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1.
Nat Commun ; 12(1): 870, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558523

RESUMO

Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.


Assuntos
Dissulfetos/química , Dissulfetos/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Cromatografia Líquida de Alta Pressão , Peptídeos/síntese química , Peptídeos/química , Proteínas/síntese química , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo
2.
Nat Commun ; 12(1): 249, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431872

RESUMO

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma.


Assuntos
Dissulfetos/metabolismo , Hipersensibilidade/fisiopatologia , Pulmão/fisiopatologia , Muco/metabolismo , Adolescente , Adulto , Animais , Asma/metabolismo , Asma/fisiopatologia , Modelos Animais de Doenças , Expectorantes/farmacologia , Feminino , Glicoproteínas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade
3.
Gene ; 776: 145445, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33484758

RESUMO

Glioblastom Multiforme (GBM) is the most invasive and malignant member of the IV grade of the subclass Astrocytoma according to the last assessment of the 2016 WHO report. Due to the resistance to treatment and weak response, as well as the topographical structure of the blood brain barrier, the treatment is also difficult due to the severe clinical manifestation, and new treatment methods and new therapeutic agents are needed. Temozolomide (TMZ) is widely used in the treatment of glioblastoma and is considered as the primary treatment modality. TMZ, a member of the class of cognitive agents, is currently considered the most effective drug because it can easily pass through the blood brain barrier. Glucose metabolism is a complex energy producing machine that, a glucose molecule produces 38 molecules of ATP after full glycolytic catabolism. According to Otto Warburg's numerous studies cancer cells perform the first glycolytic step without entering the mitochondrial step. These cells produce lactic acid and make the micro-media more acidic even in aerobic conditions. This phenomenon is attributed to the Warburg hypothesis and either as aerobic glycolysis. Although glycolysis enzymes are the primary actors of this phenotypic expression, some genetic and epigenetic factors are no exception. We experimentally used KC7F2 active ingredient to target cancer metabolism. In our study, we evaluated cancer metabolism in combination with the effect of TMZ chemotherapeutic agent, examining the effect of two different agents separately and in combination to observe the effects of cancer cell proliferation, survival, apoptosis and expression of metabolism genes on expression. We observed that the combined effect of reduced the effective dose of the TMZ alkylating agent and that the effect was increased and the effect of the combined teraphy is assessed from a metabolic point of view and that it suppresses aerobic glycolysis.


Assuntos
Dissulfetos/farmacologia , Glioma/tratamento farmacológico , Sulfonamidas/farmacologia , Temozolomida/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dissulfetos/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/patologia , Glioma/patologia , Glucose/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Sulfonamidas/metabolismo , Temozolomida/metabolismo
4.
Cell Rep ; 33(12): 108528, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33326798

RESUMO

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.


Assuntos
/metabolismo , Antivirais/farmacologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Microcorpos/metabolismo , /efeitos dos fármacos , Sequência de Aminoácidos , Animais , /virologia , Modelos Animais de Doenças , Dissulfetos/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Domínios Proteicos , Multimerização Proteica , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacos
5.
Niger J Clin Pract ; 23(10): 1401-1406, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33047697

RESUMO

Aims: This study aimed to compare the serum thiol-disulfide homeostasis, total antioxidant status (TAS), and total oxidant status (TOS) in patients with pseudoexfoliation glaucoma (PEG) patients, primary open-angle glaucoma (POAG) patients, and healthy individuals (control). Methods: Ninety subjects were included in this study. Three groups were separated as PEG, POAG, and control. All groups were chosen to be similar in terms of age and gender. Blood samples were obtained following an overnight fasting state and were collected on the ice at 4°C. The serum samples were separated from the cells by centrifugation at 3000 rpm for 15 min and were stored at -80°C. Serum samples analyzed for TAS and TOS, native thiol, total thiol, disulfide, and native thiol/disulfide ratio. Results: TAS and TOS levels of PEG patients were 1.2892 ± 0.0905 mmol/L; 5.0191 ± 2.7722 µmol/L, respectively. TAS and TOS levels of POAG patients were 1.2741 ± 0.1252 mmol/L; 4.1674 ± 1.7723 µmol/L, respectively. TAS and TOS levels of the control group were 2.3414 ± 0.1409 mmol/L; 4.0931 ± 0.1107 µmol/L, respectively. The TAS level was significantly lower in PEG and POAG groups compared to control. TOS level showed no significant differ ¬ ence between PEG, POAG, and control groups (P > 0.05). The mean serum total thiol and native thiol levels were significantly lower in patients with PEG compared to POAG and control group; there was no significant difference between the POAG and control group (P > 0.05). The mean serum disulfide level was significantly lower in patients with PEG compared to POAG (P = 0.018). Conclusion: Low levels of TAS were observed in patients with glaucoma, which was likely a response to the increased oxidative stress observed in these patients. While total thiol and native thiol levels were higher in the PEG group, the disulfide level was higher in the POAG group. TAS and TOS levels showed no significant difference between POAG and PEG groups.


