RESUMO
The p75 neurotrophin receptor (p75(NTR)) is a known mediator of ß-amyloid (Aß)-induced neurotoxicity implicated in Alzheimer's disease (AD). Here, we demonstrate that death receptor 6 (DR6) binds to p75(NTR) and is a component of the p75(NTR) signaling complex responsible for Aß-induced cortical neuron death. Cortical neurons isolated from either DR6 or p75(NTR) null mice are resistant to Aß-induced neurotoxicity. Blocking DR6 function in cortical neurons by anti-DR6 antibodies that block the binding of DR6 to p75(NTR) receptor complex or by a dominant negative DR6 construct lacking the cytoplasmic signaling death domain attenuates Aß-induced caspase 3 activation and cell death. DR6 expression is upregulated in AD cortex and correlates with elevated neuronal death. Targeting the disruption of the DR6/p75(NTR) complex to prevent Aß cytotoxicity represents a new approach for the treatment of neurodegenerative disorders such as AD.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/genética , Receptores do Fator de Necrose Tumoral/genética , Peptídeos beta-Amiloides/farmacologia , Animais , Anticorpos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Ligação Proteica , Receptores de Fator de Crescimento Neural/deficiência , Receptores do Fator de Necrose Tumoral/deficiência , Transdução de Sinais/efeitos dos fármacosRESUMO
Chicken gizzard smooth muscle myosin light chain phosphatase is composed of a approximately 37 kDa catalytic subunit, a approximately 110 kDa myosin binding or targeting subunit and a approximately 20 kDa subunit (MPs) whose function is as yet undefined. It was reported previously that a cloned chicken gizzard MPs cDNA encodes a protein of 186 amino acids (aa) [Y.H. Chen, M.X. Chen, D.R. Alessi, D.G. Gampbell, C. Shanahan, P. Cohen, P.T.W. Cohen, FEBS Lett. 356 (1994) 51-55]. More recently, we obtained by PCR amplification another MPs cDNA that encodes a protein of only 161 aa [Y. Zhang, K. Mabuchi, T. Tao, Biochim. Biophys. Acta 1343 (1997) 51-58]. In this work we obtained cDNAs corresponding to both sequences using a different set of PCR primers, indicating that the two sequences correspond to isoforms that most likely arose from alternative splicing of the same gene. Using two polyclonal antibodies, one raised against the recombinant 161 aa isoform of chicken gizzard MPs and the other against a C-terminal polypeptide that is present only in the 186 aa isoform, we found that while the 161 aa isoform is the predominant one in chicken gizzard, in chicken aorta it is the 186 aa one; in chicken stomach both isoforms are present, and in mammalian tissues such as ferret and rat only the 186 aa isoform is detected. Furthermore, we purified the MPs associated with the chicken gizzard myosin light chain phosphatase holoenzyme and determined its molecular weight, amino acid composition and six residues of its C-terminal sequence. The results from these analyses showed conclusively that the predominant isoform in chicken gizzard is the 161 aa one.
Assuntos
Músculo Liso/enzimologia , Fosfoproteínas Fosfatases/química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Galinhas , DNA Complementar/química , Furões , Moela das Aves/enzimologia , Immunoblotting , Isoenzimas/química , Isoenzimas/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Fosfatase de Miosina-de-Cadeia-Leve , Fosfoproteínas Fosfatases/isolamento & purificação , Reação em Cadeia da Polimerase , Ratos , Estômago/enzimologiaRESUMO
Various thio-reactive bifunctional crosslinkers as well as 5,5'-dithiobis(2-nitrobenzoate)-mediated disulfide bond formation were used to crosslink troponin-C and troponin-I, the Ca(2+)-binding and inhibitory subunits of troponin, respectively. In all cases, substantial crosslinking was obtained when the reactions were carried out in the absence of Ca2+. No disulfide crosslinking occurred if either Cys98 of TnC, or Cys133 of TnI were blocked, indicating that these thiols are involved in the crosslinking. Troponin containing the disulfide crosslink is no longer capable of regulating actomyosin ATPase activity in a Ca(2+)-dependent manner. Our results suggest that the relative movement between the Cys98 region of TnC and the Cys133 region of TnI is required for the Ca(2+)-regulatory process in skeletal muscle.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Cisteína , Ácido Ditionitrobenzoico/farmacologia , Músculos/fisiologia , Troponina/metabolismo , Actomiosina/metabolismo , Sequência de Aminoácidos , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Dissulfetos , Eletroforese em Gel de Poliacrilamida , Cinética , Coelhos , Fatores de Tempo , Troponina/efeitos dos fármacos , Troponina/isolamento & purificação , Troponina IRESUMO
Calponin is a thin filament-associated protein that is implicated in the regulation and maintenance of smooth muscle contraction. Molecular cloning of chicken gizzard calponin indicated the presence of two isoforms, alpha and beta, the expression of the alpha-isoform being uniformly more abundant in various smooth muscle tissues [Takahashi, K. & Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288]. For the long-range goal of understanding of the structure and function of calponin, we have started bacterial expression and site-directed mutagenesis of alpha calponin. The amino acid composition and N-terminal sequence of the recombinant alpha calponin were found to be identical to those deduced from its nucleotide sequence. Recombinant alpha calponin is capable of binding to calmodulin, troponin C, tropomyosin, and actin, and of inhibiting skeletal muscle acto-subfragment-1 ATPase activity. A mutant alpha calponin with a replacement in the putative inhibitory region (residues 146-171) has impaired ability to inhibit the acto-subfragment-1 ATPase activity, suggesting that this region of calponin may be involved in the modulation of the actin-myosin interactions.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Escherichia coli/genética , Expressão Gênica , Moela das Aves/química , Actinas/antagonistas & inibidores , Actinas/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/metabolismo , Galinhas , Clonagem Molecular , Immunoblotting , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/antagonistas & inibidores , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Tropomiosina/metabolismo , Troponina/metabolismo , Troponina C , CalponinasRESUMO
The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.