RESUMO
Cyclic peptides with an even number of alternating d,l-alpha-amino acid residues are known to self-assemble into organic nanotubes. Such peptides previously have been shown to be stable upon protease treatment, membrane active, and bactericidal and to exert antimicrobial activity against Staphylococcus aureus and other gram-positive bacteria. The present report describes the in vitro and in vivo pharmacology of selected members of this cyclic peptide family. The intravenous (i.v.) efficacy of six compounds with MICs of less than 12 microg/ml was tested in peritonitis and neutropenic-mouse thigh infection models. Four of the six peptides were efficacious in vivo, with 50% effective doses in the peritonitis model ranging between 4.0 and 6.7 mg/kg against methicillin-sensitive S. aureus (MSSA). In the thigh infection model, the four peptides reduced the bacterial load 2.1 to 3.0 log units following administration of an 8-mg/kg i.v. dose. Activity against methicillin-resistant S. aureus was similar to MSSA. The murine pharmacokinetic profile of each compound was determined following i.v. bolus injection. Interestingly, those compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties. S. aureus was unable to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations, in agreement with the proposed interaction with multiple components of the bacterial membrane canopy. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our results suggest that these amphipathic cyclic d,l-alpha-peptides have potential for systemic administration and treatment of otherwise antibiotic-resistant infections.
Assuntos
Antibacterianos , Doenças Musculares/tratamento farmacológico , Peptídeos Cíclicos , Peritonite/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Animais , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Feminino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Doenças Musculares/microbiologia , Neutropenia/induzido quimicamente , Biblioteca de Peptídeos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacocinética , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Peritonite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Coxa da Perna/microbiologia , Resultado do TratamentoRESUMO
Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD40/biossíntese , Receptores do Fator de Necrose Tumoral/química , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos CD40/química , Linhagem Celular , Sobrevivência Celular , Cristalografia por Raios X , DNA Complementar/metabolismo , Elétrons , Glutationa Transferase/metabolismo , Humanos , Receptor beta de Linfotoxina , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that participates in cell death during normal development and in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax.
Assuntos
Apoptose , Peptídeos/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/genética , Grupo dos Citocromos c/metabolismo , Citosol/metabolismo , DNA Mitocondrial/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Transporte Proteico , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína X Associada a bcl-2RESUMO
Here we report on the identification of peptides targeting the X-inhibitor of apoptosis protein (XIAP). XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. IAPs are often overexpressed in cancers and leukemias and are associated with an unfavorable clinical prognosis. We have selected peptides from a phage library by using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from two independent screenings by using different libraries. Phage-displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. The interaction was specific as it could be blocked by the cognate synthetic peptides in a dose-dependent manner. Phage displaying the XIAP-binding motif CEFESC bound to the BIR2 domain of XIAP with an estimated dissociation constant of 1.8 nm as determined by surface plasmon resonance. Protein-protein interaction assays revealed that caspase-3 and caspase-7 (but not caspase-8) blocked the binding of the CEFESC phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. In fact, the sequence EFES is homologous to a loop unique to the executioner caspase-3 and caspase-7 that are targeted by XIAP. Finally, we demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings (PFKQ) can induce programmed cell death in leukemia cells. Peptides interacting with XIAP could serve as prototypes for the design of low molecular weight modulators of apoptosis.
Assuntos
Inibidores de Caspase , Peptídeos/farmacologia , Proteínas/fisiologia , Motivos de Aminoácidos , Apoptose , Sítios de Ligação , Caspase 3 , Caspase 7 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Caspases/farmacologia , Sobrevivência Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Biossíntese Peptídica , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
BAG (Bcl-2-associated athanogene) proteins are molecular chaperone regulators that affect diverse cellular pathways. All members share a conserved motif, called the BAG domain (BD), which binds to Hsp70/Hsc70 family proteins and modulates their activity. We have determined the solution structure of BD from BAG4/SODD (silencer of death domains) by multidimensional nuclear magnetic resonance methods and compared it to the corresponding domain in BAG1 (Briknarová, K., Takayama, S., Brive, L., Havert, M. L., Knee, D. A., Velasco, J., Homma, S., Cabezas, E., Stuart, J., Hoyt, D. W., Satterthwait, A. C., Llinás, M., Reed, J. C., and Ely, K. R. (2001) Nat. Struct. Biol. 8, 349-352). The difference between BDs from these two BAG proteins is striking, and the structural comparison defines two subfamilies of mammalian BD-containing proteins. One subfamily includes the closely related BAG3, BAG4, and BAG5 proteins, and the other is represented by BAG1, which contains a structurally and evolutionarily distinct BD. BDs from both BAG1 and BAG4 are three-helix bundles; however, in BAG4, each helix in this bundle is three to four turns shorter than its counterpart in BAG1, which reduces the length of the domain by one-third. BAG4 BD thus represents a prototype of the minimal functional fragment that is capable of binding to Hsc70 and modulating its chaperone activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/química , Proteínas de Membrana , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/fisiologia , Sequência Conservada , Proteínas de Ligação a DNA , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Relação Estrutura-Atividade , Fatores de Transcrição/químicaRESUMO
TRAFs (tumor necrosis factor receptor [TNFR]-associated factors) bind to the cytoplasmic portion of liganded TNFRs and stimulate activation of NF-kappaB or JNK pathways. A modulator of TRAF signaling, TANK, serves as either an enhancer or an inhibitor of TRAF-mediated signaling pathways. The crystal structure of a region of TANK bound to TRAF3 has been determined and compared to a similar CD40/TRAF3 complex. TANK and CD40 bind to the same crevice on TRAF3. The recognition motif PxQxT is presented in a boomerang-like structure in TANK that is markedly different from the hairpin loop that forms in CD40 upon binding to TRAF3. Critical TANK contact residues were confirmed by mutagenesis to be required for binding to TRAF3 or TRAF2. Binding affinity, measured by isothermal titration calorimetry and competition assays, demonstrated that TANK competes with CD40 for the TRAF binding site.