RESUMO
Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu2+ and the other for Cu1+, the intestinal mucin, MUC2, prevents copper toxicity by blocking futile redox cycling and the squandering of dietary antioxidants, while nevertheless permitting uptake of this important trace metal into cells. These findings emphasize the value of molecular structure in advancing mucosal biology, while introducing mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones.
Assuntos
Cobre , Mucinas , Mucinas/metabolismo , Mucina-2 , Cobre/análise , Cobre/metabolismo , Intestinos , Muco/metabolismo , Mucosa Intestinal/metabolismoRESUMO
Copper-binding metallophores, or chalkophores, play a role in microbial copper homeostasis that is analogous to that of siderophores in iron homeostasis. The best-studied chalkophores are members of the methanobactin (Mbn) family-ribosomally produced, posttranslationally modified natural products first identified as copper chelators responsible for copper uptake in methane-oxidizing bacteria. To date, Mbns have been characterized exclusively in those species, but there is genomic evidence for their production in a much wider range of bacteria. This review addresses the current state of knowledge regarding the function, biosynthesis, transport, and regulation of Mbns. While the roles of several proteins in these processes are supported by substantial genetic and biochemical evidence, key aspects of Mbn manufacture, handling, and regulation remain unclear. In addition, other natural products that have been proposed to mediate copper uptake as well as metallophores that have biologically relevant roles involving copper binding, but not copper uptake, are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Quelantes/metabolismo , Cobre/metabolismo , Imidazóis/metabolismo , Oligopeptídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fenômenos Biofísicos , Quelantes/química , Genoma Bacteriano , Homeostase , Imidazóis/química , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Modelos Biológicos , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/genética , Óperon , Transporte ProteicoRESUMO
The strength of materials depends on the rate at which they are tested, as defects, for example dislocations, that move in response to applied strains have intrinsic kinetic limitations1-4. As the deformation strain rate increases, more strengthening mechanisms become active and increase the strength4-7. However, the regime in which this transition happens has been difficult to access with traditional micromechanical strength measurements. Here, with microballistic impact testing at strain rates greater than 106 s-1, and without shock conflation, we show that the strength of copper increases by about 30% for a 157 °C increase in temperature, an effect also observed in pure titanium and gold. This effect is counterintuitive, as almost all materials soften when heated under normal conditions. This anomalous thermal strengthening across several pure metals is the result of a change in the controlling deformation mechanism from thermally activated strengthening to ballistic transport of dislocations, which experience drag through phonon interactions1,8-10. These results point to a pathway to better model and predict materials properties under various extreme strain rate conditions, from high-speed manufacturing operations11 to hypersonic transport12.
Assuntos
Cobre , Ouro , Temperatura , Titânio , Ouro/química , Titânio/química , Estresse Mecânico , Teste de Materiais , Fônons , Metais/química , Temperatura AltaRESUMO
Cytochrome P450 enzymes are known to catalyse bimodal oxidation of aliphatic acids via radical intermediates, which partition between pathways of hydroxylation and desaturation1,2. Developing analogous catalytic systems for remote C-H functionalization remains a significant challenge3-5. Here, we report the development of Cu(I)-catalysed bimodal dehydrogenation/lactonization reactions of synthetically common N-methoxyamides through radical abstractions of the γ-aliphatic C-H bonds. The feasibility of switching from dehydrogenation to lactonization is also demonstrated by altering reaction conditions. The use of a readily available amide as both radical precursor and internal oxidant allows for the development of redox-neutral C-H functionalization reactions with methanol as the sole side product. These C-H functionalization reactions using a Cu(I) catalyst with loading as low as 0.5 mol.% is applied to the diversification of a wide range of aliphatic acids including drug molecules and natural products. The exceptional compatibility of this catalytic system with a wide range of oxidatively sensitive functionality demonstrates the unique advantage of using a simple amide substrate as a mild internal oxidant.
Assuntos
Carbono , Cobre , Hidrogênio , Lactonas , Amidas/química , Amidas/metabolismo , Carbono/química , Catálise , Cobre/química , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/química , Hidrogênio/química , Hidrogenação , Lactonas/química , Metanol/química , Oxidantes/química , Oxidantes/metabolismo , OxirreduçãoRESUMO
Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.
