RESUMEN
Proteolytic cleavage of the cell adhesion molecule L1 (L1) in brain tissue and in cultured cerebellar neurons results in the generation and nuclear import of a 30 kDa fragment comprising most of L1's C-terminal, intracellular domain. In search of molecules that interact with this domain, we performed affinity chromatography with the recombinant intracellular L1 domain and a nuclear extract from mouse brains, and identified potential nuclear L1 binding partners involved in transcriptional regulation, RNA processing and transport, DNA repair, chromatin remodeling, and nucleocytoplasmic transport. By co-immunoprecipitation and enzyme-linked immunosorbent assay using recombinant proteins, we verified the direct interaction between L1 and the nuclear binding partners non-POU domain containing octamer-binding protein and splicing factor proline/glutamine-rich. The proximity ligation assay confirmed this close interaction in cultures of cerebellar granule cells. Our findings suggest that L1 fragments regulate multiple nuclear functions in the nervous system. We discuss possible physiological and pathological roles of these interactions in regulation of chromatin structure, gene expression, RNA processing, and DNA repair.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/fisiología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Dominios ProteicosRESUMEN
Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , ARN Helicasas DEAD-box/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa/biosíntesis , Yersiniosis/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Cristalografía por Rayos X , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/inmunología , Técnica del Anticuerpo Fluorescente , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Parásitos/fisiología , Humanos , Tolerancia Inmunológica/fisiología , Inmunoprecipitación , Macrófagos/microbiología , Espectrometría de Masas , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Transporte de Proteínas/fisiología , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Yersiniosis/metabolismo , Yersinia enterocoliticaRESUMEN
Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica/fisiología , Glutatión/metabolismo , Insuficiencia Cardíaca/metabolismo , Adulto , Animales , Fármacos Cardiovasculares/uso terapéutico , Proteínas Portadoras/genética , Femenino , Insuficiencia Cardíaca/tratamiento farmacológico , Ventrículos Cardíacos/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Oxidación-Reducción , Fosforilación , Adulto JovenRESUMEN
Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP-deficient neurons are more sensitive to oxidative stress than wild-type neurons and present the novel findings that wild-type, but not PrP-deficient astrocytes protect wild-type cerebellar neurons against oxidative stress and that exosomes released from stressed wild-type, but not from stressed PrP-deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte-derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte-derived exosomes. Upon exposure of wild-type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild-type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP-deficient astrocytes relative to unstressed PrP-deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions.
Asunto(s)
Astrocitos/metabolismo , Muerte Celular/fisiología , Exosomas/metabolismo , Hipoxia/metabolismo , Proteínas Priónicas/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismoRESUMEN
The analysis of ADP-ribosylated proteins is a challenging task, on the one hand because of the diversity of the target proteins and the modification sites, on the other hand because of the particular problems posed by the analysis of ADP-ribosylated peptides. ADP-ribosylated proteins can be detected in in vitro experiments after the incorporation of radioactively labeled or chemically modified ADP-ribose. Endogenously ADP-ribosylated proteins may be detected and enriched by antibodies directed against the ADP-ribosyl moiety or by ADP-ribosyl binding macro domains. The determination of the exact attachment site of the modification, which is a prerequisite for the understanding of the specificity of the various ADP-ribosyl transferases and the structural consequences of ADP-ribosylation, necessitates the proteolytic cleavage of the proteins. The resulting peptides can afterwards be enriched either by IMAC (using the affinity of the pyrophosphate group for heavy metal ions) or by immobilized boronic acid beads (using the affinity of the vicinal ribose hydroxy groups for boronic acid). The identification of the modified peptides usually requires tandem mass spectrometric measurements. Problems that hamper the mass spectrometric analysis by collision-induced decay (CID) can be circumvented either by the application of different fragmentation techniques (electron transfer or electron capture dissociation; ETD or ECD) or by enzymatic cleavage of the ADP-ribosyl group to ribosyl-phosphate.