RESUMO
The human tumor suppressor p16INK4a is a small monomeric protein that can form amyloid structures. Formation of p16INK4a amyloid fibrils is induced by oxidation which creates an intermolecular disulfide bond. The conversion into amyloid is associated with a change from an all α-helical structure into ß-sheet fibrils. Currently, structural insights into p16INK4a amyloid fibrils are lacking. Here, we investigate the amyloid-forming regions of this tumor suppressor using isotope-labeling limited-digestion mass spectrometry analysis. We discover two key regions that likely form the structured core of the amyloid. Further investigations using thioflavin-T fluorescence assays, electron microscopy, and solution nuclear magnetic resonance spectroscopy of shorter peptide regions confirm the self-assembly of the identified sequences that include methionine and leucine repeat regions. This work describes a simple approach for studying protein motifs involved in the conversion of monomeric species into aggregated fibril structures. It provides insight into the polypeptide sequence underlying the core structure of amyloid p16INK4a formed after a unique oxidation-driven structural transition.
RESUMO
Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.
Assuntos
Caspase 8 , Necroptose , Oxidantes , Fatores de Necrose Tumoral , Animais , Camundongos , Caspase 8/química , Caspase 8/metabolismo , Inflamação/metabolismo , Necroptose/efeitos dos fármacos , Oxidantes/metabolismo , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Fatores de Necrose Tumoral/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Peroxidase , Lactoperoxidase , Domínio CatalíticoRESUMO
The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.
Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/químicaRESUMO
ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Vibrio cholerae/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Volta-Hélice , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by â¼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.
Assuntos
Cisteína/química , Cisteína/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Peróxido de Hidrogênio/química , Mutação , Oxidantes/química , Oxidantes/metabolismo , Oxirredução , Relação Estrutura-AtividadeRESUMO
Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.
Assuntos
Angiotensinogênio/química , Angiotensinogênio/metabolismo , Simulação de Dinâmica Molecular , Ressonância de Plasmônio de Superfície/métodos , Angiotensinogênio/genética , Angiotensinogênio/imunologia , Anticorpos Monoclonais/imunologia , Pressão Sanguínea/fisiologia , Cisteína/metabolismo , Dissulfetos/metabolismo , Epitopos/imunologia , Humanos , Cinética , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sistema Renina-Angiotensina/fisiologiaRESUMO
Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.
Assuntos
Anfirregulina/genética , Proteína BRCA1/genética , Neoplasias da Mama/genética , Receptores de Hidrocarboneto Arílico/genética , Adulto , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Receptores ErbB/genética , Cloridrato de Erlotinib/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica , Homeostase/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/genéticaRESUMO
In order to understand the conformational behavior of intrinsically disordered proteins (IDPs) and their biological interaction networks, the detection of residual structure and long-range interactions is required. However, the large number of degrees of conformational freedom of disordered proteins require the integration of extensive sets of experimental data, which are difficult to obtain. Here, we provide a straightforward approach for the detection of residual structure and long-range interactions in IDPs under near-native conditions using solvent paramagnetic relaxation enhancement (sPRE). Our data indicate that for the general case of an unfolded chain, with a local flexibility described by the overwhelming majority of available combinations, sPREs of non-exchangeable protons can be accurately predicted through an ensemble-based fragment approach. We show for the disordered protein α-synuclein and disordered regions of the proteins FOXO4 and p53 that deviation from random coil behavior can be interpreted in terms of intrinsic propensity to populate local structure in interaction sites of these proteins and to adopt transient long-range structure. The presented modification-free approach promises to be applicable to study conformational dynamics of IDPs and other dynamic biomolecules in an integrative approach.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Humanos , Conformação Proteica , Proteínas Recombinantes/química , Solventes/químicaRESUMO
Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.
