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1.
BMC Biochem ; 14: 34, 2013 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-24274906

RESUMO

BACKGROUND: Huntington's disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD. RESULTS: An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course. CONCLUSIONS: The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.


Assuntos
Análise Química do Sangue/métodos , Ensaio de Imunoadsorção Enzimática , Leucócitos Mononucleares/metabolismo , Proteínas do Tecido Nervoso/sangue , Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/normas , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Espectrometria de Massas , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/normas , Controle de Qualidade , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/normas
2.
J Biomol Screen ; 17(7): 933-45, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22644269

RESUMO

The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Inibidores do Fator de Necrose Tumoral , Doenças Autoimunes/metabolismo , Linhagem Celular , Citocina TWEAK , Células HEK293 , Humanos , Inflamação/metabolismo , Neoplasias/metabolismo , Oligopeptídeos , Peptídeos/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor de TWEAK , Fatores de Necrose Tumoral/metabolismo
3.
ISRN Mol Biol ; 2012: 823875, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-27398238

RESUMO

Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation.

4.
Proteomics ; 8(5): 930-8, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18219698

RESUMO

Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent-based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment-specific manner. The sequential use of detergent-containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane-bound or enclosed, nuclear, and cytoskeletal proteins. Fractionation of neurons was validated by comparison of the distribution of known subcellular marker proteins in the four extracts using Western blotting. Comparison of extracts by DIGE showed a clear difference in protein composition demonstrating significant variations with a fold change (FC) of at least 1.20 for 82% of the detected spots. Using proteins identified in these spots that could be assigned a subcellular localization based on descriptions in the Uniprot database, an extraction efficiency of 85% was calculated for cytosolic proteins in extract 1, 90% for membrane-bound and membrane-enclosed proteins in extract 2, 82% for nuclear proteins in extract 3 and 38% for cytoskeletal and RAFT proteins in extract 4.


Assuntos
Fracionamento Celular/métodos , Detergentes , Neurônios/química , Proteômica , Animais , Técnicas de Cultura de Células , Eletroforese em Gel Bidimensional , Ratos
5.
J Biol Chem ; 278(9): 7199-205, 2003 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-12486138

RESUMO

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that can stimulate the action of tolloid metalloproteinases, such as bone morphogenetic protein-1, on a procollagen substrate, by up to 20-fold. The PCPE molecule consists of two CUB domains followed by a C-terminal NTR (netrin-like) domain. In order to obtain structural insights into the function of PCPE, the recombinant protein was characterized by a range of biophysical techniques, including analytical ultracentrifugation, transmission electron microscopy, and small angle x-ray scattering. All three approaches showed PCPE to be a rod-like molecule, with a length of approximately 150 A. Homology modeling of both CUB domains and the NTR domain was consistent with the low-resolution structure of PCPE deduced from the small angle x-ray scattering data. Comparison with the low-resolution structure of the procollagen C-terminal region supports a recently proposed model (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869) for the mechanism of action of PCPE.


Assuntos
Glicoproteínas/química , Sequência de Aminoácidos , Linhagem Celular , Proteínas da Matriz Extracelular , Glicoproteínas/ultraestrutura , Humanos , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Ultracentrifugação , Raios X
6.
J Biol Chem ; 277(37): 33864-9, 2002 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-12105202

RESUMO

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Glicoproteínas/fisiologia , Metaloendopeptidases/fisiologia , Pró-Colágeno/metabolismo , Sítios de Ligação , Proteína Morfogenética Óssea 1 , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/metabolismo , Proteínas da Matriz Extracelular , Humanos , Metaloendopeptidases/metabolismo
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