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Proteínas/química , Proteínas/metabolismo , ADP Ribosa Transferasas/metabolismo , Animales , Humanos , Espectrometría de Masas , Péptidos/química , Péptidos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Many pathogenic microorganisms have evolved tactics to modulate host cell death or survival pathways for establishing infection. The enteropathogenic bacterium Yersinia enterocolitica deactivates TLR-induced signaling pathways, which triggers apoptosis in macrophages. In this article, we show that Yersinia-induced apoptosis of human macrophages involves caspase-dependent cleavage of the TLR adapter protein MyD88. MyD88 was also cleaved when apoptosis was mediated by overexpression of the Toll-IL-1R domain-containing adapter inducing IFN-ß in epithelial cells. The caspase-processing site was mapped to aspartate-135 in the central region of MyD88. MyD88 is consequently split by caspases in two fragments, one harboring the death domain and the other the Toll-IL-1R domain. Caspase-3 was identified as the protease that conferred the cleavage of MyD88 in in vitro caspase assays. In line with a broad role of caspase-3 in the execution of apoptosis, the processing of MyD88 was not restricted to Yersinia infection and to proapoptotic Toll-IL-1R domain-containing adapter inducing IFN-ß signaling, but was also triggered by staurosporine treatment. The cleavage of MyD88 therefore seems to be a common event in the advanced stages of apoptosis, when caspase-3 is active. We propose that the processing of MyD88 disrupts its scaffolding function and uncouples the activation of TLR and IL-1Rs from the initiation of proinflammatory signaling events. The disruption of MyD88 may consequently render dying cells less sensitive to proinflammatory stimuli in the execution phase of apoptosis. The cleavage of MyD88 could therefore be a means of conferring immunogenic tolerance to apoptotic cells to ensure silent, noninflammatory cell demise.
Asunto(s)
Apoptosis/fisiología , Interacciones Huésped-Patógeno/fisiología , Macrófagos/microbiología , Factor 88 de Diferenciación Mieloide/fisiología , Yersinia enterocolitica/fisiología , Secuencia de Aminoácidos , Animales , Caspasa 3/metabolismo , Células Epiteliales/metabolismo , Células HEK293 , Humanos , Interferón beta/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Macrófagos/inmunología , Glicoproteínas de Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Factor 88 de Diferenciación Mieloide/química , Factor 88 de Diferenciación Mieloide/deficiencia , FN-kappa B/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores de Interleucina-1/fisiología , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Especificidad de la Especie , Receptores Toll-Like/fisiologíaRESUMEN
The cell adhesion molecule close homolog of L1 (CHL1) plays important functional roles in the developing and adult nervous system. In search of the binding partners that mediate the diverse and sometimes opposing functions of CHL1, the extracellular matrix-associated proteins vitronectin and plasminogen activator inhibitor-2 (PAI-2) were identified as novel CHL1 interaction partners and tested for involvement in CHL1-dependent functions during mouse cerebellar development. CHL1-induced cerebellar neurite outgrowth and cell migration at postnatal days 6-8 were inhibited by a CHL1-derived peptide comprising the integrin binding RGD motif, and by antibodies against vitronectin or several integrins, indicating a vitronectin-dependent integrin-mediated pathway. A PAI-2-derived peptide, or antibodies against PAI-2, urokinase type plasminogen activator (uPA), uPA receptor, and several integrins reduced cell migration. CHL1 colocalized with vitronectin, PAI-2, and several integrins in cerebellar granule cells, suggesting an association among these proteins. Interestingly, at the slightly earlier age of 4-5 d, cerebellar neurons did not depend on CHL1 for neuritogenesis and cell migration. However, differentiation of progenitor cells into neurons at this stage was dependent on homophilic CHL1-CHL1 interactions. These observations indicate that homophilic CHL1 trans-interactions regulate differentiation of neuronal progenitor cells at early postnatal stages, while heterophilic trans-interactions of CHL1 with vitronectin, integrins, and the plasminogen activator system regulate neuritogenesis and neuronal cell migration at a later postnatal stage of cerebellar morphogenesis. Thus, within very narrow time windows in postnatal cerebellar development, distinct types of molecular interactions mediated by CHL1 underlie the diverse functions of this protein.