RESUMO
The hydroxymethylpyrimidine phosphate kinases (HMPPK) encoded by the thiD gene are involved in the thiamine biosynthesis pathway, can perform two consecutive phosphorylations of 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) and are found in thermophilic and mesophilic bacteria, but only a few characterizations of mesophilic enzymes are available. The presence of another homolog enzyme (pyridoxal kinase) that can only catalyze the first phosphorylation of HMP and encoded by pdxK gene, has hampered a precise annotation in this enzyme family. Here we report the kinetic characterization of two HMPPK with structure available, the mesophilic and thermophilic enzyme from Salmonella typhimurium (StHMPPK) and Thermus thermophilus (TtHMPPK), respectively. Also, given their high structural similarity, we have analyzed the structural determinants of protein thermal stability in these enzymes by molecular dynamics simulation. The results show that pyridoxal kinases (PLK) from gram-positive bacteria (PLK/HMPPK-like enzymes) constitute a phylogenetically separate group from the canonical PLK, but closely related to the HMPPK, so the PLK/HMPPK-like and canonical PLK, both encoded by pdxK genes, are different and must be annotated distinctly. The kinetic characterization of StHMPPK and TtHMPPK, shows that they perform double phosphorylation on HMP, both enzymes are specific for HMP, not using pyridoxal-like molecules as substrates and their kinetic mechanism involves the formation of a ternary complex. Molecular dynamics simulation shows that StHMPPK and TtHMPPK have striking differences in their conformational flexibility, which can be correlated with the hydrophobic packing and electrostatic interaction network given mainly by salt bridge bonds, but interestingly not by the number of hydrogen bond interactions as reported for other thermophilic enzymes. ENZYMES: EC 2.7.1.49, EC 2.7.4.7, EC 2.7.1.35, EC 2.7.1.50.
Assuntos
Proteínas de Bactérias/química , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Proteínas de Bactérias/isolamento & purificação , Ensaios Enzimáticos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Dinâmica Molecular , Fosfotransferases (Aceptor do Grupo Fosfato)/isolamento & purificação , Conformação Proteica , Estabilidade Proteica , Pirimidinas/química , Salmonella typhimurium/enzimologia , Eletricidade Estática , Especificidade por Substrato , Thermus thermophilus/enzimologiaRESUMO
The type II chaperonin CCT is involved in the prevention of the pathogenesis of numerous human misfolding disorders, as it sequesters misfolded proteins, blocks their aggregation and helps them to achieve their native state. In addition, it has been reported that CCT can prevent the toxicity of non-client amyloidogenic proteins by the induction of non-toxic aggregates, leading to new insight in chaperonin function as an aggregate remodeling factor. Here we add experimental evidence to this alternative mechanism by which CCT actively promotes the formation of conformationally different aggregates of γ-tubulin, a non-amyloidogenic CCT client protein, which are mediated by specific CCT-γ-tubulin interactions. The in vitro-induced aggregates were in some cases long fiber polymers, which compete with the amorphous aggregates. Direct injection of unfolded purified γ-tubulin into single-cell zebra fish embryos allowed us to relate this in vitro activity with the in vivo formation of intracellular aggregates. Injection of a CCT-binding deficient γ-tubulin mutant dramatically diminished the size of the intracellular aggregates, increasing the toxicity of the misfolded protein. These results point to CCT having a role in the remodeling of aggregates, constituting one of its many functions in cellular proteostasis.
Assuntos
Chaperonina com TCP-1 , Agregação Patológica de Proteínas , Desdobramento de Proteína , Deficiências na Proteostase , Tubulina (Proteína) , Animais , Chaperonina com TCP-1/química , Chaperonina com TCP-1/genética , Chaperonina com TCP-1/metabolismo , Humanos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Deficiências na Proteostase/genética , Deficiências na Proteostase/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Peixe-Zebra/metabolismoRESUMO
The increasing detection of virulent and/or multidrug resistant bacterial strains makes necessary the development of new antimicrobial agents acting through novel mechanisms and cellular targets. A good choice are molecules aimed to interfere with the cell division machinery or divisome, which is indispensable for bacterial survival and propagation. A key component of this machinery, and thus a good target, is FtsZ, a highly conserved GTPase protein that polymerizes in the middle of the cell on the inner face of the cytoplasmic membrane forming the Z ring, which acts as a scaffold for the recruitment of the divisome proteins at the division site. In this work, we tested the inhibitory effect of five diaryl naphtyl ketone (dNAK) molecules on the in vitro polymerization of both Escherichia coli and Bacillus subtilis FtsZ (EcFtsZ and BsFtsZ, respectively). Among these compounds, dNAK 4 showed the strongest inhibition of FtsZ polymerization in vitro, with an IC50 of 2.3 ± 0.06 µM for EcFtsZ and 9.13 ± 0.66 µM for BsFtsZ. We found that dNAK 4 binds to GDP-FtsZ polymers but not to the monomer in GTP or GDP state. This led to the polymerization of short and curved filaments, rings, open rings forming clusters, and in the case of BsFtsZ, a novel cylindrical structure of stacked open rings. In vivo, dNAK 4 had almost no effect on the growth of E. coli in liquid culture, in contrast to the strong inhibitory effect observed over B. subtilis growth. The insensitivity of E. coli to this compound is probably related to the impermeability of dNAK 4 to the outer membrane. The low amount of this compound required to inhibit several of the bacterial strains tested and the lack of a cytotoxic effect at the concentrations used, makes dNAK 4 a very good candidate as a starting molecule for the development of a new antibiotic.
RESUMO
The sustained increase in the prevalence of protein aggregation related diseases requires the development of feasible methods for the design of therapeutic alternatives. The procedure traditionally used for the search of drugs or therapeutic mutations includes in vitro experiments, designed to prevent the aggregation of model proteins, which are then complemented with cellular toxicity studies in vivo, slowing down the finding of solutions. To address this, we have developed a protocol that facilitates the search of molecules and anti-aggregation mutations since it allows to evaluate their therapeutic capabilities directly in in vivo experiments with the use of zebrafish early embryos. Avoiding the necessity of performing in vitro and in vivo procedures separately. Giving a more realistic method for the results interpretation. Zebrafish embryos were induced to produce intracellular aggregates of proteins by simple microinjections of known quantities of an aggregation prone protein previously labeled. The toxicity was evaluated by the survival of the embryos, while the formation of aggregates was quantified by fluorescence microscopy. The size distribution of the protein aggregates was revealed by means of ultracentrifuge sedimentation analysis. For the development of the present method, the human γ-tubulin protein was used as model protein, which generated intracellular aggregates in more than 60% of the injected embryos. To evaluate the method, a mutation was performed that altered the state of intracellular aggregation of γ-tubulin, obtaining a significant decrease in the amount and size of the intracellular aggregates. The present method can be used for any suitable intracellular aggregation protein model. The current method present important advantages such as: Easy induction of intracellular aggregates. Simple detection of intracellular protein aggregates through fluorescence microscopy and subcellular fractionation. Overall view of the effect of drugs or mutations by combining the toxicity, the development behavior and the size distribution of intracellular protein aggregates.
RESUMO
FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROOâ¢) generated from AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROOâ¢. Quantification of the number of moles of amino acid consumed per mole of ROO⢠shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROOâ¢-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.