Assuntos
Antioxidantes/metabolismo , Dissulfetos/sangue , Síndrome de Exfoliação/sangue , Glaucoma de Ângulo Aberto/sangue , Homeostase/fisiologia , Compostos de Sulfidrila/sangue , Adulto , Antioxidantes/análise , Estudos de Casos e Controles , Dissulfetos/metabolismo , Síndrome de Exfoliação/metabolismo , Feminino , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/sangue , Oxidantes/sangue , Estresse Oxidativo/fisiologia , Compostos de Sulfidrila/metabolismo
6.
Yakugaku Zasshi ; 140(9): 1119-1128, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32879244

RESUMO

Humans are exposed to various xenobiotic electrophiles on a daily basis. Electrophiles form covalent adducts with nucleophilic residues of proteins. Redox signaling, which consists of effector molecules (e.g., kinases and transcription factors) and redox sensor proteins with low pKa cysteine residues, is involved in cell survival, cell proliferation, quality control of cellular proteins and oxidative stress response. Herein, we showed that at a low dose, xenobiotic electrophiles selectively modified redox sensor proteins through covalent modification of their reactive thiols, resulting in activation of a variety of redox signaling pathways. However, increasing the dose of xenobiotic electrophiles caused non-selective and extensive modification of cellular proteins involved in toxicity. Of interest, reactive sulfur species (RSS), such as hydrogen sulfide (H2S), cysteine persulfide (CysSSH), glutathione persulfide (GSSH) and even synthetic polysulfide (e.g., Na2S4), readily captured xenobiotic electrophiles, forming their sulfur adducts, which was associated with inactivation of the electrophiles. Our findings suggest that an adaptive response through redox signaling activation and RSS-mediated electrophile capturing is involved in the regulation of electrophilic stress.


Assuntos
Cisteína/análogos & derivados , Dissulfetos/metabolismo , Glutationa/análogos & derivados , Sulfeto de Hidrogênio/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Cisteína/metabolismo , Glutationa/metabolismo , Humanos , Compostos de Sulfidrila/metabolismo , Xenobióticos/metabolismo
7.
Nat Commun ; 11(1): 4512, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908147

RESUMO

Hydrogen peroxide (H2O2) is recognized to act as a signaling molecule. Peroxiredoxins (Prxs) have the ability to transfer H2O2-derived oxidizing equivalents to redox-regulated target proteins, thus facilitating the transmission of H2O2 signals. It has remained unclear how Prxs and their target proteins are brought together to allow for target-specific protein thiol oxidation. Addressing the specific case of Prx2-dependent STAT3 oxidation, we here show that the association of the two proteins occurs prior to Prx oxidation and depends on a scaffolding protein, the membrane chaperone annexin A2. Deletion or depletion of annexin A2 interrupts the transfer of oxidizing equivalents from Prx2 to STAT3, which is observed to take place on membranes. These findings support the notion that the Prx2-STAT3 redox relay is part of a highly organized membrane signaling domain.


Assuntos
Anexina A2/metabolismo , Peroxirredoxinas/metabolismo , Fator de Transcrição STAT3/metabolismo , Anexina A2/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Dissulfetos/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Ligação Proteica , Domínios Proteicos , Transdução de Sinais
8.
PLoS One ; 15(8): e0237884, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841243

RESUMO

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.


