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1.
PLoS One ; 15(6): e0234192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479562

RESUMO

Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family.


Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supressão Genética , Ubiquinona/biossíntese , Substituição de Aminoácidos , Asparagina , Meios de Cultura/química , Regulação Fúngica da Expressão Gênica , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquinona/genética
2.
Biomed Khim ; 66(2): 105-123, 2020 Feb.
Artigo em Russo | MEDLINE | ID: mdl-32420891

RESUMO

L-asparaginase is extensively used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is also of prospective use in food industry to reduce the formation of acrylamide, which is classified as probably neurotoxic and carcinogenic to human, and in biosensors for determination of L-asparagine level in medicine and food chemistry. The importance of L-asparaginases in different fields, disadvantages of commercial ferments, and the fact that they are widespread in nature stimuli the search for biobetter L-asparaginases from new producing microorganisms. In this regard, extremofile microorganisms exhibit unique physiological properties such as thermal stability, adaptability to extreme cold conditions, salt and pH tolerance and so provide one of the most valuable sources for novel L-asparaginases. The present review summarizes the recent results on studying the structural, functional, physicochemical and kinetic properties, stability of extremophilic L-asparaginases in comparison with their mesophilic homologues.


Assuntos
Asparaginase/química , Extremófilos/enzimologia , Asparagina
3.
Food Chem ; 326: 126982, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32413762

RESUMO

Glutathione (GSH) is a potential inhibitor for acrylamide (AA) in heated food. In the present study, the inhibition pathways of GSH on AA were investigated in the asparagine(Asn)/glucose(Glc)/GSH model system. In comparison to the Asn/Glc model system, three unique molecular ions (m/z 470, 379, and 193) were identified in the Asn/Glc/GSH model system. Those molecular ions were confirmed as the Amadori rearrangement products which formed in the reaction between Glc and GSH, as well as the addition reaction products between AA and the sulfhydryl group (-SH) of GSH and cysteine (Cys). The competition between Asn and GSH for Glc in the competitive reactions was assumed to be the major pathway. Additionally, the elimination reaction between AA and GSH or between AA and Cys also played a minor role in the inhibition of AA. The variances of precursors, intermediates, and final products provided quantitative evidence for the above pathways.


Assuntos
Acrilamida/antagonistas & inibidores , Glutationa/química , Acrilamida/metabolismo , Asparagina/química , Glucose/química , Glutationa/metabolismo , Compostos de Sulfidrila/química , Espectrometria de Massas em Tandem , Temperatura
4.
Nat Commun ; 11(1): 1755, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273511

RESUMO

Asparagine synthetase (ASNS) catalyses the ATP-dependent conversion of aspartate to asparagine. However, both the regulation and biological functions of asparagine in tumour cells remain largely unknown. Here, we report that p53 suppresses asparagine synthesis through the transcriptional downregulation of ASNS expression and disrupts asparagine-aspartate homeostasis, leading to lymphoma and colon tumour growth inhibition in vivo and in vitro. Moreover, the removal of asparagine from culture medium or the inhibition of ASNS impairs cell proliferation and induces p53/p21-dependent senescence and cell cycle arrest. Mechanistically, asparagine and aspartate regulate AMPK-mediated p53 activation by physically binding to LKB1 and oppositely modulating LKB1 activity. Thus, we found that p53 regulates asparagine metabolism and dictates cell survival by generating an auto-amplification loop via asparagine-aspartate-mediated LKB1-AMPK signalling. Our findings highlight a role for LKB1 in sensing asparagine and aspartate and connect asparagine metabolism to the cellular signalling transduction network that modulates cell survival.


Assuntos
Asparagina/metabolismo , Ácido Aspártico/metabolismo , Neoplasias do Colo/metabolismo , Homeostase , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Asparagina/genética , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Ácido Aspártico/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/terapia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
5.
J Biosci Bioeng ; 129(6): 672-678, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32088137

RESUMO

l-Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l-asparaginase was screened. Type I l-asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l-asparaginase had an activity of 42.0 IU mL-1 and showed no activity toward l-glutamine and d-asparagine. The recombinant l-asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and ß-mercaptoethanol. The Km and Vmax values of the l-asparaginase for the l-asparagine were 3.22 mmol L-1 and 1.55 IU µg-1, respectively. Moreover, the recombinant l-asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l-asparaginase has the potential for application in the food processing industry.