Assuntos
Proteínas de Bactérias/química , Bacteriocinas/química , Lactococcus lactis/química , Lipoproteínas/química , Proteínas de Membrana/química , Nisina/química , Bacillus subtilis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nisina/genética , Nisina/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Anti-HIV/química , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Imunoglobulina M/química , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Células CHO , Cricetulus , Expressão Gênica , Células HEK293 , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/crescimento & desenvolvimento , Humanos , Switching de Imunoglobulina/genética , Imunoglobulina M/biossíntese , Imunoglobulina M/imunologia , Imunoglobulina M/farmacologia , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Lanthanide complexes based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) cage are commonly used as phase contrast agents in magnetic resonance imaging, but can also be utilized in structural NMR applications due to their ability to induce either paramagnetic relaxation enhancement or a pseudocontact shift (PCS) depending on the choice of the lanthanide. The size and sign of the PCS for any given atom is determined by its coordinates relative to the metal center, and the characteristics of the lanthanide's magnetic susceptibility tensor. Using a polymethylated DOTA tag (Ln-M8-SPy) conjugated to ubiquitin, we calculated the position of the metal center and characterized the susceptibility tensor for a number of lanthanides (dysprosium, thulium, and ytterbium) under a range of pH and temperature conditions. We found that there was a difference in temperature sensitivity for each of the complexes studied, which depended on the size of the lanthanide ion as well as the isomeric state of the cage. Using 17O-NMR, we confirmed that the temperature sensitivity of the compounds was enhanced by the presence of an apically bound water molecule. Since amide-containing lanthanide complexes are known to be pH sensitive and can be used as probes of physiological pH, we also investigated the effect of pH on the Ln-M8-SPy susceptibility tensor, but we found that the changes in this pH range (5.0-7.4) were not significant.
Assuntos
Compostos Heterocíclicos com 1 Anel/química , Elementos da Série dos Lantanídeos/química , Espectroscopia de Ressonância Magnética , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Metais/química , Metilação , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , TemperaturaRESUMO
Deposition of the nuclear DNA/RNA-binding protein Fused in sarcoma (FUS) in cytosolic inclusions is a common hallmark of some cases of frontotemporal lobar degeneration (FTLD-FUS) and amyotrophic lateral sclerosis (ALS-FUS). Whether both diseases also share common pathological mechanisms is currently unclear. Based on our previous finding that FUS deposits are hypomethylated in FTLD-FUS but not in ALS-FUS, we have now investigated whether genetic or pharmacological inactivation of Protein arginine methyltransferase 1 (PRMT1) activity results in unmethylated FUS or in alternatively methylated forms of FUS. To do so, we generated FUS-specific monoclonal antibodies that specifically recognize unmethylated arginine (UMA), monomethylated arginine (MMA) or asymmetrically dimethylated arginine (ADMA). Loss of PRMT1 indeed not only results in an increase of UMA FUS and a decrease of ADMA FUS, but also in a significant increase of MMA FUS. Compared to ADMA FUS, UMA and MMA FUS exhibit much higher binding affinities to Transportin-1, the nuclear import receptor of FUS, as measured by pull-down assays and isothermal titration calorimetry. Moreover, we show that MMA FUS occurs exclusively in FTLD-FUS, but not in ALS-FUS. Our findings therefore provide additional evidence that FTLD-FUS and ALS-FUS are caused by distinct disease mechanisms although both share FUS deposits as a common denominator.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Anticorpos/farmacologia , Arginina/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Células-Tronco Embrionárias , Inibidores Enzimáticos/farmacologia , Feminino , Degeneração Lobar Frontotemporal/genética , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína FUS de Ligação a RNA/imunologia , Ratos , beta Carioferinas/imunologiaRESUMO
An approach to the de novo structure prediction of proteins is described that relies on surface accessibility data from NMR paramagnetic relaxation enhancements by a soluble paramagnetic compound (sPRE). This method exploits the distance-to-surface information encoded in the sPRE data in the chemical shift-based CS-Rosetta de novo structure prediction framework to generate reliable structural models. For several proteins, it is demonstrated that surface accessibility data is an excellent measure of the correct protein fold in the early stages of the computational folding algorithm and significantly improves accuracy and convergence of the standard Rosetta structure prediction approach.
