RESUMO
Inborn error of metabolism disorders (IEMs) are a family of diseases resulting from single-gene mutations that lead to the accumulation of metabolites that are usually toxic or interfere with normal cell function. The etiological link between metabolic alteration and the symptoms of IEMs is still elusive. Several metabolites, which accumulate in IEMs, were shown to self-assemble to form ordered structures. These structures display the same biophysical, biochemical, and biological characteristics as proteinaceous amyloid fibrils. Here, we have demonstrated, for the first time, the ability of each of the branched-chain amino acids (BCAAs) that accumulate in maple syrup urine disease (MSUD) to self-assemble into amyloid-like fibrils depicted by characteristic morphology, binding to indicative amyloid-specific dyes and dose-dependent cytotoxicity by a late apoptosis mechanism. We could also detect the presence of the assemblies in living cells. In addition, by employing several in vitro techniques, we demonstrated the ability of known polyphenols to inhibit the formation of the BCAA fibrils. Our study implies that BCAAs possess a pathological role in MSUD, extends the paradigm-shifting concept regarding the toxicity of metabolite amyloid-like structures, and suggests new pathological targets that may lead to highly needed novel therapeutic opportunities for this orphan disease.
Assuntos
Doença da Urina de Xarope de Bordo , Doenças Metabólicas , Humanos , Doença da Urina de Xarope de Bordo/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Amiloide/genética , Mutação , Proteínas Amiloidogênicas/genéticaRESUMO
Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenina/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Amiloidose/genética , Amiloidose/patologia , Luciferases , Chaperonas Moleculares/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Reação em Cadeia da Polimerase , Príons/genética , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de ProteínaRESUMO
The opportunistic pathogen Staphylococcus aureus is recognized as one of the most frequent causes of biofilm-associated infections. The recently discovered phenol soluble modulins (PSMs) are small α-helical amphipathic peptides that act as the main molecular effectors of staphylococcal biofilm maturation, promoting the formation of an extracellular fibril structure with amyloid-like properties. Here, we combine computational, biophysical and in cell analysis to address the specific contribution of individual PSMs to biofilm structure. We demonstrate that despite their highly similar sequence and structure, contrary to what it was previously thought, not all PSMs participate in amyloid fibril formation. A balance of hydrophobic/hydrophilic forces and helical propensity seems to define the aggregation propensity of PSMs and control their assembly and function. This knowledge would allow to target specifically the amyloid properties of these peptides. In this way, we show that Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, prevents the assembly of amyloidogenic PSMs and disentangles their preformed amyloid fibrils.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Toxinas Bacterianas/química , Biofilmes/efeitos dos fármacos , Catequina/análogos & derivados , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Biofilmes/crescimento & desenvolvimento , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/ultraestrutura , Chá/químicaRESUMO
The aggregation of proteins or peptides in amyloid fibrils is associated with a number of clinical disorders, including Alzheimer's, Huntington's and prion diseases, medullary thyroid cancer, renal and cardiac amyloidosis. Despite extensive studies, the molecular mechanisms underlying the initiation of fibril formation remain largely unknown. Several lines of evidence revealed that short amino-acid segments (hot spots), located in amyloid precursor proteins act as seeds for fibril elongation. Therefore, hot spots are potential targets for diagnostic/therapeutic applications, and a current challenge in bioinformatics is the development of methods to accurately predict hot spots from protein sequences. In this paper, we combined existing methods into a meta-predictor for hot spots prediction, called MetAmyl for METapredictor for AMYLoid proteins. MetAmyl is based on a logistic regression model that aims at weighting predictions from a set of popular algorithms, statistically selected as being the most informative and complementary predictors. We evaluated the performances of MetAmyl through a large scale comparative study based on three independent datasets and thus demonstrated its ability to differentiate between amyloidogenic and non-amyloidogenic polypeptides. Compared to 9 other methods, MetAmyl provides significant improvement in prediction on studied datasets. We further show that MetAmyl is efficient to highlight the effect of point mutations involved in human amyloidosis, so we suggest this program should be a useful complementary tool for the diagnosis of these diseases.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Algoritmos , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas Amiloidogênicas/genética , Amiloidose/diagnóstico , Amiloidose/genética , Humanos , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Mutação Puntual/genéticaRESUMO
Hydrogen peroxide (H2O2) has been shown to promote neurodegeneration by inducing the activation of nuclear factor-κB (NF-κB). In this study, NF-κB activation was induced by H2O2 in human neuroblastoma SH-SY5Y cells. Whether paeonol, one of the phenolic phytochemicals isolated from the Chinese herb Paeonia suffruticosa Andrews (MC), would attenuate the H2O2-induced NF-κB activity was investigated. Western blot results showed that paeonol inhibited the phosphorylation of IκB and the translocation of NF-κB into the nucleus. The ability of paeonol to reduce DNA binding ability and suppress the H2O2-induced NF-κB activation was confirmed by an electrophoretic mobility shift assay and a luciferase reporter assay. Using a microarray combined with gene set analysis, we found that the suppression of NF-κB was associated with mature T cell up-regulated genes, the c-jun N-terminal kinase pathway, and two hypoxia-related gene sets, including the hypoxia up-regulated gene set and hypoxia inducible factor 1 targets. Moreover, using network analysis to investigate genes that were altered by H2O2 and reversely regulated by paeonol, we found that NF-κB was the primary center of the network and amyloid precursor protein (APP) was the secondary center. Western blotting showed that paeonol inhibited APP at the protein level. In conclusion, our work suggests that paeonol down-regulates H2O2-induced NF-κB activity, as well as NF-κB-associated APP expression. Furthermore, the gene expression profile accompanying the suppression of NF-κB by paeonol was identified. The new gene set that can be targeted by paeonol provided a potential use for this drug and a possible pharmacological mechanism for other phenolic compounds that protect against oxidative-related injury.