Assuntos
Acinetobacter/enzimologia , Acrilamida/metabolismo , Asparaginase/metabolismo , Solanum tuberosum/metabolismo , Acinetobacter/genética , Asparaginase/genética , Asparagina/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamina/metabolismo , Lanches
6.
Nat Commun ; 11(1): 791, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034141

RESUMO

The conserved hemagglutinin (HA) stem has been a focus of universal influenza vaccine efforts. Influenza A group 1 HA stem-nanoparticles have been demonstrated to confer heterosubtypic protection in animals; however, the protection does not extend to group 2 viruses, due in part to differences in glycosylation between group 1 and 2 stems. Here, we show that introducing the group 2 glycan at Asn38HA1 to a group 1 stem-nanoparticle (gN38 variant) based on A/New Caledonia/20/99 (H1N1) broadens antibody responses to cross-react with group 2 HAs. Immunoglobulins elicited by the gN38 variant provide complete protection against group 2 H7N9 virus infection, while the variant loses protection against a group 1 H5N1 virus. The N38HA1 glycan thus is pivotal in directing antibody responses by controlling access to group-determining stem epitopes. Precise targeting of stem-directed antibody responses to the site of vulnerability by glycan repositioning may be a step towards achieving cross-group influenza protection.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Nanopartículas/química , Polissacarídeos/química , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Asparagina/química , Asparagina/metabolismo , Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas , Epitopos/imunologia , Feminino , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Imunoglobulinas/imunologia , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle
7.
Arch Microbiol ; 202(5): 967-981, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32052094

RESUMO

L-asparaginase (E.C.3.5.1.1) is an important enzyme that has been purified and characterized for over decades to study and evaluate its anti-carcinogenic activity against different lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma. The ability of the enzyme to convert L-asparagine into aspartic acid and ammonia is the reason behind its anti-cancerous activity. Apart from its medicinal uses, it is widely used in food industry to tackle acrylamide, a probable human carcinogen and, production in carbohydrate-rich foods cooked at high temperatures. There are variety of organisms including microorganisms such as bacteria, fungi, algae, and plants that produce L-asparaginase. The enzyme obtained from different microbial and plant sources have different physiochemical properties and kinetic parameters. L-asparaginases have an optimum pH range between 6 and 10 and an optimum temperature between 37 and 85 °C. This article has reviewed the lowest molecular mass for L-asparaginase in Yersinia pseudotuberculosis Q66CJ2 which is 36.27 kDa, while the highest for Pseudomonas otitidis which has a molecular mass of 205 ± 3 kDa. This review is an attempt to summarize most of the available sources, their phylogenetic relationships, purification methods, data regarding different physiochemical and kinetic properties of L-asparaginase.


Assuntos
Asparaginase/química , Bactérias/enzimologia , Fungos/enzimologia , Doença de Hodgkin/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Amônia/metabolismo , Asparaginase/genética , Asparaginase/isolamento & purificação , Asparagina/química , Ácido Aspártico/metabolismo , Humanos , Filogenia , Plantas
8.
Biochem Pharmacol ; 174: 113850, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32044355

RESUMO

The human cytochrome P450 enzyme CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer. Hence, CYP4Z1 has previously been suggested as an anti-breast cancer target. In the present study we employed extended mutation analysis to increase our understanding of the substrate binding mode of this enzyme. In a combined in vitro and in silico approach we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1. Using a large array of recombinant CYP4Z1 mutants we show that, apart from Asn381, all other postulated binding residues only play an auxiliary role in substrate recognition and binding. Different substrate interaction motifs were identified via dynamic pharmacophores (dynophores) and their impact on catalytically competent substrate binding was classified. These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1.


Assuntos
Arginina/metabolismo , Asparagina/metabolismo , Simulação por Computador , Família 4 do Citocromo P450/metabolismo , Arginina/química , Arginina/genética , Asparagina/química , Asparagina/genética , Sítios de Ligação/fisiologia , Família 4 do Citocromo P450/química , Família 4 do Citocromo P450/genética , Humanos , Mutação de Sentido Incorreto/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato/fisiologia
9.
mBio ; 11(1)2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937642

