RESUMO
HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Vírion/ultraestrutura , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestrutura , Produtos do Gene gag do Vírus da Imunodeficiência Humana/ultraestrutura , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Sequência de Aminoácidos , Dissulfetos/farmacologia , Epitopos/química , Células HEK293 , Proteína gp41 do Envelope de HIV/química , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Modelos Moleculares , Testes de Neutralização , Peptídeos/química , Polissacarídeos/química , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/químicaRESUMO
Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Animais , Dissulfetos/metabolismo , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Receptores Acoplados a Proteínas G/isolamento & purificação , Receptores Acoplados a Proteínas G/ultraestrutura , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Proteínas Wnt/química , Proteínas Wnt/isolamento & purificação , Proteínas Wnt/ultraestruturaRESUMO
The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.
Assuntos
Biopolímeros/metabolismo , Mucinas/metabolismo , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , Dissulfetos/metabolismo , Feminino , Glicosilação , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mucinas/química , Mucinas/ultraestrutura , Peptídeos/química , Domínios Proteicos , Multimerização Proteica , Fator de von Willebrand/química , Fator de von Willebrand/ultraestruturaRESUMO
Non-native conformations drive protein-misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.
Assuntos
Proteínas de Escherichia coli , Dobramento de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Conformação Proteica , Dissulfetos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.
Assuntos
Peroxirredoxinas , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Cisteína/metabolismo , Dissulfetos , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Oxirredução , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.
Assuntos
Eritrócitos , Malária Falciparum , Complexos Multiproteicos , Parasitos , Plasmodium falciparum , Proteínas de Protozoários , Animais , Humanos , Anticorpos Neutralizantes/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Microscopia Crioeletrônica , Dissulfetos/química , Dissulfetos/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Malária Falciparum/patologia , Merozoítos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Parasitos/metabolismo , Parasitos/patogenicidade , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/ultraestruturaRESUMO
Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Trifosfato de Adenosina , Animais , Cálcio/metabolismo , Canais de Cálcio , Proteínas de Ligação ao Cálcio/genética , Dissulfetos/metabolismo , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mucus is made of enormous mucin glycoproteins that polymerize by disulfide crosslinking in the Golgi apparatus. QSOX1 is a catalyst of disulfide bond formation localized to the Golgi. Both QSOX1 and mucins are highly expressed in goblet cells of mucosal tissues, leading to the hypothesis that QSOX1 catalyzes disulfide-mediated mucin polymerization. We found that knockout mice lacking QSOX1 had impaired mucus barrier function due to production of defective mucus. However, an investigation on the molecular level revealed normal disulfide-mediated polymerization of mucins and related glycoproteins. Instead, we detected a drastic decrease in sialic acid in the gut mucus glycome of the QSOX1 knockout mice, leading to the discovery that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases. Sialylation defects in the colon are known to cause colitis in humans. Here we show that QSOX1 redox control of sialylation is essential for maintaining mucosal function.
Assuntos
Glicosiltransferases , Complexo de Golgi , Mucosa Intestinal , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Animais , Camundongos , Colo/metabolismo , Dissulfetos/metabolismo , Glicoproteínas , Glicosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Mucinas/química , Mucinas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Mucosa Intestinal/metabolismoRESUMO
PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.
Assuntos
Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Dissulfetos , Microscopia de Força Atômica , Modelos Moleculares , Oxirredução , Proteínas/química , Proteínas/metabolismoRESUMO
A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Redobramento de Proteína , Rotavirus/metabolismo , Rotavirus/ultraestrutura , Internalização do Vírus , Animais , Antígenos Virais/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dissulfetos/química , Dissulfetos/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestrutura , Mutação , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Rotavirus/química , Rotavirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Vírion/química , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
The joining (J) chain regulates polymerization of multimeric Immunoglobulin(Ig)M and IgA, forming a disulfide bond to the C termini of their Ig heavy chains, and it controls IgM/IgA transport across mucosal epithelia. Like Ig itself and human-like adaptive immunity, J chain emerged in jawed vertebrates (gnathostomes), but its origin has remained mysterious since its discovery over 50 y ago. Here, we show unexpectedly that J chain is a member of the CXCL chemokine family. The J chain gene (JCHAIN) is linked to clustered CXCL chemokine loci in all gnathostomes except actinopterygians that lost JCHAIN. JCHAIN and most CXCL genes have four exons with the same intron phases, including the same cleavage site for the signal peptide/mature protein. The second exon of both genes encodes a CXC motif at the same position, and the lengths of exons 1 to 3 are similar. No other gene in the human secretome shares all of these characteristics. In contrast, intrachain disulfide bonds of the two proteins are completely different, likely due to modifications in J chain to direct Ig polymerization and mucosal transport. Crystal structures of CXCL8 and J chain share a conserved beta-strand core but diverge otherwise due to different intrachain disulfide bonds and extension of the J chain C terminus. Identification of this ancestral affiliation between J chain and CXCL chemokines addresses an age-old problem in immunology.
