RESUMEN
Amyloid aggregation is a key feature of Alzheimer's disease (AD) and a primary target for past and present therapeutic efforts. Recent research is making it increasingly clear that the heterogeneity of amyloid deposits, extending past the commonly targeted amyloid-ß (Aß), must be considered for successful therapy. We recently demonstrated that amyloid-α (Aα or p3), a C-terminal peptidic fragment of Aß, aggregates rapidly to form amyloids and can expedite the aggregation of Aß through seeding. Here, we advance the understanding of Aα biophysics and biology in several important ways. We report the first cryogenic electron microscopy (cryo-EM) structure of an Aα amyloid fibril, proving unambiguously that the peptide is fibrillogenic. We demonstrate that Aα induces Aß to form amyloid aggregates that are less toxic than pure Aß aggregates and use nuclear magnetic resonance spectroscopy (NMR) to provide insights into specific interactions between Aα and Aß in solution. This is the first evidence that Aα can coassemble with Aß and alter its biological effects at relatively low concentrations. Based on the above, we urge researchers in the field to re-examine the significance of Aα in AD.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Amiloide/química , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Fragmentos de Péptidos/químicaRESUMEN
The 21st amino acid, selenocysteine (Sec), is synthesized on its dedicated transfer RNA (tRNASec). In bacteria, Sec is synthesized from Ser-tRNA[Ser]Sec by Selenocysteine Synthase (SelA), which is a pivotal enzyme in the biosynthesis of Sec. The structural characterization of bacterial SelA is of paramount importance to decipher its catalytic mechanism and its role in the regulation of the Sec-synthesis pathway. Here, we present a comprehensive single-particle cryo-electron microscopy (SPA cryoEM) structure of the bacterial SelA with an overall resolution of 2.69 Å. Using recombinant Escherichia coli SelA, we purified and prepared samples for single-particle cryoEM. The structural insights from SelA, combined with previous in vivo and in vitro knowledge, underscore the indispensable role of decamerization in SelA's function. Moreover, our structural analysis corroborates previous results that show that SelA adopts a pentamer of dimers configuration, and the active site architecture, substrate binding pocket, and key K295 catalytic residue are identified and described in detail. The differences in protein architecture and substrate coordination between the bacterial enzyme and its counterparts offer compelling structural evidence supporting the independent molecular evolution of the bacterial and archaea/eukarya Ser-Sec biosynthesis present in the natural world.
RESUMEN
Evolving antimicrobial resistance has motivated the search for novel targets and alternative therapies. Caseinolytic protease (ClpP) has emerged as an enticing new target since its function is conserved and essential for bacterial fitness, and because its inhibition or dysregulation leads to bacterial cell death. ClpP protease function controls global protein homeostasis and is, therefore, crucial for the maintenance of the bacterial proteome during growth and infection. Previously, acyldepsipeptides (ADEPs) were discovered to dysregulate ClpP, leading to bactericidal activity against both actively growing and dormant Gram-positive pathogens. Unfortunately, these compounds had very low efficacy against Gram-negative bacteria. Hence, we sought to develop non-ADEP ClpP-targeting compounds with activity against Gram-negative species and called these activators of self-compartmentalizing proteases (ACPs). These ACPs bind and dysregulate ClpP in a manner similar to ADEPs, effectively digesting bacteria from the inside out. Here, we performed further ACP derivatization and testing to improve the efficacy and breadth of coverage of selected ACPs against Gram-negative bacteria. We observed that a diverse collection of Neisseria meningitidis and Neisseria gonorrhoeae clinical isolates were exquisitely sensitive to these ACP analogues. Furthermore, based on the ACP-ClpP cocrystal structure solved here, we demonstrate that ACPs could be designed to be species specific. This validates the feasibility of drug-based targeting of ClpP in Gram-negative bacteria.
Asunto(s)
Antibacterianos , Depsipéptidos , Péptido Hidrolasas , Antibacterianos/farmacología , Bacterias , Depsipéptidos/farmacología , Bacterias GramnegativasRESUMEN
Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.
Asunto(s)
Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/metabolismo , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/parasitología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Esófago/química , Esófago/enzimología , Femenino , Tracto Gastrointestinal/química , Tracto Gastrointestinal/enzimología , Proteínas del Helminto/genética , Humanos , Cinética , Masculino , Modelos Moleculares , Nucleósido-Difosfato Quinasa/genética , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Alineación de SecuenciaRESUMEN
Schistosoma mansoni depends upon the purine salvage pathway to obtain purine nucleotides; therefore, enzymes from this pathway are essential for parasite survival. Here, we focused on the adenine phosphoribosyltransferase (APRT) enzyme, which catalyzes the condensation reaction between adenine and PRPP (5-phosphoribosylpyrophosphate) to produce AMP and PPi. Kinetic experiments using the heterologously expressed protein of one APRT isoform from S. mansoni indicate that it is catalytically active, and whole-mount in situ hybridization studies indicate that the transcripts of this protein are concentrated in the posterior region of the ovary and vitellaria of female adult worms. Moreover, a phylogenetic analysis has shown that APRT exists in multiple copies originating from gene duplications at the base of the Schistosoma genus. Other enzymes from the purine and pyrimidine salvage pathways have also been found to present multiple copies in schistosomes, suggesting that evolutionary pressure to diversify these genes' families may be related to a specialized role in parasite reproduction.