RESUMO
In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth in response to nutrients. The small molecule dipicolinic acid (DPA) plays a central role in both the stress resistance of the dormant spore and its exit from dormancy during germination. The spoVA locus is required for DPA import during sporulation and has been implicated in its export during germination, but the molecular bases are unclear. Here, we define the minimal set of proteins encoded in the Bacillus subtilis spoVA operon required for DPA import and demonstrate that these proteins form a membrane complex. Structural modeling of these components combined with mutagenesis and in vivo analysis reveal that the C and Eb subunits form a membrane channel, while the D subunit functions as a cytoplasmic plug. We show that point mutations that impair the interactions between D and the C-Eb membrane complex reduce the efficiency of DPA import during sporulation and reciprocally accelerate DPA release during germination. Our data support a model in which DPA transport into spores involves cycles of unplugging and then replugging the C-Eb membrane channel, while nutrient detection during germination triggers DPA release by unplugging it.
Assuntos
Proteínas de Bactérias , Esporos Bacterianos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos Picolínicos/metabolismo , Esporos Bacterianos/genéticaRESUMO
The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 µM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.
Assuntos
Neuralgia , Receptores sigma , Animais , Ligantes , Camundongos , Neuralgia/tratamento farmacológico , Receptores sigma/metabolismo , Relação Estrutura-AtividadeRESUMO
The σ2 receptor is an enigmatic protein that has attracted significant attention because of its involvement in diseases as diverse as cancer and neurological disorders. Unlike virtually all other receptors of medical interest, it has eluded molecular cloning since its discovery, and the gene that codes for the receptor remains unknown, precluding the use of modern biological methods to study its function. Using a chemical biology approach, we purified the σ2 receptor from tissue, revealing its identity as TMEM97, an endoplasmic reticulum-resident transmembrane protein that regulates the sterol transporter NPC1. We show that TMEM97 possesses the full suite of molecular properties that define the σ2 receptor, and we identify Asp29 and Asp56 as essential for ligand recognition. Cloning the σ2 receptor resolves a longstanding mystery and will enable therapeutic targeting of this potential drug target.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/genética , Receptores sigma/genética , Doença de Alzheimer/metabolismo , Animais , Ácido Aspártico/química , Proteínas de Transporte/metabolismo , Bovinos , Colesterol/química , Retículo Endoplasmático/metabolismo , Humanos , Insetos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Fígado/metabolismo , Células MCF-7 , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteína C1 de Niemann-Pick , Células PC12 , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ratos , Receptores sigma/metabolismo , Proteínas Recombinantes/metabolismo , Esquizofrenia/metabolismoRESUMO
Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 Å apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.
Assuntos
Dissulfetos/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Trypanosoma brucei brucei/enzimologia , Motivos de Aminoácidos , Animais , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Oxirredução , Conformação Proteica , RotaçãoRESUMO
Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.
Assuntos
Biologia Computacional/métodos , Modelos Moleculares , Proteínas/química , Homologia Estrutural de Proteína , Cristalografia por Raios X , Bases de Dados de Proteínas , Elétrons , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The sigma-1 receptor is an enigmatic ER-resident transmembrane protein linked to a variety of human diseases. Although the receptor was first cloned 20 years ago, the molecular structure of the protein and the mechanistic basis for its interaction with drug-like small molecules have remained unclear until recently. The determination of the first crystal structure of human sigma-1 offered the first detailed views of the sigma-1 architecture, and revealed an unusual overall fold with a single transmembrane helix in each protomer. The structure shows an overall trimeric receptor arrangement, and each protomer binds a single ligand molecule at the center of its carboxy-terminal domain. These results offer detailed molecular views of receptor structure, oligomerization, and ligand recognition, providing a framework for the next era of sigma-1 research.
Assuntos
Receptores sigma/química , Receptores sigma/metabolismo , Animais , Cristalografia por Raios X/métodos , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Receptor Sigma-1RESUMO
Computational design of protein function has made substantial progress, generating new enzymes, binders, inhibitors, and nanomaterials not previously seen in nature. However, the ability to design new protein backbones for function--essential to exert control over all polypeptide degrees of freedom--remains a critical challenge. Most previous attempts to design new backbones computed the mainchain from scratch. Here, instead, we describe a combinatorial backbone and sequence optimization algorithm called AbDesign, which leverages the large number of sequences and experimentally determined molecular structures of antibodies to construct new antibody models, dock them against target surfaces and optimize their sequence and backbone conformation for high stability and binding affinity. We used the algorithm to produce antibody designs that target the same molecular surfaces as nine natural, high-affinity antibodies; in five cases interface sequence identity is above 30%, and in four of those the backbone conformation at the core of the antibody binding surface is within 1 Å root-mean square deviation from the natural antibodies. Designs recapitulate polar interaction networks observed in natural complexes, and amino acid sidechain rigidity at the designed binding surface, which is likely important for affinity and specificity, is high compared to previous design studies. In designed anti-lysozyme antibodies, complementarity-determining regions (CDRs) at the periphery of the interface, such as L1 and H2, show greater backbone conformation diversity than the CDRs at the core of the interface, and increase the binding surface area compared to the natural antibody, potentially enhancing affinity and specificity.
Assuntos
Regiões Determinantes de Complementaridade/química , Biologia Computacional/métodos , Conformação Proteica , Engenharia de Proteínas/métodos , Análise de Sequência de Proteína/métodos , Algoritmos , Sequência de Aminoácidos , Lógica Fuzzy , Humanos , Dados de Sequência MolecularRESUMO
AlphaFold2 (AF2) models have had wide impact but mixed success in retrospective ligand recognition. We prospectively docked large libraries against unrefined AF2 models of the σ2 and serotonin 2A (5-HT2A) receptors, testing hundreds of new molecules and comparing results with those obtained from docking against the experimental structures. Hit rates were high and similar for the experimental and AF2 structures, as were affinities. Success in docking against the AF2 models was achieved despite differences between orthosteric residue conformations in the AF2 models and the experimental structures. Determination of the cryo-electron microscopy structure for one of the more potent 5-HT2A ligands from the AF2 docking revealed residue accommodations that resembled the AF2 prediction. AF2 models may sample conformations that differ from experimental structures but remain low energy and relevant for ligand discovery, extending the domain of structure-based drug design.
Assuntos
Aprendizado Profundo , Descoberta de Drogas , Simulação de Acoplamento Molecular , Receptor 5-HT2A de Serotonina , Agonistas do Receptor 5-HT2 de Serotonina , Antagonistas do Receptor 5-HT2 de Serotonina , Humanos , Microscopia Crioeletrônica , Desenho de Fármacos , Descoberta de Drogas/métodos , Ligantes , Conformação Proteica , Dobramento de Proteína , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2A de Serotonina/ultraestrutura , Receptores sigma/química , Receptores sigma/metabolismo , Bibliotecas de Moléculas Pequenas/química , Agonistas do Receptor 5-HT2 de Serotonina/química , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/química , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologiaRESUMO
AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the σ2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors' experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.
RESUMO
Bacteria from the orders Bacillales and Clostridiales differentiate into stress-resistant spores that can remain dormant for years, yet rapidly germinate upon nutrient sensing. How spores monitor nutrients is poorly understood but in most cases requires putative membrane receptors. The prototypical receptor from Bacillus subtilis consists of three proteins (GerAA, GerAB, GerAC) required for germination in response to L-alanine. GerAB belongs to the Amino Acid-Polyamine-Organocation superfamily of transporters. Using evolutionary co-variation analysis, we provide evidence that GerAB adopts a structure similar to an L-alanine transporter from this superfamily. We show that mutations in gerAB predicted to disrupt the ligand-binding pocket impair germination, while mutations predicted to function in L-alanine recognition enable spores to respond to L-leucine or L-serine. Finally, substitutions of bulkier residues at these positions cause constitutive germination. These data suggest that GerAB is the L-alanine sensor and that B subunits in this broadly conserved family function in nutrient detection.
Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Esporos Bacterianos/fisiologia , Alanina/química , Alanina/metabolismo , Aminoácidos/química , Bacillus subtilis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , MutaçãoRESUMO
In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5' exon derived from the spliced leader (SL) RNA. Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA-binding protein TRF4, leading to the shutoff of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structures of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all of the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments. IMPORTANCE In this study, we found that SLS is induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. We also report on the autophosphorylation of PK3 during SLS induction. This study has implications for our understanding of how trypanosomes keep the homeostasis between the ER and the mitochondria and suggests that PK3 may participate in the connection between these two organelles. The pathway, when induced, leads to the suicide of these parasites, and its induction offers a potential novel drug target against these parasites.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , RNA Líder para Processamento/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/parasitologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Humanos , Proteínas Mitocondriais/genética , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteínas de Protozoários/genética , Interferência de RNA , Splicing de RNA , RNA de Protozoário/metabolismo , RNA Líder para Processamento/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/metabolismoRESUMO
Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost.
RESUMO
SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. SARS-CoV-2 infection is necessary but not sufficient for development of clinical COVID-19 disease. Currently, there are no approved pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We have investigated several plausible hypotheses for famotidine activity including antiviral and host-mediated mechanisms of action. We propose that the principal mechanism of action of famotidine for relieving COVID-19 symptoms involves on-target histamine receptor H2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release. Based on these findings and associated hypothesis, new COVID-19 multi-drug treatment strategies based on repurposing well-characterized drugs are being developed and clinically tested, and many of these drugs are available worldwide in inexpensive generic oral forms suitable for both outpatient and inpatient treatment of COVID-19 disease.
RESUMO
Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. Currently, there are no pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We explore several plausible avenues of activity including antiviral and host-mediated actions. We propose that the principal famotidine mechanism of action for COVID-19 involves on-target histamine receptor H2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release.
RESUMO
The thioredoxin superfamily has expanded and diverged extensively throughout evolution such that distant members no longer show appreciable sequence homology. Nevertheless, redox-active thioredoxin-fold proteins functioning in diverse physiological contexts often share canonical amino acids near the active-site (di-)cysteine motif. Quiescin sulfhydryl oxidase 1 (QSOX1), a catalyst of disulfide bond formation secreted by fibroblasts, is a multi-domain thioredoxin superfamily enzyme with certain similarities to the protein disulfide isomerase (PDI) enzymes. Among other potential functions, QSOX1 supports extracellular matrix assembly in fibroblast cultures. We introduced mutations at a cis-proline in QSOX1 that is conserved across the thioredoxin superfamily and was previously observed to modulate redox interactions of the bacterial enzyme DsbA. The resulting QSOX1 variants showed a striking detrimental effect when added exogenously to fibroblasts: they severely disrupted the extracellular matrix and cell adhesion, even in the presence of naturally secreted, wild-type QSOX1. The specificity of this phenomenon for particular QSOX1 mutants inspired an investigation of the effects of mutation on catalytic and redox properties. For a series of QSOX1 mutants, the detrimental effect correlated with the redox potential of the first redox-active site, and an X-ray crystal structure of one of the mutants revealed the reorganization of the cis-proline loop caused by the mutations. Due to the conservation of the mutated residues across the PDI family and beyond, insights obtained in this study may be broadly applicable to a variety of physiologically important redox-active enzymes. IMPACT STATEMENT: We show that mutation of a conserved cis-proline amino acid, analogous to a mutation used to trap substrates of a bacterial disulfide catalyst, has a dramatic effect on the physiological function of the mammalian disulfide catalyst QSOX1. As the active-site region of QSOX1 is shared with the large family of protein disulfide isomerases in humans, the effects of such mutations on redox properties, enzymatic activity, and biological targeting may be relevant across the family.