Assuntos
Imunoglobulina A , Cadeias J de Imunoglobulina , Animais , Humanos , Cadeias J de Imunoglobulina/metabolismo , Éxons , Imunoglobulina A/genética , Dissulfetos , Quimiocinas/genética , Imunoglobulina MRESUMO
The assembly of ß-barrel proteins into membranes is mediated by the evolutionarily conserved ß-barrel assembly machine (BAM) complex. In Escherichia coli, BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of ß-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in the substrate ß-strands, we can compare the residence time of each substrate strand within the BamA lumen. We validated this method using two defective, slow-folding substrates. We used this method to characterize stable intermediates which occur during folding of two structurally different native substrates. Strikingly, these intermediates occur during identical stages of folding for both substrates: soon after folding has begun and just before folding is completed. We suggest that these intermediates arise due to barriers to folding that are common between ß-barrel substrates, and that the BAM catalyst is able to fold so many different substrates because it addresses these common challenges.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Dobramento de Proteína , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Modelos Moleculares , Dissulfetos/química , Dissulfetos/metabolismo , Especificidade por Substrato , Cisteína/química , Cisteína/metabolismoRESUMO
Mutations in the Kunitz-type serine protease inhibitor HAI-2, encoded by SPINT2, are responsible for the pathogenesis of syndromic congenital sodium diarrhea (SCSD), an intractable secretory diarrhea of infancy. Some of the mutations cause defects in the functionally required Kunitz domain 1 and/or subcellular targeting signals. Almost all SCSD patients, however, harbor SPINT2 missense mutations that affect the functionally less important Kunitz domain 2. How theses single amino acid substitutions inactivate HAI-2 was, here, investigated by the doxycycline-inducible expression of three of these mutants in HAI-2-knockout Caco-2 human colorectal adenocarcinoma cells. Examining protein expressed from these HAI-2 mutants reveals that roughly 50% of the protein is synthesized as disulfide-linked oligomers that lose protease inhibitory activity due to the distortion of the Kunitz domains by disarrayed disulfide bonding. Although the remaining protein is synthesized as monomers, its glycosylation status suggests that the HAI-2 monomer remains in the immature, lightly glycosylated form, and is not converted to the heavily glycosylated mature form. Heavily glycosylated HAI-2 possesses full anti-protease activity and appropriate subcellular targeting signals, including the one embedded in the complex-type N-glycan. As predicted, these HAI-2 mutants cannot suppress the excessive prostasin proteolysis caused by HAI-2 deletion. The oligomerization and glycosylation defects have also been observed in a colorectal adenocarcinoma line that harbors one of these SPINT2 missense mutations. Our study reveals that the abnormal protein folding and N-glycosylation can cause widespread HAI-2 inactivation in SCSD patents.
Assuntos
Adenocarcinoma , Neoplasias Colorretais , Serina Endopeptidases , Humanos , Glicoproteínas de Membrana/metabolismo , Células CACO-2 , Glicosilação , Mutação , Diarreia/congênito , Dobramento de Proteína , Neoplasias Colorretais/genética , Dissulfetos , Proteínas Secretadas Inibidoras de Proteinases/genéticaRESUMO
Antibodies of the immunoglobulin M (IgM) class represent the frontline of humoral immune responses. They are secreted as planar polymers in which flanking µ2 L2 "monomeric" subunits are linked by two disulfide bonds, one formed by the penultimate cysteine (C575) in the tailpiece of secretory µ chains (µs tp) and the second by C414 in the Cµ3. The latter bond is not present in membrane IgM. Here, we show that C575 forms a non-native, intra-subunit disulfide bond as a key step in the biogenesis of secretory IgM. The abundance of this unexpected intermediate correlates with the onset and extent of polymerization. The rearrangement of the C-terminal tails into a native quaternary structure is guaranteed by the engagement of protein disulfide isomerase ERp44, which attacks the non-native C575 bonds. The resulting conformational changes promote polymerization and formation of C414 disulfide linkages. This unusual assembly pathway allows secretory polymers to form without the risk of disturbing the role of membrane IgM as part of the B cell antigen receptor.
