RESUMO
Sprifermin, recombinant human fibroblast growth factor 18 (rhFGF18), induces cartilage regeneration in knees of patients with osteoarthritis (OA). We hypothesized that a temporal multiphasic process of extracellular matrix (ECM) degradation and formation underlie this effect. We aimed to characterize the temporal ECM remodeling of human knee OA articular cartilage in response to sprifermin treatment. Articular cartilage explants from patients with knee OA (npatients = 14) were cultured for 70 days, with permanent exposure to sprifermin (900, 450, 225 ng/mL), FGF18 (450 ng/mL), insulin-like growth factor-1 (100 ng/mL, positive control) or vehicle (nreplicates/treatment/patient = 2). Metabolic activity (AlamarBlue) and biomarkers of type IIB collagen (PIIBNP) formation (Pro-C2 enzyme-linked immunosorbent assay [ELISA]) and aggrecanase-mediated aggrecan neo-epitope NITEGE (AGNx1 ELISA) were quantified once a week. At end of culture (day 70), gene expression (quantitative reverse transcription polymerase chain reaction) and proteoglycan content (Safranin O/Fast green staining) were quantified. The cartilage had continuously increased metabolic activity, when treated with sprifermin/FGF18 compared to vehicle. During days 7-28 PIIBNP was decreased and NITEGE was increased, and during days 35-70 PIIBNP was increased. At end of culture, the cartilage had sustained proteoglycan content and relative expression of ACAN < COL2A1 < SOX9 < COL1A1, indicating that functional chondrocytes remained in the explants. Sprifermin induces a temporal biphasic cartilage remodeling in human knee OA articular cartilage explants, with early-phase increased aggrecanase activity and late-phase increased type II collagen formation.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/uso terapêutico , Osteoartrite do Joelho/tratamento farmacológico , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/análise , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Masculino , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Proteoglicanas/análise , Proteoglicanas/metabolismoRESUMO
Following nerve injury in neonatal rats, a large proportion of motoneurons die, possibly as a consequence of an increase in vulnerability to the excitotoxic effects of glutamate. Calcium-dependent glutamate excitotoxicity is thought to play a significant role not only in injury-induced motoneuron death, but also in motoneuron degeneration in diseases such as amyotrophic lateral sclerosis (ALS). Motoneurons are particularly vulnerable to calcium influx following glutamate receptor activation, as they lack a number of calcium binding proteins, such as calbindin-D(28k) and parvalbumin. Therefore, it is possible that increasing the ability of motoneurons to buffer intracellular calcium may protect them from cell death and prevent the decline in motor function that usually occurs as a consequence of motoneuron loss. In this study we have tested this possibility by examining the effect of neonatal axotomy on motoneuron survival and muscle force production in normal and transgenic mice that over-express parvalbumin in their motoneurons.The sciatic nerve was crushed in one hindlimb of new-born transgenic and wildtype mice. The effect on motoneuron survival was assessed 8 weeks later by retrograde labelling of motoneurons innervating the tibialis anterior muscle. Following nerve injury in wildtype mice, only 20.2% (+/-2.2, S.E.M.; n=4) of injured motoneurons survive long term compared with 47.2% (+/-4.4, S.E.M.; n=4) in parvalbumin over-expressing mice. Surprisingly, this dramatic increase in motoneuron survival was not reflected in a significant improvement in muscle function, since 8 weeks after injury there was no improvement in either maximal twitch and tetanic force, or muscle weights.Thus, inducing spinal motoneurons to express parvalbumin protects a large proportion of motoneurons from injury-induced cell death, but this is not sufficient to restore muscle function.