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Integrinas/metabolismo , Neuritas/metabolismo , Inhibidor 2 de Activador Plasminogénico/metabolismo , Vitronectina/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/farmacología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Ratones , Ratones Noqueados , Neuritas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Lectinas Tipo C/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/metabolismo , Animales , Antígenos de Carbohidratos Asociados a Tumores/química , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Unión ProteicaRESUMEN
Dendritic targeting of mRNAs encoding the microtubule-associated protein 2 (MAP2) in neurons involves a cis-acting dendritic targeting element. Two rat brain proteins, MAP2-RNA trans-acting protein (MARTA)1 and MARTA2, bind to the cis-element with both high affinity and specificity. In this study, affinity-purified MARTA2 was identified as orthologue of human far-upstream element binding protein 3. In neurons, it resides in somatodendritic granules and dendritic spines and associates with MAP2 mRNAs. Expression of a dominant-negative variant of MARTA2 disrupts dendritic targeting of endogenous MAP2 mRNAs, while not noticeably altering the level and subcellular distribution of polyadenylated mRNAs as a whole. Finally, MAP2 transcripts associate with the microtubule-based motor KIF5 and inhibition of KIF5, but not cytoplasmic dynein function disrupts extrasomatic trafficking of MAP2 mRNA granules. Thus, in neurons MARTA2 appears to represent a key trans-acting factor involved in KIF5-mediated dendritic targeting of MAP2 mRNAs.
Asunto(s)
Dendritas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Western Blotting , Dendritas/ultraestructura , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Espectrometría de Masas , Microscopía Inmunoelectrónica , Neuronas/ultraestructura , Transporte de Proteínas/fisiología , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.
Asunto(s)
Anexina A2/antagonistas & inhibidores , Hiperfosfatemia/fisiopatología , Animales , Anexina A2/análisis , Anexina A2/fisiología , Apoptosis , Movimiento Celular , Células Cultivadas , Embrión de Pollo , Regulación hacia Abajo , Humanos , Masculino , Neovascularización Fisiológica , Proteómica , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/complicaciones , Rigidez VascularRESUMEN
Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm-negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp- and eDNA-dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.
Asunto(s)
Adhesinas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriólisis , Biopelículas , ADN Bacteriano/metabolismo , Regulación hacia Abajo , Regulación Bacteriana de la Expresión Génica , Staphylococcus epidermidis/fisiología , Transactivadores/metabolismo , Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Humanos , Staphylococcus epidermidis/genética , Transactivadores/genéticaRESUMEN
Drastic enrichment of potential disease-specific glycoprotein markers in human plasma can be achieved by the combination of affinity- and immuno-depletion. In the affinity-fractionation step all glycoproteins carrying a certain glycostructure are isolated by lectin affinity chromatography, thus depleting other components. Against the respective glycoprotein fraction isolated from the plasma of healthy individuals antibodies are raised in llamas. The llama heavy chain antibodies (which are particularly stable) directed at the isolated plasma glycoprotein fraction are immobilized and the immunoaffinity column thus obtained is used to deplete the respective glycoprotein fraction of patient plasma samples. Depletion of proteins normally found in human plasma by 99.8-99.9% can be achieved, resulting in a 800-1000-fold enrichment of potential disease-specific proteins in the flow-through of the immunoaffinity column.