Assuntos
Catecol Oxidase , Proteínas de Drosophila , Drosophila melanogaster , Precursores Enzimáticos , Hemócitos , Oxigênio , Transição de Fase , Respiração , Animais , Feminino , Masculino , Transporte Biológico , Anidrases Carbônicas/metabolismo , Catecol Oxidase/metabolismo , Cobre/metabolismo , Cristalização , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Precursores Enzimáticos/metabolismo , Hemocianinas/metabolismo , Hemócitos/imunologia , Hemócitos/metabolismo , Homeostase , Concentração de Íons de Hidrogênio , Hiperóxia/metabolismo , Hipóxia/metabolismo , Larva/anatomia & histologia , Larva/citologia , Larva/imunologia , Larva/metabolismo , Oxigênio/metabolismoRESUMO
Tsvetkov et al. (2022) discovered a new form of cell death triggered by targeted accumulation of Cu in mitochondria that drives lipoylated TCA cycle enzyme aggregation via direct Cu binding.
Assuntos
Apoptose , Cobre , Mitocôndrias , Morte Celular , Cobre/metabolismo , Lipoilação , Mitocôndrias/metabolismoRESUMO
Carbon-oxygen bonds are commonplace in organic molecules, including chiral bioactive compounds; therefore, the development of methods for their construction with simultaneous control of stereoselectivity is an important objective in synthesis. The Williamson ether synthesis, first reported in 18501, is the most widely used approach to the alkylation of an oxygen nucleophile, but it has significant limitations (scope and stereochemistry) owing to its reaction mechanism (SN2 pathway). Transition-metal catalysis of the coupling of an oxygen nucleophile with an alkyl electrophile has the potential to address these limitations, but progress so far has been limited2-7, especially with regard to controlling enantioselectivity. Here we establish that a readily available copper catalyst can achieve an array of enantioconvergent substitution reactions of α-haloamides, a useful family of electrophiles, by oxygen nucleophiles; the reaction proceeds under mild conditions in the presence of a wide variety of functional groups. The catalyst is uniquely effective in being able to achieve enantioconvergent alkylations of not only oxygen nucleophiles but also nitrogen nucleophiles, giving support for the potential of transition-metal catalysts to provide a solution to the pivotal challenge of achieving enantioselective alkylations of heteroatom nucleophiles.
Assuntos
Alquilação , Catálise , Cobre , Oxigênio , Carbono/química , Cobre/química , Oxigênio/químicaRESUMO
Chiral amines are commonly used in the pharmaceutical and agrochemical industries1. The strong demand for unnatural chiral amines has driven the development of catalytic asymmetric methods1,2. Although the N-alkylation of aliphatic amines with alkyl halides has been widely adopted for over 100 years, catalyst poisoning and unfettered reactivity have been preventing the development of a catalyst-controlled enantioselective version3-5. Here we report the use of chiral tridentate anionic ligands to enable the copper-catalysed chemoselective and enantioconvergent N-alkylation of aliphatic amines with α-carbonyl alkyl chlorides. This method can directly convert feedstock chemicals, including ammonia and pharmaceutically relevant amines, into unnatural chiral α-amino amides under mild and robust conditions. Excellent enantioselectivity and functional-group tolerance were observed. The power of the method is demonstrated in a number of complex settings, including late-stage functionalization and in the expedited synthesis of diverse amine drug molecules. The current method indicates that multidentate anionic ligands are a general solution for overcoming transition-metal-catalyst poisoning.
Assuntos
Alquilação , Aminas , Catálise , Cobre , Amidas/química , Aminas/química , Cobre/química , Ligantes , Preparações Farmacêuticas/químicaRESUMO
Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.
Assuntos
Plasticidade Celular , Cobre , Inflamação , Transdução de Sinais , Animais , Camundongos , Cobre/metabolismo , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , NAD/metabolismo , Transdução de Sinais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Peróxido de Hidrogênio/metabolismo , Epigênese Genética/efeitos dos fármacos , Metformina/análogos & derivados , Oxirredução , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genética , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genéticaRESUMO
Protein lipoylation, a crucial post-translational modification (PTM), plays a pivotal role in mitochondrial function and emerges as a key player in cell death through cuproptosis. This novel copper-driven cell death pathway is activated by excessive copper ions binding to lipoylated mitochondrial proteins, disrupting energy production and causing lethal protein aggregation and cell death. The intricate relationship among protein lipoylation, cellular energy metabolism, and cuproptosis offers a promising avenue for regulating essential cellular functions. This review focuses on the mechanisms of lipoylation and its significant impact on cell metabolism and cuproptosis, emphasizing the key genes involved and their implications for human diseases. It offers valuable insights into targeting dysregulated cellular metabolism for therapeutic purposes.