RESUMO

The survival of commensal bacteria in the human gut partially depends on their ability to metabolize host-derived molecules. The use of the glycosidic moiety of N-glycoproteins by bacteria has been reported, but the role of N-glycopeptides or glycoamino acids as the substrates for bacterial growth has not been evaluated. We have identified in Lactobacillus casei strain BL23 a gene cluster (alf-2) involved in the catabolism of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn (6'FN-Asn), a constituent of the core-fucosylated structures of mammalian N-glycoproteins. The cluster consists of the genes alfHC, encoding a major facilitator superfamily (MFS) permease and the α-l-fucosidase AlfC, and the divergently oriented asdA (aspartate 4-decarboxylase), alfR2 (transcriptional regulator), pepV (peptidase), asnA2 (glycosyl-asparaginase), and sugK (sugar kinase) genes. Knockout mutants showed that alfH, alfC, asdA, asnA2, and sugK are necessary for efficient 6'FN-Asn utilization. The alf-2 genes are induced by 6'FN-Asn, but not by its glycan moiety, via the AlfR2 regulator. The constitutive expression of alf-2 genes in an alfR2 strain allowed the metabolism of a variety of 6'-fucosyl-glycans. However, GlcNAc-Asn did not support growth in this mutant background, indicating that the presence of a 6'-fucose moiety is crucial for substrate transport via AlfH. Within bacteria, 6'FN-Asn is defucosylated by AlfC, generating GlcNAc-Asn. This glycoamino acid is processed by the glycosylasparaginase AsnA2. GlcNAc-Asn hydrolysis generates aspartate and GlcNAc, which is used as a fermentable source by L. casei These data establish the existence in a commensal bacterial species of an exclusive metabolic pathway likely to scavenge human milk and mucosal fucosylated N-glycopeptides in the gastrointestinal tract.IMPORTANCE The gastrointestinal tract accommodates more than 1014 microorganisms that have an enormous impact on human health. The mechanisms enabling commensal bacteria and administered probiotics to colonize the gut remain largely unknown. The ability to utilize host-derived carbon and energy resources available at the mucosal surfaces may provide these bacteria with a competitive advantage in the gut. Here, we have identified in the commensal species Lactobacillus casei a novel metabolic pathway for the utilization of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn, which is present in the core-fucosylated N-glycoproteins from mammalians. These results give insight into the molecular interactions between the host and commensal/probiotic bacteria and may help to devise new strategies to restore gut microbiota homeostasis in diseases associated with dysbiotic microbiota.


Assuntos
Asparagina/análogos & derivados , Fucose/análogos & derivados , Trato Gastrointestinal/microbiologia , Interações entre Hospedeiro e Microrganismos , Lactobacillus casei/metabolismo , Redes e Vias Metabólicas , Asparagina/metabolismo , Fucose/metabolismo , Humanos , Lactobacillus casei/genética , Família Multigênica , Probióticos , Simbiose
11.
Chemistry ; 26(32): 7219-7225, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31984562

RESUMO

Lack of new antibiotics and increasing antimicrobial resistance are among the main concerns of healthcare communities nowadays, and these concerns necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids-a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe 1) a concise total synthesis of cystobactamid 507, 2) the identification of the bioactive conformation using noncovalently bonded rigid analogues, and 3) the first structure-activity relationship (SAR) study for cystobactamid 507 leading to new analogues with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor-groove binding as part of the drug-target interaction without showing significant intercalation. By designing a new analogue of cystobactamid 919-2, we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.


Assuntos
Antibacterianos/síntese química , Asparagina/análogos & derivados , Bactérias Gram-Negativas/efeitos dos fármacos , Nitrocompostos/química , Antibacterianos/química , Antibacterianos/farmacologia , Asparagina/química , Asparagina/farmacologia , Bactérias Gram-Negativas/química , Testes de Sensibilidade Microbiana , Conformação Molecular , Nitrocompostos/farmacologia , Relação Estrutura-Atividade
12.
J Chromatogr A ; 1615: 460740, 2020 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-31796250

RESUMO

The identification and quantification of post-translational modifications (PTMs) is a crucial step required during the development of therapeutic proteins. In particular, the characterization of charge variants separated by cation exchange chromatography (CEX) is a tedious process commonly performed with an off-line manual fraction collection followed by peptide mapping. To improve the efficiency of this time-consuming approach, a generic on-line multi-dimensional LC/MS approach was developed for the characterization of various monoclonal antibody (mAb) isotypes and a bi-specific antibody (BsAb). Fractions collected from 1D CEX analysis were consecutively reduced on a 2D reversed phase liquid chromatography (RPLC) column (polyphenyl), digested within 1-2 min using a 3D immobilized trypsin cartridge, and finally the obtained peptides were separated on another 4D RPLC column (C18), and simultaneously identified with a Q Exactive™ mass spectrometer. 2D RPLC columns and 3D trypsin cartridges from different suppliers were compared, as well as the effects of reducing agents. The effect of 2D and 4D RPLC column temperature, and 2D RPLC column mass load were also systematically studied. Under optimal conditions, the multi-dimensional LC/MS system described in this paper is a robust tool for the on-line digestion of proteins and shows high repeatability. Similar levels of oxidation and deamidation were measured using the off-line and on-line approaches for the same stressed samples. The lower amounts of deamidation and isomerization measured at some asparagine and aspartic acid residues by the on-line approach compared to the manual off-line procedure suggest reduced artifacts using the on-line methodology. The multi-dimensional LC/MS described here enables fast, on-line, automated characterization of therapeutic antibodies without the need for off-line fraction collection and sample pre-treatment (manual approach). The entire workflow can be completed within less than one day, compared to weeks with the manual off-line procedure.