Asunto(s)
Biomarcadores/sangre , Cromatografía de Afinidad/métodos , Glicoproteínas/sangre , Inmunoensayo/métodos , Animales , Anticuerpos Inmovilizados/química , Antígenos/administración & dosificación , Antígenos/química , Antígenos/inmunología , Proteínas Sanguíneas/química , Camélidos del Nuevo Mundo/inmunología , Cromatografía de Afinidad/instrumentación , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoensayo/instrumentación , Cadenas Pesadas de Inmunoglobulina/química , Lectinas/química , VacunaciónRESUMEN
Oligomannosidic glycans play important roles in nervous system development and function. By performing a phage display screening with oligomannose-specific antibodies, we identified an oligomannose-mimicking peptide that was functionally active in modulating neurite outgrowth and neuron-astrocyte adhesion. Using the oligomannose-mimicking peptide in crosslinking experiments, synapsin I was identified as a novel oligomannose-binding protein in mouse brain. Further analyses not only verified that synapsin I is an oligomannose-binding lectin, but also indicated that it is a glycoprotein carrying oligomannose and Lewis(x). We also found that synapsin I is expressed in glia-enriched cultures and is released from glial cells via exosomes. Incubation of glial-derived exosomes in the presence of high KCl concentrations or subjecting glial cell cultures to either oxygen/glucose deprivation or hydrogen peroxide resulted in release of synapsin I from exosomes. Application of synapsin I promoted neurite outgrowth from hippocampal neurons and increased survival of cortical neurons upon hydrogen peroxide treatment or oxygen/glucose deprivation. Coculture experiments using wild-type hippocampal neurons and wild-type or synapsin-deficient glial cells showed enhanced neurite outgrowth when synapsin was expressed by glial cells. Synapsin-induced neurite outgrowth was dependent on oligomannose on synapsin I and the neural cell adhesion molecule NCAM at the neuronal cell surface. The data indicate that, under conditions of high neuronal activity and/or oxidative stress, synapsin can be released from glial-derived exosomes and promotes neurite outgrowth and neuronal survival by modulating the interactions between glia and neurons.
Asunto(s)
Exosomas/metabolismo , Lectinas/metabolismo , Neuritas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Oligosacáridos/metabolismo , Sinapsinas/metabolismo , Animales , Comunicación Celular/genética , Comunicación Celular/fisiología , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Femenino , Glicoproteínas/metabolismo , Masculino , Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/deficiencia , Moléculas de Adhesión de Célula Nerviosa/genética , Embarazo , Unión Proteica/fisiologíaRESUMEN
Crotamine, a highly basic myotoxic polypeptide (molecular mass 4881 Da) isolated from the venom of the Brazilian rattlesnake Crotalus durissus terrificus, causes skeletal muscle contraction and spasms, affects the functioning of voltage-sensitive sodium channels by inducing sodium influx and possesses antitumour activity, suggesting potential pharmaceutical applications. Crotamine was purified from C. durissus terrificus venom; the crystals diffracted to 1.9 Å resolution and belonged to the orthorhombic space group I2(1)2(1)2(1) or I222, with unit-cell parameters a = 67.75, b = 74.4, c = 81.01 Å. The self-rotation function indicated that the asymmetric unit contained three molecules. However, structure determination by molecular replacement using NMR-determined coordinates was unsuccessful and a search for potential derivatives has been initiated.