Assuntos
Neurônios Motores/patologia , Músculo Esquelético/fisiopatologia , Parvalbuminas/biossíntese , Animais , Animais Recém-Nascidos , Morte Celular , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Músculo Esquelético/inervação , Compressão Nervosa , Degeneração Neural , Regeneração Nervosa , Parvalbuminas/genética , Reação em Cadeia da Polimerase , Recuperação de Função Fisiológica , Nervo Isquiático/fisiologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
The mechanism responsible for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is poorly understood. Several lines of evidence indicate that susceptibility of motor neurons to Ca(2+) overload induced by excitotoxic stimuli is involved. In this study, we investigated whether the high density of Ca(2+)-permeable AMPA receptors on motor neurons gives rise to higher Ca(2+) transients in motor neurons compared to dorsal horn neurons. Dorsal horn neurons were chosen as controls as these cells do not degenerate in ALS. In cultured spinal motor neurons, the rise of the cytosolic Ca(2+) concentration induced by kainic acid (KA) and mediated by the AMPA receptor was almost twice as high as in spinal neurons from the dorsal horn. Furthermore, we investigated whether increasing the motor neuron's cytosolic Ca(2+)-buffering capacity protects them from excitotoxic death. To obtain motor neurons with increased Ca(2+) buffering capacity, we generated transgenic mice overexpressing parvalbumin (PV). These mice have no apparent phenotype. PV overexpression was present in the central nervous system, kidney, thymus, and spleen. Motor neurons from these transgenic mice expressed PV in culture and were partially protected from KA-induced death as compared to those isolated from nontransgenic littermates. PV overexpression also attenuated KA-induced Ca(2+) transients, but not those induced by depolarization. We conclude that the high density of Ca(2+)-permeable AMPA receptors on the motor neuron's surface results in high Ca(2+) transients upon stimulation and that the low cytosolic Ca(2+)-buffering capacity of motor neurons may contribute to the selective vulnerability of these cells in ALS. Overexpression of a high-affinity Ca(2+) buffer such as PV protects the motor neuron from excitotoxicity and this protective effect depends upon the mode of Ca(2+) entry into the cell.
Assuntos
Agonistas de Aminoácidos Excitatórios/toxicidade , Neurônios Motores/efeitos dos fármacos , Neurotoxinas/toxicidade , Parvalbuminas/farmacologia , Esclerose Lateral Amiotrófica/etiologia , Animais , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Ácido Caínico/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Especificidade de Órgãos , Parvalbuminas/genética , Parvalbuminas/metabolismo , Fenótipo , Células do Corno Posterior/citologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/metabolismo , Receptores de AMPA/metabolismoRESUMO
Among the EF-hand Ca(2+)-binding proteins, parvalbumin (PV) and calbindin D9k (CaB) have the function of Ca(2+) buffers. They evolved from an ancestor protein through two phylogenetic pathways, keeping one pair of EF-hands. They differ by the extra helix-loop-helix (AB domain) found in PV and by the linker between the binding sites. To investigate whether the deletion of AB in PV restores a CaB-like structure, we prepared and solved the structure of the truncated rat PV (PVratDelta37) by X-ray and NMR. PVratDelta37 keeps the PV fold, but is more compact, having a well-structured linker, which differs remarkably from CaB. PvratDelta37 has no stable apo-form, has lower affinity for Ca(2+) than full-length PV, and does not bind Mg(2+), in contrast to CaB. Structural differences of the hydrophobic core are partially responsible for lowering the calcium-binding affinity of the truncated protein. It can be concluded that the AB domain, like the linker of CaB, plays a role in structural stabilization. The AB domain of PV protects the hydrophobic core, and is required to maintain high affinity for divalent cation binding. Therefore, the AB domain possibly modulates PV buffer function.
Assuntos
Proteínas de Ligação ao Cálcio/química , Motivos EF Hand/fisiologia , Evolução Molecular , Parvalbuminas/química , Sequência de Aminoácidos , Animais , Cálcio/química , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Cátions/metabolismo , Cristalização , Cristalografia por Raios X , Deleção de Genes , Modelos Moleculares , Dados de Sequência Molecular , Parvalbuminas/genética , Parvalbuminas/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
Calmodulin (CaM) is the main intracellular Ca2+ sensor protein responsible for mediating Ca2+ triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII-/- cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B-cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII-/- cells. Despite higher Ca2+ resting levels, the CaMII-/- cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes.