Assuntos
Anticorpos Monoclonais/química , Técnicas de Química Analítica/métodos , Cromatografia Líquida , Espectrometria de Massas , Asparagina/química , Mapeamento de Peptídeos , Peptídeos/química , Tripsina , Fluxo de Trabalho
13.
Food Chem ; 303: 125372, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31446360

RESUMO

The formation and partial degradation of acrylamide (AA), asparagine and low molecular weight sugars were evaluated during an industrial coffee roasting process, in which the temperature increased from 90° to about 215 °C. Arabica and Robusta varieties were roasted individually. AA content reached the maximum value at 10 min, corresponding to a temperature of 175-177 °C (1045 ±â€¯28 and 795 ±â€¯25 µg kg-1 for Arabica and Robusta, respectively). Successively, AA content decreased very quickly and at 14 min (203-205 °C) its concentration was lower than the benchmark level of 400 µg kg-1 for roast coffee set by the EU Commission Regulation (2017/2158). In the final product, AA content was close to 300 µg kg-1. Asparagine quickly decreased; contrary, the concentration of fructose and glucose increased reaching their maximum value at 12 min. Then, a quick degradation occurred; their increase could be mainly due to the hydrolysis of sucrose, which decreased in the same period.


Assuntos
Acrilamida/análise , Café/química , Manipulação de Alimentos , Temperatura Alta , Asparagina/química , Sacarose , Açúcares/química
14.
Proc Natl Acad Sci U S A ; 117(1): 717-726, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871197

RESUMO

Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+ channel (ENaC) formed by α-, ß-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC's ability to mediate SF responsiveness relies on the "force-from-filament" principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively their N-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal of N-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.


Assuntos
Asparagina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Matriz Extracelular/metabolismo , Domínios Proteicos/genética , Animais , Asparagina/química , Modelos Animais de Doenças , Células Endoteliais , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Feminino , Glicosilação , Células HEK293 , Humanos , Hipertensão/etiologia , Hipertensão/patologia , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Oócitos , Técnicas de Patch-Clamp , Mutação Puntual , Polissacarídeos/química , Estresse Mecânico , Xenopus laevis
15.
Mater Sci Eng C Mater Biol Appl ; 107: 110260, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761157

RESUMO

The current work describes the successful synthesis of a magnetic CoFe2O4 centered asparagine functionalized noble metal (M = Cu, Ni) anchored nanocomposite. The novel materials, synthesized by post-functionalization approach, have been characterized by XRD, SEM, EDX, X-ray elemental mapping, FT-IR and VSM studies. The materials are proved to be efficient heterogeneous catalyst in the synthesis of diarylthioethers by C-S cross coupling reaction and 5-substituted 1H-tetrazoles by azide-alkyne cycloaddition reaction under green conditions. The current methodology is advantageous in terms of simplicity of procedure, facile synthesis, high yield in short reaction time, easy magnetic isolation and reusability of catalysts in consecutive runs with insignificant change in catalytic activity.


Assuntos
Asparagina/química , Nanopartículas de Magnetita/química , Nanocompostos/química , Sulfetos/química , Tetrazóis/química , Catálise , Cobalto/química , Cobre/química , Reação de Cicloadição , Compostos Férricos/química , Química Verde , Níquel/química , Sulfetos/síntese química , Tetrazóis/síntese química
16.
Int J Mol Sci ; 20(23)2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810196

RESUMO

Oligosaccharyltransferase (OST) is a multi-span membrane protein complex that catalyzes the addition of glycans to selected Asn residues within nascent polypeptides in the lumen of the endoplasmic reticulum. This process, termed N-glycosylation, is a fundamental post-translational protein modification that is involved in the quality control, trafficking of proteins, signal transduction, and cell-to-cell communication. Given these crucial roles, N-glycosylation is essential for homeostasis at the systemic and cellular levels, and a deficiency in genes that encode for OST subunits often results in the development of complex genetic disorders. A growing body of evidence has also demonstrated that the expression of OST subunits is cell context-dependent and is frequently altered in malignant cells, thus contributing to tumor cell survival and proliferation. Importantly, a recently developed inhibitor of OST has revealed this enzyme as a potential target for the treatment of incurable drug-resistant tumors. This review summarizes our current knowledge regarding the functions of OST in the light of health and tumor progression, and discusses perspectives on the clinical relevance of inhibiting OST as a tumor treatment.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Hexosiltransferases/genética , Proteínas de Membrana/genética , Neoplasias/genética , Processamento de Proteína Pós-Traducional/genética , Sequência de Aminoácidos/genética , Asparagina/genética , Progressão da Doença , Retículo Endoplasmático/genética , Glicosilação , Hexosiltransferases/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Neoplasias/tratamento farmacológico , Polissacarídeos/genética
17.
Commun Biol ; 2: 392, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31667366