Asunto(s)
Venenos de Crotálidos/química , Crotalus , Animales , Venenos de Crotálidos/aislamiento & purificación , Cristalización , Cristalografía por Rayos XRESUMEN
Virulence of nosocomial pathogen Staphylococcus epidermidis is essentially related to formation of adherent biofilms, assembled by bacterial attachment to an artificial surface and subsequent production of a matrix that mediates interbacterial adhesion. Growing evidence supports the idea that proteins are functionally involved in S. epidermidis biofilm accumulation. We found that in S. epidermidis 1585v overexpression of a 460 kDa truncated isoform of the extracellular matrix-binding protein (Embp) is necessary for biofilm formation. Embp is a giant fibronectin-binding protein harbouring 59 Found In Various Architectures (FIVAR) and 38 protein G-related albumin-binding (GA) domains. Studies using defined Embp-positive and -negative S. epidermidis strains proved that Embp is sufficient and necessary for biofilm formation. Further data showed that the FIVAR domains of Embp mediate binding of S. epidermidis to solid-phase attached fibronectin, constituting the first step of biofilm formation on conditioned surfaces. The binding site in fibronectin was assigned to the fibronectin domain type III12. Embp-mediated biofilm formation also protected S. epidermidis from phagocytosis by macrophages. Thus, Embp is a multifunctional cell surface protein that mediates attachment to host extracellular matrix, biofilm accumulation and escape from phagocytosis, and therefore is well suited for promoting implant-associated infections.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Proteínas Portadoras/metabolismo , Fibronectinas/metabolismo , Staphylococcus epidermidis/fisiología , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Portadoras/genética , Eliminación de Gen , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Fagocitosis , Unión Proteica , Mapeo de Interacción de Proteínas , Staphylococcus epidermidis/inmunología , Staphylococcus epidermidis/metabolismo , Staphylococcus epidermidis/patogenicidadRESUMEN
A chitinase has been isolated and purified from Crocus vernus corms. N-terminal amino-acid sequence analysis of the approximately 30â kDa protein showed 33% identity to narbonin, a seed protein from Vicia narbonensis L. The C. vernus chitinase was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as the main precipitant. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=172.3, b=37.1, c=126.4â Å, ß=127° and two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.1â Å.
Asunto(s)
Quitinasas/química , Quitinasas/aislamiento & purificación , Crocus/enzimología , Secuencia de Aminoácidos , Animales , Quitinasas/genética , Cristalización , Cristalografía por Rayos X , Globulinas/genética , Datos de Secuencia Molecular , Proteínas de Vegetales Comestibles/genética , Alineación de Secuencia , Difracción de Rayos XRESUMEN
Members of the 14-3-3 protein family have been implicated in neuronal migration, synaptic plasticity and learning. Using affinity chromatography followed by mass spectrometry analysis, we show here that the cytoskeletal protein alphaII spectrin is a novel ligand of 14-3-3beta. We found that 14-3-3beta interacts with alphaII spectrin via the mode 2 14-3-3 binding motif RLIQS(1302)HP. Binding required phosphorylation of Ser(1302) by casein kinase II and was enhanced in the presence of calmodulin. Co-immunoprecipitation of alphaII spectrin and 14-3-3beta with the neural cell adhesion molecule NCAM suggested that the 14-3-3-spectrin-interaction affects NCAM function. Indeed, disruption of the 14-3-3beta/alphaII spectrin interaction by mutating Ser(1302) to Ala enhanced NCAM-dependent neurite outgrowth. Our results indicate that the phosphorylation-dependent interaction between 14-3-3beta and alphaII spectrin acts as a switch between positive and negative regulation of neurite outgrowth stimulated by NCAM, representing a novel and acute mechanism preventing uncontrolled elongation of neuronal processes.
Asunto(s)
Proteínas 14-3-3/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/metabolismo , Isoformas de Proteínas/metabolismo , Espectrina/metabolismo , Proteínas 14-3-3/genética , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Calmodulina/metabolismo , Quinasa de la Caseína II/metabolismo , Células Cultivadas , Hipocampo/citología , Humanos , Neuronas/citología , Neuronas/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrina/genéticaRESUMEN
Protein biomarker discovery in the low concentration range of human body fluids requires the enrichment of the proteins of interest. Here we report on a tandem affinity strategy: In the first step, we isolated a human plasma glyco-subproteome of healthy individuals by wheat germ agglutinin (WGA) lectin affinity chromatography. In the second step, the proteins of this subproteome were used to raise antibodies in llama (Lama glama). The heavy-chain fraction of the llama antibodies was used to deplete from the WGA lectin binding fraction all proteins normally found in human plasma. In this way, we selectively enriched the glycoprotein, CEA, a known cancer marker which had been spiked into normal plasma. As a proof of concept, we applied this method to the analysis of plasma sample from colon cancer patients. We could demonstrate the selective enrichment of CEA by a factor of 600-800.