Assuntos
Apoptose/fisiologia , Calmodulina/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Apoptose/imunologia , Cálcio/metabolismo , Calmodulina/genética , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Galinhas , Deleção de Genes , Fosforilação , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
The use of the chicken DT40 B cell line is increasing in popularity due to the ease with which it can be manipulated genetically. It offers a targeted to random DNA integration ratio of more than 1:2, by far exceeding that of any mammalian cell line. The facility with which knockout cell lines can be generated, combined with a short generation time, makes the DT40 cell line attractive for phenotype analysis of single and multiple gene disruptions. Advantage has been taken of this to investigate such diverse fields as B cell antigen receptor (BCR) signaling, cell cycle regulation, gene conversion and apoptosis. In this review, we give a historical introduction and a practical guide to the use of the DT40 cell line, along with an overview of the main topics being researched using the DT40 cell line as a model system. These topics include B cell-specific subjects such as B cell signaling and Ig rearrangement, and subjects common to all cell types such as apoptosis, histones, mRNA modification, chromosomal maintenance and DNA repair. Attention is in each case brought to peculiarities of the DT40 cell line that are of relevance for the subject. Novel applications of the cell line, e.g., as a vector for gene targeting of human chromosomes, are also discussed in this review.
Assuntos
Linhagem Celular , Perfilação da Expressão Gênica , Deleção de Sequência , Animais , Galinhas , Humanos , Recombinação GenéticaRESUMO
Mammalian skeletal muscle shows an enormous variability in its functional features such as rate of force production, resistance to fatigue, and energy metabolism, with a wide spectrum from slow aerobic to fast anaerobic physiology. In addition, skeletal muscle exhibits high plasticity that is based on the potential of the muscle fibers to undergo changes of their cytoarchitecture and composition of specific muscle protein isoforms. Adaptive changes of the muscle fibers occur in response to a variety of stimuli such as, e.g., growth and differentition factors, hormones, nerve signals, or exercise. Additionally, the muscle fibers are arranged in compartments that often function as largely independent muscular subunits. All muscle fibers use Ca(2+) as their main regulatory and signaling molecule. Therefore, contractile properties of muscle fibers are dependent on the variable expression of proteins involved in Ca(2+) signaling and handling. Molecular diversity of the main proteins in the Ca(2+) signaling apparatus (the calcium cycle) largely determines the contraction and relaxation properties of a muscle fiber. The Ca(2+) signaling apparatus includes 1) the ryanodine receptor that is the sarcoplasmic reticulum Ca(2+) release channel, 2) the troponin protein complex that mediates the Ca(2+) effect to the myofibrillar structures leading to contraction, 3) the Ca(2+) pump responsible for Ca(2+) reuptake into the sarcoplasmic reticulum, and 4) calsequestrin, the Ca(2+) storage protein in the sarcoplasmic reticulum. In addition, a multitude of Ca(2+)-binding proteins is present in muscle tissue including parvalbumin, calmodulin, S100 proteins, annexins, sorcin, myosin light chains, beta-actinin, calcineurin, and calpain. These Ca(2+)-binding proteins may either exert an important role in Ca(2+)-triggered muscle contraction under certain conditions or modulate other muscle activities such as protein metabolism, differentiation, and growth. Recently, several Ca(2+) signaling and handling molecules have been shown to be altered in muscle diseases. Functional alterations of Ca(2+) handling seem to be responsible for the pathophysiological conditions seen in dystrophinopathies, Brody's disease, and malignant hyperthermia. These also underline the importance of the affected molecules for correct muscle performance.
