, at least in part, by promoting NEDD8-dependent TAp73 cytoplasmic localization and provide the first evidence of a covalent post-translational modification exclusively targeting the TA isoforms of p73.