RESUMEN
Anthrax edema factor (EF) is a calmodulin-dependent adenylate cyclase that converts adenosine triphosphate (ATP) into 3'-5'-cyclic adenosine monophosphate (cAMP), contributing to the establishment of Bacillus anthracis infections and the resulting pathophysiology. We show that EF adenylate cyclase toxin activity is strongly mediated by the N-end rule, and thus is dependent on the identity of the N-terminal amino acid. EF variants having different N-terminal residues varied by more than 100-fold in potency in cultured cells and mice. EF variants having unfavorable, destabilizing N-terminal residues showed much greater activity in cells when the E1 ubiquitin ligase was inactivated or when proteasome inhibitors were present. Taken together, these results show that EF is uniquely affected by ubiquitination and/or proteasomal degradation.
Asunto(s)
Adenilil Ciclasas/metabolismo , Antígenos Bacterianos/metabolismo , Bacillus anthracis/enzimología , Toxinas Bacterianas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Adenilil Ciclasas/genética , Animales , Antígenos Bacterianos/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Línea Celular , RatonesRESUMEN
The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , ADN/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , ARN/metabolismo , Anticuerpos de Cadena Única/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/aislamiento & purificación , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Tampones (Química) , Cationes Bivalentes/química , ADN/química , Epítopos/química , Epítopos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Concentración Osmolar , ARN/química , Anticuerpos de Cadena Única/aislamiento & purificación , Resonancia por Plasmón de SuperficieRESUMEN
Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.