RESUMO

Programmed cell death 1 (PD-1) is inhibitory receptor and immune checkpoint protein. Blocking the interaction of PD-1 and its ligands PD-L1/ L2 is able to active T-cell-mediated antitumor response. Monoclonal antibody-based drugs targeting PD-1 pathway have exhibited great promise in cancer therapy. Here we show that MW11-h317, an anti-PD-1 monoclonal antibody, displays high affinity for PD-1 and blocks PD-1 interactions with PD-L1/L2. MW11-h317 can effectively induce T-cell-mediated immune response and inhibit tumor growth in mouse model. Crystal structure of PD-1/MW11-h317 Fab complex reveals that both the loops and glycosylation of PD-1 are involved in recognition and binding, in which Asn58 glycosylation plays a critical role. The unique glycan epitope in PD-1 to MW11-h317 is different from the first two approved clinical PD-1 antibodies, nivolumab and pembrolizumab. These results suggest MW11-h317 as a therapeutic monoclonal antibody of PD-1 glycosylation-targeting which may become efficient alternative for cancer therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Antineoplásicos Imunológicos/química , Asparagina/metabolismo , Antígeno B7-H1/metabolismo , Ligação Competitiva , Cristalografia por Raios X , Epitopos/química , Feminino , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neoplasias/tratamento farmacológico , Nivolumabe/química , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Domínios e Motivos de Interação entre Proteínas
18.
Int J Mol Sci ; 20(20)2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31635169

RESUMO

The nature of renal amyloidosis involving Bence-Jones proteins in multiple myeloma is still unclear. The development of amyloidosis in neurodegenerative diseases is often associated with a high content of asparagine and glutamine residues in proteins forming amyloid deposits. To estimate the influence of Asn and Gln residues on the aggregation of Bence-Jones protein BIF, we obtained recombinant BIF and its mutants with the substitution of Tyr187→Asn (Y187N) in α-helix of CL domain, Lys170→Asn (K170N) and Ser157→Gln (S157Q) in CL domain loops, Arg109→Asn in VL-CL linker (R109N) and Asp29→Gln in VL domain loop (D29Q). The morphology of protein aggregates was studied at pH corresponding to the conditions in bloodstream (pH 7.2), distal (pH 6.5) and proximal renal tubules (pH 4.5) by atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS). The Lys170→Asn replacement almost completely inhibits amyloidogenic activity. The Y187N forms fibril-like aggregates at all pH values. The Arg109→Asn replacement resulted in formation of fibril-like structures at pH 7.2 and 6.5 while the substitutions by Gln provoked formation of those structures only at pH 7.2. Therefore, the amyloidogenic properties are highly dependent on the location of Asn or Gln.


Assuntos
Asparagina/química , Proteína de Bence Jones/química , Glutamina/química , Proteínas Mutantes/química , Mutação , Agregados Proteicos , Sequência de Aminoácidos , Substituição de Aminoácidos , Asparagina/genética , Proteína de Bence Jones/genética , Proteína de Bence Jones/metabolismo , Glutamina/genética , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Difração de Raios X
19.
PLoS Biol ; 17(10): e3000104, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600193

RESUMO

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.


Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Processamento de RNA , Proteínas de Ligação a RNA/genética , Spliceossomos/metabolismo , Asparagina/química , Asparagina/metabolismo , Sítios de Ligação , Clonagem Molecular , Cobre/química , Cobre/metabolismo , Cryptococcus neoformans/metabolismo , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Inteínas , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Spliceossomos/ultraestrutura , Homologia Estrutural de Proteína , Zinco/química , Zinco/metabolismo
20.
Nat Commun ; 10(1): 4910, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659163

RESUMO

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Membrana/química , Oxigenases de Função Mista/química , Proteínas Musculares/química , Sequência de Aminoácidos , Asparagina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Domínio Catalítico , Cristalografia , Dissulfetos/química , Dissulfetos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Conformação Proteica
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