RESUMEN
Aggregation of initially stably structured proteins is involved in more than 20 human amyloid diseases. Despite intense research, however, how this class of proteins assembles into amyloid fibrils remains poorly understood, principally because of the complex effects of amino acid substitutions on protein stability, solubility, and aggregation propensity. We address this question using ß2-microglobulin (ß2m) as a model system, focusing on D76N-ß2m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting ß2m from aggregation by coupling aggregation with antibiotic resistance in E. coli using a tripartite ß-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-ß2m) we show that residue 76 has a unique ability to drive ß2m aggregation in vivo and in vitro. Using a randomly mutated D76N-ß2m variant library, we show that all of the mutations found to improve protein behavior involve residues in a single aggregation-prone region (APR) (residues 60 to 66). Surprisingly, no correlation was found between protein stability and protein aggregation rate or yield, with several mutations in the APR decreasing aggregation without affecting stability. Together, the results demonstrate the power of the TPBLA to develop proteins that are resilient to aggregation and suggest a model for D76N-ß2m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state.
Asunto(s)
Amiloidosis , Agregación Patológica de Proteínas , Microglobulina beta-2 , Sustitución de Aminoácidos , Proteínas Amiloidogénicas/genética , Amiloidosis/genética , Pruebas de Enzimas , Escherichia coli , Humanos , Mutación Puntual , Agregación Patológica de Proteínas/genética , Pliegue de Proteína , Microglobulina beta-2/química , Microglobulina beta-2/genética , beta-LactamasasRESUMEN
OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most ß-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of blaOXA-535 and blaOXA-436 genetic environments suggests that an ISCR1 may be responsible for blaOXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.
Asunto(s)
Shewanella/enzimología , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbapenémicos/farmacología , Cromosomas Bacterianos/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Shewanella/efectos de los fármacos , Shewanella/genética , beta-Lactamasas/genética , beta-Lactamas/farmacologíaRESUMEN
The synthesis of one of the most potent dual inhibitors of the anti-apoptotic proteins Bcl-xL and Mcl-1 is reported. This analogue of a natural sesquiterpenoid dimer meiogynin A was elaborated by a convergent asymmetric synthesis with 36% yield in ten steps.