Assuntos
Sinalização do Cálcio/fisiologia , ATPases Transportadoras de Cálcio/fisiologia , Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Animais , Humanos , Troponina/fisiologiaRESUMO
The apoptosis-linked gene ALG-2 encodes a Ca(2+)-binding protein of the penta EF-hand family. To investigate the Ca(2+) binding properties of the recombinant ALG-2 protein, we have cloned ALG-2 cDNA from mouse liver mRNA. Sequence analysis showed that two types of clones were present. One (named ALG-2,5) corresponds to the published ALG-2 sequence (Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521-525); the second (named ALG-2,1) is 6 nucleotides shorter, and the corresponding protein lacks the amino acid residues Gly(121) and Phe(122). Both transcripts are present in mouse tissues in the same 2:1 molar ratio. The ALG-2,5 and ALG-2,1 recombinant proteins are fully soluble in the metal-free form but can be precipitated from bacterial lysates by Ca(2+). In the presence of Tween the Ca(2+) binding profiles display two high affinity sites with [Ca(2+)](0.5) values of 1.2 and 3.1 microM for ALG-2,5 and ALG-2,1, respectively, plus one low affinity site. Using the yeast two-hybrid system we demonstrate that both proteins have a strong tendency to form homo- and heterodimers. In contrast to ALG-2, 5, the ALG-2,1 isoform does not interact with the target protein AIP-1, earlier described to play a role in apoptosis (Vito, P., Pellegrini, L., Guiet, C., and D'Adamio, L. (1999) J. Biol. Chem. 274, 1533-1540). We propose that the minor sequence difference between ALG-2,5 and ALG-2,1 affects the Ca(2+) binding properties and function of the proteins.
Assuntos
Apoptose , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Variação Genética , Fígado/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Encéfalo/metabolismo , Clonagem Molecular , Feminino , Rim/metabolismo , Camundongos , Ovário/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Transcrição GênicaRESUMO
Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 microM), compared with [Ca2+]0.5 = 37 nM and nH = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nH from 2 to 0.65, but without modification of the [Ca2+]0. 5-value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-third of their alpha helix content compared with the Ca2+ form of wild-type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolded conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 microM and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19-28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF-hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal-free forms.
Assuntos
Proteínas de Ligação ao Cálcio/química , Parvalbuminas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Dicroísmo Circular , Técnicas In Vitro , Magnésio/metabolismo , Dados de Sequência Molecular , Mutação , Parvalbuminas/genética , Parvalbuminas/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Dynamics of the rat alpha-parvalbumin calcium-loaded form have been determined by measurement of 15N nuclear relaxation using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed using spectral density functions and the Lipari-Szabo formalism. The major dynamic features for the rat alpha-parvalbumin calcium-loaded form are (1) the extreme rigidity of the helix-loop-helix EF-hand motifs and the linker segment connecting them, (2) the N and C termini of the protein being restricted in their mobility, (3) a conformational exchange occurring at the kink of helix D, and (4) the residue at relative position 2 in the Ca2+-binding sites having an enhanced mobility. Comparison of the Ca2+-binding EF-hand domains of alpha-parvalbumin-Ca2+, calbindin-Ca2+, and calmodulin-Ca2+ shows that parvalbumin is probably the most rigid of the EF-hand proteins. It also illustrates the dynamical properties which are conserved in the EF-hand domains from different members of this superfamily: (1) a tendency toward higher mobility of NH vectors at relative position 2 in the Ca2+-binding loop, (2) a restricted mobility for the other residues in the binding loop, and (3) an overall rigidity for the helices of EF-hand motifs. The differences in mobility between parvalbumin and the two EF-hand proteins occur mainly at the linker connecting the pair of EF hands and also at the C terminus of the last helix. In parvalbumin-Ca2+, these two regions are characterized by a pronounced rigidity compared to the corresponding more mobile regions in calbindin-Ca2+ and calmodulin-Ca2+.
Assuntos
Proteínas de Ligação ao Cálcio/química , Parvalbuminas/química , Conformação Proteica , Termodinâmica , Amidas , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Moleculares , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Parvalbuminas/metabolismo , Estrutura Secundária de Proteína , Prótons , RatosRESUMO
In this study we report that phosphatidylinositol 3-kinase (PI 3-kinase), a lipid kinase which participates in downstream signalling events of heterotrimeric G protein-coupled receptors and receptor tyrosine kinases, contains a high affinity binding site for calmodulin (CaM). The putative CaM-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 nM. Furthermore, a sequence comparison among different PI 3-kinase isoforms revealed that the sequence which can bind CaM is highly conserved within different PI 3-kinase isoforms. These results indicate a novel mechanism for regulating PI 3-kinase and provide a new direct link between Ca2+ and phospholipid signalling pathways.