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Biomarcadores/sangre , Cromatografía de Afinidad/métodos , Proteoma/análisis , Proteómica/métodos , Animales , Afinidad de Anticuerpos/inmunología , Western Blotting , Camélidos del Nuevo Mundo/inmunología , Antígeno Carcinoembrionario/sangre , Antígeno Carcinoembrionario/inmunología , Cromatografía Líquida de Alta Presión , Neoplasias del Colon/sangre , Humanos , Espectrometría de Masas , Proteoma/inmunología , Proteómica/instrumentación , Reproducibilidad de los Resultados , Aglutininas del Germen de Trigo/inmunologíaRESUMEN
Metastases arise from disseminated tumor cells (DTC) that colonize secondary organs. However, DTC survival strategies to start metastatic outgrowth are unclear. The hostile (hypoxic, hypoglycemic) microenvironmental conditions of the bone marrow serve as an ideal model environment for investigation of DTC survival strategies under environmental stress. We investigated the breast cancer DTC cell line BC-M1 established from the bone marrow of a cancer patient by 2-D DIGE and MS analysis. We observed specific overexpression of the unfolded protein response (UPR) proteins Grp78, Grp94, and protein disulfide-isomerase in breast, lung, and prostate cancer DTC cell lines from the bone marrow. The UPR contributes to survival under adverse environmental conditions including chemotherapy. We show in cellular models that Grp78 expression of the UPR is regulated by tyrosine 1248 of ErbB-2. The breast cancer DTC cell lines shared stem/progenitor cell cancer phenotypes (CD44(high)/CD24(low)). Immunocytochemical staining of bone marrow samples from breast cancer patients confirmed in situ high expression of Grp78 and Grp94 in DTC of breast cancer patients, indicating the potential of both proteins as novel markers for DTC detection. Our results suggest the presence of a previously not recognized stress resistant DTC population that combines stem/progenitor attributes with an UPR phenotype.
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Neoplasias de la Mama/metabolismo , Células Madre Neoplásicas/metabolismo , Proteoma/metabolismo , Respuesta de Proteína Desplegada/fisiología , Western Blotting , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Neoplasias de la Mama/patología , Hipoxia de la Célula , Línea Celular Tumoral , Movimiento Celular , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Células Madre Neoplásicas/patología , Mapeo Peptídico , Fenotipo , Proteoma/química , Proteómica/métodos , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVE: Considerable uncertainty remains regarding the veracity of measuring myokine irisin more than seven years after its original description. Unresolved issues include the nature of transcription of the irisin precursor fibronectin type III domain containing 5 (FNDC5) gene across species, the reliability of irisin levels measured with commercial enzyme-linked immunosorbent assays (ELISAs), and the overall validity of the recently published reference values for human serum measured with quantitative mass spectrometry. We utilized multiple species and measures to evaluate the robustness of commonly used reagents and methods for reporting irisin. METHODS: Amplification of cDNA was used to assess the FNDC5 transcript patterns in humans and mice. The specificity and sensitivity of different irisin antibodies were examined via western blotting. Quantification of circulating native irisin was conducted with mass spectrometry using an absolute quantification peptide for irisin. RESULTS: We show that there is a greater transcript diversity of human FNDC5 than currently annotated, but no indication of the expression of transcripts leading to a truncated form of irisin. Available irisin antibodies still bind to patterns of unspecific serum proteins, which compromise reliable measurements of irisin with ELISAs. Absolute quantification of irisin with labeled peptides by mass spectrometry is an advanced method but requires a multi-step sample preparation introducing uncontrollable variations in the measurement. CONCLUSION: Our data represent an explicit warning against measuring circulating irisin using available methods. Measuring irisin is akin to chasing shadows.