Assuntos
Dissulfetos/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Plantas/metabolismo , Saposinas/metabolismo , Solanum tuberosum/metabolismo , Cisteína/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Plantas/química , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Sais/química , Enxofre/metabolismo
9.
Arch Biochem Biophys ; 692: 108515, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32791141

RESUMO

Apoptosis-inducing factor (AIF) is a flavoprotein and essential partner of the CHCHD4 redox protein during the mitochondrial intermembrane space import machinery. Mammalian AIF has three cysteine residues, which have received little attention. Previous reports have evidenced a redox interaction between AIF and thioredoxin 1 (Trx1), particularly after oxidant conditions. Therefore, we asked whether the cysteine residues of the human AIF could be oxidized. Our data showed that endogenous AIF could be oxidized to disulfide-linked conjugates (DLC). Overexpressed WT AIF in HEK293T cells, as well as recombinant WT AIF, formed DLC. Expression of C256S, C317S or C441S AIF mutants severely inhibited DLC formation in cells exposed to oxidants. In vitro, DLC formation was completely precluded with C256S and C441S AIF mutants and partially inhibited with the C317S mutant. DLC was shown to enhance cellular susceptibility to apoptosis induced by staurosporine, likely by preventing AIF to maintain mitochondrial oxidative phosphorylation. Cells with decreased expression of Trx1 produced more AIF DLC than those with normal Trx1 levels, and in vitro, Trx1 was able to decrease the amount of AIF DLC. Finally, confocal analysis, as well as immunoblotting of mitochondrial fraction, indicated that a fraction of Trx1 is present in mitochondria. Overall, these data provide evidence that all three cysteine residues of AIF can be oxidized to DLC, which can be disrupted by mitochondrial Trx1.


Assuntos
Fator de Indução de Apoptose , Apoptose , Dissulfetos , Substituição de Aminoácidos , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação de Sentido Incorreto , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologia
10.
Nat Struct Mol Biol ; 27(9): 781-789, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661419

RESUMO

Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.


Assuntos
Apoptose , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Células HEK293 , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor/química , Mitocôndrias/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Proto-Oncogênicas c-bcl-2/química
11.
Nat Commun ; 11(1): 3219, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591542

RESUMO

The receptor-linked protein tyrosine phosphatases (RPTPs) are key regulators of cell-cell communication through the control of cellular phosphotyrosine levels. Most human RPTPs possess an extracellular receptor domain and tandem intracellular phosphatase domains: comprising an active membrane proximal (D1) domain and an inactive distal (D2) pseudophosphatase domain. Here we demonstrate that PTPRU is unique amongst the RPTPs in possessing two pseudophosphatase domains. The PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and we show that this is due to multiple structural rearrangements that destabilise the active site pocket and block the catalytic cysteine. Upon oxidation, this cysteine forms an intramolecular disulphide bond with a vicinal "backdoor" cysteine, a process thought to reversibly inactivate related phosphatases. Importantly, despite the absence of catalytic activity, PTPRU binds substrates of related phosphatases strongly suggesting that this pseudophosphatase functions in tyrosine phosphorylation by competing with active phosphatases for the binding of substrates.


Assuntos
Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Biocatálise , Linhagem Celular , Dissulfetos/metabolismo , Estabilidade Enzimática , Humanos , Modelos Moleculares , Oxirredução , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/química , Especificidade por Substrato
12.
Nat Commun ; 11(1): 2264, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385283

RESUMO

ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Antineoplásicos/química , Transporte Biológico , Dissulfetos/metabolismo , Células HEK293 , Humanos , Mesilato de Imatinib/metabolismo , Ligantes , Mitoxantrona/química , Mitoxantrona/metabolismo , Modelos Biológicos , Estrutura Secundária de Proteína
13.
Proc Natl Acad Sci U S A ; 117(24): 13329-13338, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461372

RESUMO

Two-dimensional (2D) molybdenum disulfide (MoS2) nanomaterials are an emerging class of biomaterials that are photoresponsive at near-infrared wavelengths (NIR). Here, we demonstrate the ability of 2D MoS2 to modulate cellular functions of human stem cells through photothermal mechanisms. The interaction of MoS2 and NIR stimulation of MoS2 with human stem cells is investigated using whole-transcriptome sequencing (RNA-seq). Global gene expression profile of stem cells reveals significant influence of MoS2 and NIR stimulation of MoS2 on integrins, cellular migration, and wound healing. The combination of MoS2 and NIR light may provide new approaches to regulate and direct these cellular functions for the purposes of regenerative medicine as well as cancer therapy.