Assuntos
Dissulfetos/química , Imunoglobulina M/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Células HEK293 , Humanos , Imunoglobulina M/químicaRESUMO
The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.
Assuntos
Fator de Indução de Apoptose , Complexo I de Transporte de Elétrons , Proteínas de Transporte da Membrana Mitocondrial , Fator de Indução de Apoptose/metabolismo , Dissulfetos/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução , Transporte ProteicoRESUMO
In recent years, cyclic peptides have emerged as a promising therapeutic modality due to their diverse biological activities. Understanding the structures of these cyclic peptides and their complexes is crucial for unlocking invaluable insights about protein target-cyclic peptide interaction, which can facilitate the development of novel-related drugs. However, conducting experimental observations is time-consuming and expensive. Computer-aided drug design methods are not practical enough in real-world applications. To tackles this challenge, we introduce HighFold, an AlphaFold-derived model in this study. By integrating specific details about the head-to-tail circle and disulfide bridge structures, the HighFold model can accurately predict the structures of cyclic peptides and their complexes. Our model demonstrates superior predictive performance compared to other existing approaches, representing a significant advancement in structure-activity research. The HighFold model is openly accessible at https://github.com/hongliangduan/HighFold.
Assuntos
Dissulfetos , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Dissulfetos/química , Software , Modelos Moleculares , Conformação Proteica , Algoritmos , Biologia Computacional/métodosRESUMO
BACKGROUND: Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS: We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.
Assuntos
Síndrome de Andersen , Humanos , Camundongos , Animais , Síndrome de Andersen/genética , Síndrome de Andersen/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Doença do Sistema de Condução Cardíaco , Dissulfetos , Fosfatidilinositóis/metabolismoRESUMO
Hydrogen sulfide (H2S) is not only a well-established toxic gas but also an important small molecule bioregulator in all kingdoms of life. In contemporary biology, H2S is often classified as a "gasotransmitter," meaning that it is an endogenously produced membrane permeable gas that carries out essential cellular processes. Fluorescent probes for H2S and related reactive sulfur species (RSS) detection provide an important cornerstone for investigating the multifaceted roles of these important small molecules in complex biological systems. A now common approach to develop such tools is to develop "activity-based probes" that couple a specific H2S-mediated chemical reaction to a fluorescent output. This Review covers the different types of such probes and also highlights the chemical mechanisms by which each probe type is activated by specific RSS. Common examples include reduction of oxidized nitrogen motifs, disulfide exchange, electrophilic reactions, metal precipitation, and metal coordination. In addition, we also outline complementary activity-based probes for imaging reductant-labile and sulfane sulfur species, including persulfides and polysulfides. For probes highlighted in this Review, we focus on small molecule systems with demonstrated compatibility in cellular systems or related applications. Building from breadth of reported activity-based strategies and application, we also highlight key unmet challenges and future opportunities for advancing activity-based probes for H2S and related RSS.
Assuntos
Sulfeto de Hidrogênio , Sulfeto de Hidrogênio/química , Corantes Fluorescentes/química , Diagnóstico por Imagem , Enxofre , DissulfetosRESUMO
Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.
Assuntos
Hidrolases/química , Hidrolases/metabolismo , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas , Reciclagem , Actinobacteria/enzimologia , Burkholderiales/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Ácidos Ftálicos/metabolismo , Polimerização , ThermobifidaRESUMO
S-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide. Proteins detected using the ascorbate-dependent biotin switch method are typically interpreted to be directly regulated by S-nitrosation. However, these S-nitrosated proteins are shown to predominantly comprise transient intermediates leading to disulfide bond formation. These disulfides are likely to be the dominant end effectors resulting from elevations in nitrosating cellular nitric oxide species. We propose that S-nitrosation primarily serves as a transient intermediate leading to disulfide formation. Overall, we conclude that the current widely held perception that stable S-nitrosation directly regulates the function of many proteins is significantly incorrect.