Assuntos
Cobre , Lipoilação , Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Cobre/metabolismo , Animais , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Metabolismo EnergéticoRESUMO
Multijunction solar cells can overcome the fundamental efficiency limits of single-junction devices. The bandgap tunability of metal halide perovskite solar cells renders them attractive for multijunction architectures1. Combinations with silicon and copper indium gallium selenide (CIGS), as well as all-perovskite tandem cells, have been reported2-5. Meanwhile, narrow-gap non-fullerene acceptors have unlocked skyrocketing efficiencies for organic solar cells6,7. Organic and perovskite semiconductors are an attractive combination, sharing similar processing technologies. Currently, perovskite-organic tandems show subpar efficiencies and are limited by the low open-circuit voltage (Voc) of wide-gap perovskite cells8 and losses introduced by the interconnect between the subcells9,10. Here we demonstrate perovskite-organic tandem cells with an efficiency of 24.0 per cent (certified 23.1 per cent) and a high Voc of 2.15 volts. Optimized charge extraction layers afford perovskite subcells with an outstanding combination of high Voc and fill factor. The organic subcells provide a high external quantum efficiency in the near-infrared and, in contrast to paradigmatic concerns about limited photostability of non-fullerene cells11, show an outstanding operational stability if excitons are predominantly generated on the non-fullerene acceptor, which is the case in our tandems. The subcells are connected by an ultrathin (approximately 1.5 nanometres) metal-like indium oxide layer with unprecedented low optical/electrical losses. This work sets a milestone for perovskite-organic tandems, which outperform the best p-i-n perovskite single junctions12 and are on a par with perovskite-CIGS and all-perovskite multijunctions13.
Assuntos
Compostos de Cálcio , Índio , Cobre , Óxidos , TitânioRESUMO
Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N2O) from soils and industrial processes have increased considerably over the last decades1-3. As the final step of bacterial denitrification, N2O is reduced to chemically inert N2 (refs. 1,4) in a reaction that is catalysed by the copper-dependent nitrous oxide reductase (N2OR) (ref. 5). The assembly of its unique [4Cu:2S] active site cluster CuZ requires both the ATP-binding-cassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL (refs. 4,6). Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY and its complexes with NosL and N2OR, respectively. We find that the periplasmic NosD protein contains a binding site for a Cu+ ion and interacts specifically with NosL in its nucleotide-free state, whereas its binding to N2OR requires a conformational change that is triggered by ATP binding. Mutually exclusive structures of NosDFY in complex with NosL and with N2OR reveal a sequential metal-trafficking and assembly pathway for a highly complex copper site. Within this pathway, NosDFY acts as a mechanical energy transducer rather than as a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme N2OR.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Óxido Nitroso , Oxirredutases , Pseudomonas stutzeri , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Cobre/química , Cobre/metabolismo , Citoplasma/enzimologia , Chaperonas Moleculares/metabolismo , Óxido Nitroso/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Oxirredutases/ultraestrutura , Periplasma/enzimologia , Ligação Proteica , Conformação Proteica , Pseudomonas stutzeri/citologia , Pseudomonas stutzeri/enzimologiaRESUMO
Copper is an essential nutrient for maintaining enzyme activity and transcription factor function. Excess copper results in the aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT), which correlates to the mitochondrial tricarboxylic acid (TCA) cycle, resulting in proteotoxic stress and eliciting a novel cell death modality: cuproptosis. Cuproptosis exerts an indispensable role in cancer progression, which is considered a promising strategy for cancer therapy. Cancer immunotherapy has gained extensive attention owing to breakthroughs in immune checkpoint blockade; furthermore, cuproptosis is strongly connected to the modulation of antitumor immunity. Thus, a thorough recognition concerning the mechanisms involved in the modulation of copper metabolism and cuproptosis may facilitate improvement in cancer management. This review outlines the cellular and molecular mechanisms and characteristics of cuproptosis and the links of the novel regulated cell death modality with human cancers. We also review the current knowledge on the complex effects of cuproptosis on antitumor immunity and immune response. Furthermore, potential agents that elicit cuproptosis pathways are summarized. Lastly, we discuss the influence of cuproptosis induction on the tumor microenvironment as well as the challenges of adding cuproptosis regulators to therapeutic strategies beyond traditional therapy.