Assuntos
Dissulfetos/efeitos da radiação , Células-Tronco Mesenquimais/efeitos da radiação , Molibdênio/efeitos da radiação , Nanoestruturas/efeitos da radiação , Adesão Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Sobrevivência Celular , Dissulfetos/química , Dissulfetos/metabolismo , Perfilação da Expressão Gênica , Humanos , Raios Infravermelhos , Integrinas/genética , Integrinas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Molibdênio/química , Molibdênio/metabolismo , Nanoestruturas/química , Fármacos Fotossensibilizantes , Transdução de Sinais/efeitos da radiação
14.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32434885

RESUMO

Disulfide bonds are critical for the structure and function of many proteins. Some large DNA viruses encode their own sulfhydryl oxidase for disulfide bond formation. Previous studies have demonstrated that the baculovirus-encoded sulfhydryl oxidase P33 is necessary for progeny virus production, and its enzymatic activity is important for morphogenesis and oral infectivity of baculoviruses. However, the downstream substrates of P33 in the putative redox pathway of baculoviruses are unknown. In this study, we showed that PIF5, one of the per os infectivity factors (PIFs), contained intramolecular disulfide bonds and that the disulfide bond formation was interrupted in the absence of P33. In vivo pulldown and colocalization analyses revealed that PIF5 and P33 interacted with each other during virus infection. Further, in vitro assays validated that the reduced PIF5 proteins could be oxidized by P33. To understand the contribution of disulfide bonds to the function of PIF5, several cysteine-to-serine mutants were constructed, which all interfered with the disulfide bond formation of PIF5 to different extents. All the mutants lost their oral infectivity but had no impact on infectious budding virus (BV) production or virus morphogenesis. Taken together, our results indicated PIF5 as the first identified substrate of P33. Further, the disulfide bonds in PIF5 play an essential role in its function in oral infection.IMPORTANCE Similar to some large DNA viruses that encode their own disulfide bond pathway, baculovirus encodes a viral sulfhydryl oxidase, P33. Enzyme activity of P33 is related to infectious BV production, occlusion-derived virus (ODV) envelopment, occlusion body morphogenesis, and oral infectivity, suggesting that P33 is involved in disulfide bond formation of multiple proteins. A complete disulfide bond formation pathway normally contains a sulfhydryl oxidase, a disulfide-donating enzyme, and one or more substrates. In baculovirus, apart from P33, other components of the putative pathway remain unknown. In this study, we identified PIF5 as the first substrate of P33, which is fundamental for revealing the complete disulfide bond formation pathway in baculovirus. PIF5 is essential for oral infection and is absent from the PIF complex. Our study demonstrated that native disulfide bonds in PIF5 are required for oral infection, which will help us to reveal its mode of action.


Assuntos
Dissulfetos/metabolismo , Nucleopoliedrovírus/metabolismo , Oxirredutases/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Mutação de Sentido Incorreto , Nucleopoliedrovírus/genética , Oxirredução , Oxirredutases/genética , Proteínas Virais/genética
15.
Nat Cell Biol ; 22(4): 476-486, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231310

RESUMO

SLC7A11-mediated cystine uptake is critical for maintaining redox balance and cell survival. Here we show that this comes at a significant cost for cancer cells with high levels of SLC7A11. Actively importing cystine is potentially toxic due to its low solubility, forcing cancer cells with high levels of SLC7A11 (SLC7A11high) to constitutively reduce cystine to the more soluble cysteine. This presents a significant drain on the cellular NADPH pool and renders such cells dependent on the pentose phosphate pathway. Limiting glucose supply to SLC7A11high cancer cells results in marked accumulation of intracellular cystine, redox system collapse and rapid cell death, which can be rescued by treatments that prevent disulfide accumulation. We further show that inhibitors of glucose transporters selectively kill SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results identify a coupling between SLC7A11-associated cystine metabolism and the pentose phosphate pathway, and uncover an accompanying metabolic vulnerability for therapeutic targeting in SLC7A11high cancers.


Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Carcinoma de Células Renais/genética , Cistina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Via de Pentose Fosfato/genética , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dissulfetos/metabolismo , Fármacos Gastrointestinais/farmacologia , Glucose/deficiência , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Estresse Fisiológico , Sulfassalazina/farmacologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Nature ; 580(7802): 216-219, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269349

RESUMO

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas , Reciclagem , Actinobacteria/enzimologia , Burkholderiales/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Ácidos Ftálicos/metabolismo , Polimerização
17.
Int J Mol Sci ; 21(6)2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32197489

RESUMO

Postsynaptic density-95 (PSD95), a major scaffolding protein, is critical in coupling N-methyl-D-aspartate receptor (NMDAR) to cellular signaling networks in the central nervous system. A couple of cysteine residues in the N-terminus of PSD95 are potential sites for disulfide bonding, S-nitrosylation and/or palmitoylation. Protein disulfide isomerase (PDI) reduces disulfide bonds (S-S) to free thiol (-SH) on various proteins. However, the involvement of PDI in disulfide bond formation/S-nitrosylation of PSD95 and its role in epilepsy are still unknown. In the present study, acute seizure activity significantly increased the bindings of PDI to NR2A, but not to PSD95, while it decreased the NR2A-PSD95 binding. In addition, pilocarpine-induced seizures increased the amount of nitrosylated (SNO-) thiols, not total (free and SNO-) thiols, on PSD95. Unlike acute seizure, spontaneous seizing rats showed the increases in PDI-PSD95 binding, total- and SNO-thiol levels on PSD95, and NR2A-PSD95 interaction. PDI siRNA effectively reduced spontaneous seizure activity with decreases in total thiol level on PSD95 and NR2A-PSD95 association. These findings indicate that PDI-mediated reduction of disulfide-bond formations may facilitate the NR2A-PSD95 binding and contribute to spontaneous seizure generation in epileptic animals.


Assuntos
Proteína 4 Homóloga a Disks-Large/metabolismo , Dissulfetos/metabolismo , Epilepsia/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Epilepsia/patologia , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley
18.
PLoS One ; 15(3): e0230366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32203539

RESUMO

Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.


Assuntos
Campylobacter jejuni/enzimologia , Dissulfetos/metabolismo , Proteínas Periplásmicas/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Sequência de Aminoácidos , Campylobacter jejuni/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Oxirredução , Periplasma/enzimologia , Proteínas Periplásmicas/genética , Filogenia , Proteína Dissulfeto Redutase (Glutationa)/genética
19.
Nat Commun ; 11(1): 1272, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152292

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/química , Sequência de Aminoácidos , Apoproteínas/química , Dissulfetos/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/ultraestrutura , Humanos , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Estabilidade Proteica , Receptores de Glucagon/química
20.
Int J Mol Sci ; 21(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033303

RESUMO

α-Lipoic acid, glutathione, cysteine, and cysteinylglycine can be applied as therapeutic agents in civilization diseases such as diabetes mellitus, cardiovascular diseases, and cancers. On the other hand, a higher concentration of homocysteine can result in health problems and has been indicated as an independent risk factor for cardiovascular disease and accelerated atherosclerosis. Here, the first simplified HPLC-UV assay that enables simultaneous determination of α-lipoic acid and low-molecular-mass thiols in plasma, reduces the number of steps, shortens the total time of sample preparation, and limits the amount of single-use polypropylene laboratory materials is described. The assay is based on reversed-phase high performance liquid chromatography with UV detection and simultaneous reduction of disulfide bound with tris(2-carboxyethyl)phosphine and the selective pre-column derivatization of the thiol group with 1-benzyl-2-chloropyridinium bromide. Linearity in the detector responses for plasma samples were observed in ranges: 0.12-5.0 nmol mL-1 for α-lipoic acid; 2.0-20.0 nmol mL-1 for glutathione, cysteinylglycine, and homocysteine; and 40.0-400.0 for cysteine. The LODs for α-lipoic acid and low-molecular-mass thiols were 0.08 and 0.12 nmol mL-1, respectively, while LOQs were 0.12 and 0.16 nmol mL-1, respectively. The usefulness of the proposed method has been proven by its application to real samples.


Assuntos
Plasma/metabolismo , Compostos de Sulfidrila/sangue , Compostos de Sulfidrila/metabolismo , Ácido Tióctico/sangue , Ácido Tióctico/metabolismo , Adulto , Cisteína/metabolismo , Dipeptídeos/metabolismo , Dissulfetos/metabolismo , Feminino , Glutationa/metabolismo , Homocisteína/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Peso Molecular , Oxirredução , Compostos de Piridínio/metabolismo
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