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1.
J Chromatogr A ; 1644: 462090, 2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-33823387

RESUMO

In this study, to selectively enrich N-glycans from complex biological samples, a novel Zr(IV) modified adenosine triphosphate (Zr(IV)-ATP) functionalized monolith was prepared through a facile approach. Well-defined macroporous structure was observed in the ATP functionalized monolith, which allows rapid mass transfer under low backpressure and is beneficial for the enrichment of N-glycans. After being modified with Zr(IV), the resulting Zr(IV)-ATP functionalized monolith could selectively capture N-glycans through the specific interactions between the sulfonate groups of 1-aminopyrene-3,6,8-trisulfonic acid (APTS) labeled N-glycans and Zr(IV). An APTS labeled maltooligosaccharide ladder was used to optimize the enrichment conditions for APTS labeled N-glycans, and capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detector was employed to evaluate the enrichment efficiency. The results show that the APTS labeled maltooligosaccharides could be enriched under the selected conditions and the signal amplify factors of the maltooligosaccharides were between 7.4 and 19.5 with RSDs for reproducibility from 4.0% to 8.3% (n = 3). Finally, the proposed method was successfully used for the enrichment and detection of N-glycans released from Ribonuclease B.


Assuntos
Trifosfato de Adenosina/química , Polissacarídeos/química , Zircônio/química , Eletroforese Capilar , Glucose/química , Oligossacarídeos/química , Polímeros/química , Pirenos/química , Reprodutibilidade dos Testes , Espectroscopia de Infravermelho com Transformada de Fourier
2.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799639

RESUMO

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a 'gate' for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Triptofano/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Domínio Catalítico/genética , Dicroísmo Circular , DNA/química , DNA/genética , DNA Helicases/química , DNA Helicases/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Humanos , Mutação de Sentido Incorreto , Conformação Proteica , Homologia de Sequência de Aminoácidos , Temperatura , Triptofano/química , Triptofano/genética
3.
Molecules ; 26(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671368

RESUMO

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.


Assuntos
Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Mutantes/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Benzotiazóis/química , Benzotiazóis/metabolismo , Sítios de Ligação , AMP Cíclico/química , AMP Cíclico/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Ligantes , Domínios Proteicos , Homologia Estrutural de Proteína , Termodinâmica , Triazóis/química , Triazóis/metabolismo
4.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670267

RESUMO

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Deleção de Sequência
5.
Adv Protein Chem Struct Biol ; 124: 87-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33632471

RESUMO

Protein kinases use ATP to phosphorylate other proteins. Phosphorylation (p) universally orchestrates a fine-tuned network modulating a multitude of biological processes. Moreover, the start of networks, ATP-binding site, has been recognized dual roles to impact protein kinases function: (i) orthosteric inhibition, via being blocked to interference ATP occupying and (ii) allosteric regulation, via being altered first to induce further conformational changes. The above two terminologies are widely used in drug design, which has acquired quite a significant progress in the protein kinases field over the past 2 decades. Most small molecular inhibitors directly compete with ATP to implement orthosteric inhibition, still exhibiting irreplaceable and promising therapeutic effects. Additionally, numerous inhibitors can paradoxically lead protein kinases to hyperphosphorylation, even activation, indicative of the allosteric regulation role of the ATP-binding site. Here, we review the quintessential examples that apply for the dual roles in diverse ways. Our work provides an insight into the molecular mechanisms under the dual roles and will be promisingly instructive for future drug development.


Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Humanos
6.
Eur J Med Chem ; 213: 113200, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524686

RESUMO

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.


Assuntos
Trifosfato de Adenosina/farmacologia , Antibacterianos/farmacologia , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Antibacterianos/síntese química , Antibacterianos/química , Cristalografia por Raios X , DNA Topoisomerase IV/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Relação Estrutura-Atividade
7.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530558

RESUMO

Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.


Assuntos
Proteínas Mitocondriais/química , Simulação de Dinâmica Molecular , Conformação Proteica , Proteína Desacopladora 2/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Transporte de Íons , Proteínas de Membrana/química , Camundongos , Proteínas Mitocondriais/metabolismo , Ligação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade , Proteína Desacopladora 2/metabolismo
8.
Biochemistry ; 60(6): 460-464, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33464880

RESUMO

The Escherichia coli ATP-consuming chaperonin machinery, a complex between GroEL and GroES, has evolved to facilitate folding of substrate proteins (SPs) that cannot do so spontaneously. A series of kinetic experiments show that the SPs are encapsulated in the GroEL/ES nanocage for a short duration. If confinement of the SPs is the mechanism by which GroEL/ES facilitates folding, it follows that the assisted folding rate, relative to the bulk value, should always be enhanced. Here, we show that this is not the case for the folding of rhodanese in the presence of the full machinery of GroEL/ES and ATP. The assisted folding rate of rhodanese decreases. On the basis of our finding and those reported in other studies, we suggest that the ATP-consuming chaperonin machinery has evolved to optimize the product of the folding rate and the yield of the folded SPs on the biological time scale. Neither the rate nor the yield is separately maximized.