Assuntos
Cobre , Neoplasias , Humanos , Neoplasias/terapia , Imunoterapia , Morte Celular , Homeostase , Apoptose , Microambiente TumoralRESUMO
The substitution of an alkyl electrophile by a nucleophile is a foundational reaction in organic chemistry that enables the efficient and convergent synthesis of organic molecules. Although there has been substantial recent progress in exploiting transition-metal catalysis to expand the scope of nucleophilic substitution reactions to include carbon nucleophiles1-4, there has been limited progress in corresponding reactions with nitrogen nucleophiles5-8. For many substitution reactions, the bond construction itself is not the only challenge, as there is a need to control stereochemistry at the same time. Here we describe a method for the enantioconvergent substitution of unactivated racemic alkyl electrophiles by a ubiquitous nitrogen-containing functional group, an amide. Our method uses a photoinduced catalyst system based on copper, an Earth-abundant metal. This process for asymmetric N-alkylation relies on three distinct ligands-a bisphosphine, a phenoxide and a chiral diamine. The ligands assemble in situ to form two distinct catalysts that act cooperatively: a copper/bisphosphine/phenoxide complex that serves as a photocatalyst, and a chiral copper/diamine complex that catalyses enantioselective C-N bond formation. Our study thus expands enantioselective N-substitution by alkyl electrophiles beyond activated electrophiles (those bearing at least one sp- or sp2-hybridized substituent on the carbon undergoing substitution)8-13 to include unactivated electrophiles.
Assuntos
Amidas/química , Cobre/química , Fotoquímica , Brometos/química , Carbono/química , Catálise , Ciclização , Diaminas/química , Ligantes , Nitrogênio/química , Fosfinas/químicaRESUMO
Copper is an essential trace element for the human body, and its requirement for optimistic immune functions has been recognized for decades. How copper is involved in the innate immune pathway, however, remains to be clarified. Here, we report that copper serves as a signal molecule to regulate the kinase activity of alpha-kinase 1 (ALPK1), a cytosolic pattern-recognition receptor (PRR), and therefore promotes host cell defense against bacterial infection. We show that in response to infection, host cells actively accumulate copper in the cytosol, and the accumulated cytosolic copper enhances host cell defense against evading pathogens, including intracellular and, unexpectedly, extracellular bacteria. Subsequently, we demonstrate that copper activates the innate immune pathway of host cells in an ALPK1-dependent manner. Further mechanistic studies reveal that copper binds to ALPK1 directly and is essential for the kinase activity of this cytosolic PRR. Moreover, the binding of copper to ALPK1 enhances the sensitivity of ALPK1 to the bacterial metabolite ADP-heptose and eventually prompts host cells to elicit an enhanced immune response during bacterial infection. Finally, using a zebrafish in vivo model, we show that a copper-treated host shows an increased production of proinflammatory cytokines, enhanced recruitment of phagosome cells, and promoted bacterial clearance. Our findings uncover a previously unrecognized role of copper in the modulation of host innate immune response against bacterial pathogens and advance our knowledge on the cross talk between cytosolic copper homeostasis and immune system.
Assuntos
Infecções Bacterianas , Cobre , Animais , Humanos , Peixe-Zebra , Imunidade Inata , Citocinas , Receptores de Reconhecimento de PadrãoRESUMO
Bacteria use diverse strategies and molecular machinery to maintain copper homeostasis and to cope with its toxic effects. Some genetic elements providing copper resistance are acquired by horizontal gene transfer; however, little is known about how they are controlled and integrated into the central regulatory network. Here, we studied two copper-responsive systems in a clinical isolate of Pseudomonas paraeruginosa and deciphered the regulatory and cross-regulation mechanisms. To do so, we combined mutagenesis, transcriptional fusion analyses and copper sensitivity phenotypes. Our results showed that the accessory CusRS two-component system (TCS) responds to copper and activates both its own expression and that of the adjacent nine-gene operon (the pcoA2 operon) to provide resistance to elevated levels of extracellular copper. The same locus was also found to be regulated by two core-genome-encoded TCSs-the copper-responsive CopRS and the zinc-responsive CzcRS. Although the target palindromic sequence-ATTCATnnATGTAAT-is the same for the three response regulators, transcriptional outcomes differ. Thus, depending on the operon/regulator pair, binding can result in different activation levels (from none to high), with the systems demonstrating considerable plasticity. Unexpectedly, although the classical CusRS and the noncanonical CopRS TCSs rely on distinct signaling mechanisms (kinase-based vs. phosphatase-based), we discovered cross-talk in the absence of the cognate sensory kinases. This cross-talk occurred between the proteins of these two otherwise independent systems. The cusRS-pcoA2 locus is part of an Integrative and Conjugative Element and was found in other Pseudomonas strains where its expression could provide copper resistance under appropriate conditions. The results presented here illustrate how acquired genetic elements can become part of endogenous regulatory networks, providing a physiological advantage. They also highlight the potential for broader effects of accessory regulatory proteins through interference with core regulatory proteins.