Assuntos
Calmodulina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Cálcio/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Molecular mechanisms leading to elevated calmodulin (CaM) expression in cancer have not yet been discovered. We have quantitated the levels of transcripts derived from all three CaM genes in a variety of the same origin rat fibroblasts transformed with oncogenes in combination with gene for protein kinase C using Northern blot analysis with three CaM gene specific cDNA probes. Five species of CaM mRNA were detected in all these cells. Surprisingly many of the investigated cell lines exhibited a decreased content of all CaM mRNAs as compared to control cells with CaMI and CaMII transcripts showing the most pronounced alterations. In contrast, CaM protein levels were increased in all these cell lines as determined by a radioimmunoassay. These results suggest that oncogenic up-regulation of CaM synthesis takes place posttranscriptionally. Several CaM binding proteins were found at different concentrations in the studied cell lines depending on the oncogenes used for transformation. However, CaM overexpression does not seem to affect the overall levels of CaM binding proteins.
Assuntos
Proteínas de Ligação a Calmodulina/biossíntese , Calmodulina/biossíntese , Transformação Celular Neoplásica , Isoenzimas/genética , Oncogenes , Proteína Quinase C/genética , Animais , Northern Blotting , Cálcio/farmacologia , Calmodulina/genética , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular , Linhagem Celular Transformada , Sondas de DNA , Ácido Egtázico/farmacologia , Fibroblastos , Regulação da Expressão Gênica , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C beta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transcrição Gênica , TransfecçãoRESUMO
The chicken calmodulin I (CaMI) gene has been isolated and characterized on the level of cDNA and genomic DNA. The deduced amino acid (aa) sequence is identical to the one of chicken CaMII which consists of 148 aa. The CaMI gene contains six exons. Its intron/exon organization is identical to that of the chicken CaMII and the CaMI and CaMIII genes of rat and human. Expression of the CaMI gene was detected in all chicken tissues examined, although at varying levels. The gene is transcribed into four mRNAs of 0.8, 1.4, 1.7 and 4.4 kb as determined by Northern blot analysis. Our results demonstrate that the "multigene-one-protein" principle of CaM synthesis is not only applicable to mammals whose CaM is encoded by three different genes, but also to chickens.
Assuntos
Calmodulina/genética , Galinhas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Calmodulina/biossíntese , Calmodulina/química , Clonagem Molecular , Códon , DNA Complementar , Éxons , Humanos , Íntrons , Mamíferos , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Transcrição GênicaRESUMO
The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination. Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not. Also, prokaryotic sequences markedly suppress V(D)J recombination. This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3' MAR of Emu stimulates V(D)J recombination in an episomal but not in a chromosomal context. The fact that other MARs do not share this activity suggests that the effect is no mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation. The presence of a 26 bp A-T-rich sequence motif in the 5' and 3' MARs of Emu and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Emu core.
Assuntos
Elementos Facilitadores Genéticos , Rearranjo Gênico do Linfócito B , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Diversidade de Anticorpos , Linfócitos B , Composição de Bases , Sequência de Bases , Linhagem Celular , Sequência Consenso , Humanos , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Íntrons , Camundongos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Recombinação Genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , TransfecçãoRESUMO
To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased in normal mice but virtually not affected in the transgenic animals. This suggests that ectopically expressed parvalbumin is involved in the regulation of Ca2+ signals in the sinusoidal endothelial cells. This animal model could be of interest to those working on the physiology of liver circulation.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Fígado/metabolismo , Parvalbuminas/biossíntese , Animais , Proteínas de Ligação ao Cálcio/genética , Endotélio/metabolismo , Endotélio/ultraestrutura , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Fígado/ultraestrutura , Camundongos , Camundongos Transgênicos , Microscopia Imunoeletrônica , Parvalbuminas/genética , RatosRESUMO
Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C and consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta1 and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosphorylation by CaMKII, suggesting a possible role of CaMKII in regulating the dynamics of interaction between PCNA and RF-C and thus interfering in the formation of an active sliding clamp by DNA polymerases delta and epsilon.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Animais , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Bovinos , DNA Polimerase II , DNA Polimerase III , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor , Inibidores da Síntese de Ácido Nucleico , Fosforilação , Proteína de Replicação CRESUMO
Rat parvalbumin (PV) and oncomodulin (OM) display considerable sequence similarity and structural similarity, but differ in the affinity and selectivity of metal binding to their CD site, a Ca2+/Mg(2+)-mixed site in PV and a Ca(2+)-specific site in OM. In an attempt to identify the structural basis for these differences, mutations were introduced in the previously generated [W102]PV mutant, which contains a unique tryptophan as a conformational-sensitive fluorescent probe inside the hydrophobic core. In the present report, we substituted selected amino acid residues in the CD site of PV by those present at identical positions in OM. One mutant protein, named [F66, W102]PV, has one new substitution in which isoleucine at position 66 was exchanged by phenylalanine. The second mutant protein, [I46, I50, L58, F66, W102]PV, has four new substitutions, namely V46-->I, L50-->I, I58-->L and I66-->F. Tryptophan fluorescence and difference spectrophotometry indicated that the mutations do not alter significantly the hydrophobic core. Both mutant proteins display two metal-binding sites of identical affinities with intrinsic affinity constants K'Ca2+ of 2.9 x 10(7) M-1 for [F66, W102]PV and 1.7 x 10(7) M-1 for [I46, I50, L58, F66, W102]PV and K'Mg2+ of 3.1 x 10(4) M-1 for [F66, W102]PV and 1.9 x 10(4) M-1 for [I46, I50, L58, F66, W102]PV. Thus, the five-residue substitution, but not the two-residue one, leads to a small decrease of affinity compared to [W102]PV (K'Ca2+ = 2.7 x 10(7) M-1, K'Mg2+ = 4.4 x 10(4) M-1). Despite these similarities, the Mg2+ effect on Ca2+ binding is different for the two mutant parvalbumins: the Ca(2+)-binding isotherms of [F66, W102]PV undergo a parallel shift upon increasing Mg2+ concentrations, which indicates that the Mg2+ effect on the two Ca(2+)-binding sites is the same and quantitatively very similar to that described for [W102]PV. In [I46, I50, L58, F66, W102]PV, Mg2+ antagonizes the binding of the second Ca2+ (likely at the EF site) much more than that of the first Ca2+ (likely the CD site). According to the competition equation, the two sites display KMg2+.compet values of 390 M-1 and 3.9 x 10(3) M-1, respectively. These data indicate that (a) the single I66-->F mutation does not modify the cation binding parameters. (b) Multiple modifications in the hydrophobic core still do not change the affinity for Ca2+ and Mg2+, but strongly affect the Mg2+ antagonism and probably the selectivity of the CD site.
Assuntos
Cálcio/metabolismo , Magnésio/metabolismo , Parvalbuminas/química , Parvalbuminas/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação ao Cálcio/química , Primers do DNA , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Térbio/metabolismo , TriptofanoRESUMO
Rat parvalbumin (PV) and oncomodulin (OM) differ in the affinity and selectivity of metal binding to their CD site, which is a high-affinity Ca2+/Mg(2+)-mixed site in PV and a low-affinity Ca(2+)-specific site in OM. To assess to what degree the Ca2+/Mg2+ specificity and affinity of an EF-hand motif in a protein is intrinsically determined by its sequence, the complete CD sites were exchanged, yielding two chimeras, [S41-Q71]PV and [D41-S71]OM. The optical characteristics of a Trp102, inserted in the hydrophobic core of PV, OM and the two chimeras, are very similar in all four proteins, which suggests that the hydrophobic core is qualitatively similar in the chimeras as in the parent proteins. Direct Ca2+ and Mg2+ binding monitored by flow dialysis and gel filtration revealed that [S41-Q71]PV binds only one Mg2+ with an intrinsic affinity K'Mg2+ of 3.0 x 10(4) M-1 and two Ca2+ with