Assuntos
Proteínas/química , Algoritmos , Bases de Dados de Proteínas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de ProteínaRESUMO
The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical-shift assignments, and data analysis that relies on well-separated resonances. We present a covalent paramagnetic lanthanide-binding tag (LBT) for increasing the chemical-shift dispersion and facilitating the chemical-shift assignment of challenging, repeat-containing IDPs. Linkage of the DOTA-based LBT to a cysteine residue induces pseudo-contact shifts (PCS) for resonances more than 20 residues from the spin-labeling site. This leads to increased chemical-shift dispersion and decreased signal overlap, thereby greatly facilitating chemical-shift assignment. This approach is applicable to IDPs of varying sizes and complexity, and is particularly helpful for repeat-containing IDPs and low-complexity regions. This results in improved efficiency for IDP analysis and binding studies.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Elementos da Série dos Lantanídeos/química , Espectroscopia de Ressonância MagnéticaRESUMO
The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.
Assuntos
Amiloide , Inibidor p16 de Quinase Dependente de Ciclina , Cisteína , Oxirredução , Amiloide/metabolismo , Amiloide/química , Humanos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Cisteína/metabolismo , Cisteína/química , Dissulfetos/metabolismo , Dissulfetos/química , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/química , Mutação , PolimerizaçãoRESUMO
Bacterial plasmids and chromosomes widely contain toxin-antitoxin (TA) loci, which are implicated in stress response, growth regulation and even tolerance to antibiotics and environmental stress. Type I TA systems consist of a stable toxin-expressing mRNA, which is counteracted by an unstable RNA antitoxin. The Long Direct Repeat (LDR-) D locus, a type I TA system of Escherichia Coli (E. coli) K12, encodes a 35 amino acid toxic peptide, LdrD. Despite being characterized as a bacterial toxin, causing rapid killing and nucleoid condensation, little was known about its function and its mechanism of toxicity. Here, we show that LdrD specifically interacts with ribosomes which potentially blocks translation. Indeed, in vitro translation of LdrD-coding mRNA greatly reduces translation efficiency. The structure of LdrD in a hydrophobic environment, similar to the one found in the interior of ribosomes was determined by NMR spectroscopy in 100% trifluoroethanol solution. A single compact α-helix was found which would fit nicely into the ribosomal exit tunnel. Therefore, we conclude that rather than destroying bacterial membranes, LdrD exerts its toxic activity by inhibiting protein synthesis through binding to the ribosomes.
Assuntos
Antitoxinas , Toxinas Bacterianas , Escherichia coli/genética , Escherichia coli/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico , Biossíntese de Proteínas , Antitoxinas/química , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/químicaRESUMO
Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS) in which Th17 cells have a crucial but unclear function. Here we show that choline acetyltransferase (ChAT), which synthesizes acetylcholine (ACh), is a critical driver of pathogenicity in EAE. Mice with ChAT-deficient Th17 cells resist disease progression and show reduced brain-infiltrating immune cells. ChAT expression in Th17 cells is linked to strong TCR signaling, expression of the transcription factor Bhlhe40, and increased Il2, Il17, Il22, and Il23r mRNA levels. ChAT expression in Th17 cells is independent of IL21r signaling but dampened by TGFß, implicating ChAT in controlling the dichotomous nature of Th17 cells. Our study establishes a cholinergic program in which ACh signaling primes chronic activation of Th17 cells, and thereby constitutes a pathogenic determinant of EAE. Our work may point to novel targets for therapeutic immunomodulation in MS.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Células Th17 , Virulência , Colinérgicos , Esclerose Múltipla/genética , Acetilcolina/metabolismo , Camundongos Endogâmicos C57BL , Diferenciação CelularRESUMO
Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1-Gcn2 interaction by binding to Gcn1, thus preventing Gcn2-mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.
Assuntos
Proteínas dos Microfilamentos/química , Proteínas Serina-Treonina Quinases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Meios de Contraste/química , Escherichia coli/genética , Escherichia coli/metabolismo , Gadolínio DTPA/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
The par toxin-antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin-antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin-antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding alpha-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, alpha-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.