Assuntos
Proteínas de Bactérias , Cobre , Regulação Bacteriana da Expressão Gênica , Óperon , Pseudomonas , Cobre/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Óperon/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Transdução de Sinais/genéticaRESUMO
The intrinsically disordered C-terminal peptide region of severe acute respiratory syndrome coronavirus 2 nonstructural protein-1 (Nsp1-CT) inhibits host protein synthesis by blocking messenger RNA (mRNA) access to the 40S ribosome entrance tunnel. Aqueous copper(II) ions bind to the disordered peptide with micromolar affinity, creating a possible strategy to restore protein synthesis during host infection. Electron paramagnetic resonance (EPR) and tryptophan fluorescence measurements on a 10-residue model of the disordered protein region (Nsp1-CT10), combined with advanced quantum mechanics calculations, suggest that the peptide binds to copper(II) as a multidentate ligand. Two optimized computational models of the copper(II)-peptide complexes were derived: One corresponding to pH 6.5 and the other describing the complex at pH 7.5 to 8.5. Simulated EPR spectra based on the calculated model structures are in good agreement with experimental spectra.
Assuntos
Cobre , Proteínas Intrinsicamente Desordenadas , SARS-CoV-2 , Proteínas não Estruturais Virais , Cobre/química , Cobre/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ligação Proteica , Modelos Moleculares , COVID-19/virologiaRESUMO
The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.
Assuntos
Proteínas de Bactérias , Cobre , Haemophilus influenzae , Oxazolona , Fatores de Virulência , Haemophilus influenzae/metabolismo , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Haemophilus influenzae/patogenicidade , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Cobre/metabolismo , Cobre/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Oxazolona/metabolismo , Tioamidas/metabolismo , Tioamidas/química , Ferro/metabolismo , Processamento de Proteína Pós-Traducional , Oxirredutases/metabolismo , Oxirredutases/genética , Óperon , Cisteína/metabolismoRESUMO
Developing anticancer drugs with low side effects is an ongoing challenge. Immunogenic cell death (ICD) has received extensive attention as a potential synergistic modality for cancer immunotherapy. However, only a limited set of drugs or treatment modalities can trigger an ICD response and none of them have cytotoxic selectivity. This provides an incentive to explore strategies that might provide more effective ICD inducers free of adverse side effects. Here, we report a metal-based complex (Cu-1) that disrupts cellular redox homeostasis and effectively stimulates an antitumor immune response with high cytotoxic specificity. Upon entering tumor cells, this Cu(II) complex enhances the production of intracellular radical oxidative species while concurrently depleting glutathione (GSH). As the result of heightening cellular oxidative stress, Cu-1 gives rise to a relatively high cytotoxicity to cancer cells, whereas normal cells with low levels of GSH are relatively unaffected. The present Cu(II) complex initiates a potent ferroptosis-dependent ICD response and effectively inhibits in vivo tumor growth in an animal model (c57BL/6 mice challenged with colorectal cancer). This study presents a strategy to develop metal-based drugs that could synergistically potentiate cytotoxic selectivity and promote apoptosis-independent ICD responses through perturbations in redox homeostasis.
Assuntos
Cobre , Glutationa , Homeostase , Oxirredução , Animais , Camundongos , Humanos , Glutationa/metabolismo , Camundongos Endogâmicos C57BL , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Estresse Oxidativo/efeitos dos fármacos , Sinergismo Farmacológico , Morte Celular Imunogênica/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Ferroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismoRESUMO
Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post-transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, we then identified >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. We focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many noncatalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, our results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs.