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1.
Science ; 373(6554): 541-547, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326236

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 , COVID-19/tratamento farmacológico , 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 fármacos
2.
Res Sq ; 2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32702719

RESUMO

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 H 2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release.

3.
Protein Sci ; 28(1): 228-238, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30367560

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/metabolismo
4.
Proc Natl Acad Sci U S A ; 114(27): 7160-7165, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28559337

RESUMO

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 , Células PC12 , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ratos , Receptores sigma/metabolismo , Proteínas Recombinantes/metabolismo , Esquizofrenia/metabolismo
5.
Adv Exp Med Biol ; 964: 5-13, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28315261

RESUMO

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
6.
Proteins ; 83(8): 1385-406, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25670500

RESUMO

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 Molecular
7.
J Mol Biol ; 425(22): 4366-78, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23867277

RESUMO

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/química
8.
Science ; 341(6141): 74-6, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23704371

RESUMO

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 & inibidores
9.
PLoS One ; 7(11): e50649, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209798

RESUMO

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.


Assuntos
Mimiviridae/enzimologia , Oxirredutases/química , Proteínas Virais/química , Cristalografia por Raios X , Oxirredutases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Virais/metabolismo
10.
FEBS Lett ; 586(23): 4119-25, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23068612

RESUMO

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/metabolismo
11.
Nature ; 488(7411): 414-8, 2012 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-22801504

RESUMO

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 , Rotação
12.
Nature ; 473(7348): 540-3, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21532589

RESUMO

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 Aminoácidos
13.
FEBS Lett ; 584(8): 1521-5, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20211621

RESUMO

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer "subunit" has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Oxirredutases/química , Multimerização Proteica , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Quaternária de Proteína
14.
Biochemistry ; 47(17): 4955-63, 2008 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-18393449

RESUMO

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.


Assuntos
Mutagênese , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Catálise , Bovinos , Cisteína/metabolismo , Dissulfetos/metabolismo , Proteínas do Ovo/química , Proteínas do Ovo/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Proteínas do Leite/química , Proteínas do Leite/genética , Dados de Sequência Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Tiorredoxinas/química
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