an identical K'Ca2+ of 4.4 x 10(6) M-1, whereas [D41-S71]OM binds two Mg2+ with a mean K'Mg2+ of 2 x 10(4) M-1 and two Ca2+ with a K'Ca2+ of 1.3 x 10(7) M-1. K'Ca2+ of the CD site of [S41-Q71]PV was 2.5-fold higher than of the CD site in [W102]OM, but 5-6-fold lower than that of the CD site in [W102]PV. In [D41-S71]OM, K'Ca2+ of the CD site was twofold lower than in [W102]PV, but eightfold higher than in [W102]OM. These results indicate that the sequence of the CD site determines its Ca2+/Mg(2+)-specificity, whereas its affinity for Ca2+ influenced by the protein into which the CD site is inserted. The inserted CD site in turn influences the affinity of the EF site to which it is paired in the host protein and the paired sites display an equalized affinity for Ca2+. Mg2+ decreases the affinity of the chimeras for Ca2+, but not according to a simple competition model. The Mg2+ antagonism is much more pronounced in [D41-S71]OM than in [S41-Q71]PV, but in each chimera the CD and EF site are quantitatively affected in the same manner. Thus, [S41-Q71]PV which can only bind a single Mg2+ ion, displays a Ca2+/Mg(2+)-antagonism for both sites with a KMg.compet of 2.3 x 10(2) M-1. These results confirm the 'equalizer' principle in the cation-binding parameters of [S41-Q71]PV: both sites display the same Ca2+ affinity and Mg2+ antagonism. In [D41-S71]OM with its two Ca2+/Mg2+ sites the antagonism shows qualitatively the same complexity as in wild-type PV, although it is somewhat weaker in amplitude.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Magnésio/metabolismo , Parvalbuminas/química , Parvalbuminas/metabolismo , Animais , Sítios de Ligação , DNA Complementar , Cinética , Espectrometria de Massas , Metais/análise , Mutagênese Sítio-Dirigida , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Especificidade por Substrato , Compostos de Sulfidrila/análiseRESUMO
Recently, a new subfamily of Ras-related GTP-binding proteins consisting of Rad (Ras associated with diabetes), Gem (immediate early gene expressed in mitogen-stimulated T-cells), and Kir (tyrosine kinase-inducible Ras-like) was discovered. The C terminus of these proteins contains an extension of approximately 30 amino acids not present in other members of the Ras family and which exhibits all the hallmarks typical for calmodulin (CaM)-binding domains. A peptide corresponding to the putative CaM-binding domain of the Kir/Gem protein was synthesized, and its affinity for CaM was determined by fluorescence spectrometry. Titration of dansyl-CaM with the Kir/Gem peptide gave an affinity constant of 1 nM. Furthermore, a single point mutation of the peptide, W269G, abolished this high affinity interaction. Gel-shift analysis showed that the complex formation between CaM and the Kir/Gem peptide is strictly calcium-dependent. We also demonstrate with a newly developed [32P]CaM overlay technique that full-length Kir/Gem and Rad proteins bind CaM in a Ca2+-dependent fashion. The binding of CaM to glutathione S-transferase-Kir and GST-Gem inhibited the binding of GTP to Kir/Gem significantly. These results suggest the existence of a direct link between Ca2+/CaM and growth factor signal transduction pathways at the level of small Ras-like GTPases.
Assuntos
Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Proteínas Monoméricas de Ligação ao GTP , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/farmacologia , Compostos de Dansil , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Humanos , Proteínas Imediatamente Precoces/biossíntese , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Espectrometria de FluorescênciaRESUMO
Many genes are regulated by the intracellular calcium, protein kinase C (PKC) and protein kinase A (PKA) pathways and it has been shown that these pathways synergize in some cell types, whereas they antagonize in others. Here we show that the calcium and PKC pathways suppress the effects mediated by the PKA pathway in a fibroblast cell line. The suppressing effect of elevated intracellular Ca2+ levels, but not of the PKC pathway, can be abrogated by the addition of cyclosporin A (CsA), indicating that the effect of Ca2+ is mediated by phosphatase-2B (PP-2B/calcineurin). Suppression by the PKC pathway is not mediated by the proto-oncogenes c-fos, c-jun and junB, as the co-transfection of these genes does not block the effects of the PKA stimulator 8-Br-cAMP. In addition, cotransfection with the catalytic subunit of PKA shows that the inhibitory effect of PKC occurs upstream of PKA activation.