Assuntos
Adesão Celular , Matriz Extracelular , Fibroblastos/enzimologia , Mutação de Sentido Incorreto , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Prolina , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Matriz Extracelular/química , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Prolina/química , Prolina/genética , Prolina/metabolismoRESUMO
Quiescin sulfhydryl oxidase 1 (QSOX1) is a catalyst of disulfide bond formation that undergoes regulated secretion from fibroblasts and is over-produced in adenocarcinomas and other cancers. We have recently shown that QSOX1 is required for incorporation of particular laminin isoforms into the extracellular matrix (ECM) of cultured fibroblasts and, as a consequence, for tumor cell adhesion to and penetration of the ECM. The known role of laminins in integrin-mediated cell survival and motility suggests that controlling QSOX1 activity may provide a novel means of combating metastatic disease. With this motivation, we developed a monoclonal antibody that inhibits the activity of human QSOX1. Here, we present the biochemical and structural characterization of this antibody and demonstrate that it is a tight-binding inhibitor that blocks one of the redox-active sites in the enzyme, but not the site at which de novo disulfides are generated catalytically. Sulfhydryl oxidase activity is thus prevented without direct binding of the sulfhydryl oxidase domain, confirming the model for the interdomain QSOX1 electron transfer mechanism originally surmised based on mutagenesis and protein dissection. In addition, we developed a single-chain variant of the antibody and show that it is a potent QSOX1 inhibitor. The QSOX1 inhibitory antibody will be a valuable tool in studying the role of ECM composition and architecture in cell migration, and the recombinant version may be further developed for potential therapeutic applications based on manipulation of the tumor microenvironment.
Assuntos
Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Dissulfetos/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Tolueno/análogos & derivados , Sequência de Aminoácidos , Anticorpos Bloqueadores/metabolismo , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Tolueno/químicaRESUMO
Disulfide bond formation in secretory proteins occurs primarily in the endoplasmic reticulum (ER), where multiple enzyme families catalyze cysteine cross-linking. Quiescin sulfhydryl oxidase 1 (QSOX1) is an atypical disulfide catalyst, localized to the Golgi apparatus or secreted from cells. We examined the physiological function for extracellular catalysis of de novo disulfide bond formation by QSOX1. QSOX1 activity was required for incorporation of laminin into the extracellular matrix (ECM) synthesized by fibroblasts, and ECM produced without QSOX1 was defective in supporting cell-matrix adhesion. We developed an inhibitory monoclonal antibody against QSOX1 that could modulate ECM properties and undermine cell migration.
Assuntos
Matriz Extracelular/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Anticorpos Monoclonais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Cisteína/metabolismo , Dissulfetos/metabolismo , Matriz Extracelular/enzimologia , Matriz Extracelular/ultraestrutura , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Humanos , Laminina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidoresRESUMO
Quiescin Sulfhydryl Oxidase (QSOX), a catalyst of disulfide bond formation, is found in both plants and animals. Mammalian, avian, and trypanosomal QSOX enzymes have been studied in detail, but plant QSOX has yet to be characterized. Differences between plant and animal QSOXs in domain composition and active-site sequences raise the question of whether these QSOXs function by the same mechanism. We demonstrate that Arabidopsis thaliana QSOX produced in bacteria is folded and functional as a sulfhydryl oxidase but does not exhibit the interdomain electron transfer observed for its animal counterpart. Based on this finding, further exploration into the respective roles of the redox-active sites in plant QSOX and the reason for their concatenation is warranted.
Assuntos
Proteínas de Arabidopsis/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Arabidopsis/classificação , Catálise , Dissulfetos/metabolismo , Evolução Molecular , Flavina-Adenina Dinucleotídeo/metabolismo , Espectrometria de Massas , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/classificação , Filogenia , Tiorredoxinas/metabolismoRESUMO
The mimivirus genome contains many genes that lack homologs in the sequence database and are thus known as ORFans. In addition, mimivirus genes that encode proteins belonging to known fold families are in some cases fused to domain-sized segments that cannot be classified. One such ORFan region is present in the mimivirus enzyme R596, a member of the Erv family of sulfhydryl oxidases. We determined the structure of a variant of full-length R596 and observed that the carboxy-terminal region of R596 assumes a folded, compact domain, demonstrating that these ORFan segments can be stable structural units. Moreover, the R596 ORFan domain fold is novel, hinting at the potential wealth of protein structural innovation yet to be discovered in large double-stranded DNA viruses. In the context of the R596 dimer, the ORFan domain contributes to formation of a broad cleft enriched with exposed aromatic groups and basic side chains, which may function in binding target proteins or localization of the enzyme within the virus factory or virions. Finally, we find evidence for an intermolecular dithiol/disulfide relay within the mimivirus R596 dimer, the first such extended, intersubunit redox-active site identified in a viral sulfhydryl oxidase.