RESUMO
We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/irrigação sanguínea , Peptídeos/farmacologia , Sinais Direcionadores de Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Células Cultivadas , Relação Dose-Resposta a Droga , Desenho de Fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Feminino , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/patologia , Membranas Intracelulares/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Mitocôndrias Hepáticas/ultraestrutura , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Sinais Direcionadores de Proteínas/genética , Ratos , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.
Assuntos
Apoptose/genética , Receptor de Fator de Crescimento Neural/química , Receptor de Fator de Crescimento Neural/metabolismo , Sequência de Aminoácidos/genética , Animais , Sistema Livre de Células/metabolismo , Dimerização , Vetores Genéticos/genética , Humanos , Mutação/genética , Fragmentos de Peptídeos/genética , Plasmídeos/biossíntese , Plasmídeos/genética , Estrutura Terciária de Proteína/genética , Receptor de Fator de Crescimento Neural/genética , Proteínas Recombinantes de Fusão/genética , Transfecção , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismoRESUMO
Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.