Assuntos
Chaperonina 10/química , Chaperonina 60/química , Dobramento de Proteína , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Conformação Proteica
9.
Int J Mol Sci ; 22(3)2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33499026

RESUMO

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.


Assuntos
Actinas/química , Membrana Celular/metabolismo , Conexinas/metabolismo , Fibroblastos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Pele/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Movimento Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas , RNA Interferente Pequeno/metabolismo , Cicatrização
10.
J Mater Chem B ; 9(5): 1351-1363, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33447840

RESUMO

In this study, a dual-receptor doxorubicin-targeted delivery system based on mesoporous silica nanoparticles (MSNs) modified with mucine-1 and ATP aptamers (DOX@MSNs-Apts) was developed. An amine-modified mucine-1 (MUC1) aptamer was covalently anchored on the surface of carboxyl-functionalized MSNs. Then, ATP aptamers (ATP1 and ATP2 aptamers) were immobilized on the surface of MSNs through partial hybridization with the MUC1 aptamer by forming a Y-shaped DNA structure on the MSNs surface (DOX@MSNs-Apts) as a gatekeeper. The developed DOX@MSNs-Apts exhibited high DOX loading capacity. In addition, it indicated an ATP-responsive feature, leading to the release of DOX in the environment with high ATP concentration (10 mM), similar to the intracellular environment of tumor cells. This property demonstrated that anticancer drug (DOX) could be entrapped inside the nanocarrier with nearly no leakage in blood and a very low concentration of ATP (1 µM). It was found that after the internalization of DOX@MSNs-MUC1 by cancer cells via the MUC1 receptor-mediated endocytosis, the ATP aptamers left the surface of the nanocarrier, allowing for rapid DOX release. DOX@MSNs-Apts indicated higher cellular uptake in MCF-7 and C26 cancer cells (MUC1+), rather than CHO cells (MUC1-). The in vitro cytotoxicity and the in vivo antitumor efficacy of DOX@MSNs-Apts showed greater cytotoxicity than the nanoparticles decorated with scrambled ATP aptamers (DOX@MSNs-Apts scrambled) in C26 and MCF-7 cell lines (MUC1+). The biodistribution and in vivo anticancer efficacy on the C26 tumor bearing mice indicated that the DOX@MSNs-Apts had a higher tumor accumulation and superior tumor growth inhibitory effect compared to free DOX and their scrambled aptamers, DOX@MSNs-Apts scrambled. Overall, the obtained results indicated that the prepared smart platform could reveal new insights into the treatment of cancer.


Assuntos
Trifosfato de Adenosina/química , Antibióticos Antineoplásicos/farmacologia , DNA/química , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Animais , Antibióticos Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Doxorrubicina/química , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mucina-1/química , Mucina-1/metabolismo , Nanopartículas/química , Tamanho da Partícula , Porosidade , Dióxido de Silício/química , Dióxido de Silício/farmacologia , Propriedades de Superfície
11.
Biochem Biophys Res Commun ; 541: 50-55, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477032

RESUMO

SARS-CoV-2 is a highly contagious coronavirus causing the ongoing pandemic. Very recently its genomic RNA of ∼30 kb was decoded to be packaged with nucleocapsid (N) protein into phase separated condensates. Interestingly, viruses have no ability to generate ATP but host cells have very high ATP concentrations of 2-12 mM. A key question thus arises whether ATP modulates liquid-liquid phase separation (LLPS) of the N protein. Here we discovered that ATP not only biphasically modulates LLPS of the viral N protein as we previously found on human FUS and TDP-43, but also dissolves the droplets induced by oligonucleic acid. Residue-specific NMR characterization showed ATP specifically binds the RNA-binding domain (RBD) of the N protein with the average Kd of 3.3 ± 0.4 mM. The ATP-RBD complex structure was constructed by NMR-derived constraints, in which ATP occupies a pocket within the positive-charged surface utilized for binding nucleic acids. Our study suggests that ATP appears to be exploited by SARS-CoV-2 to promote its life cycle by facilitating the uncoating, localizing and packing of its genomic RNA. Therefore the interactions of ATP with the viral RNA and N protein might represent promising targets for design of drugs and vaccines to terminate the pandemic.


Assuntos
Trifosfato de Adenosina/metabolismo , Extração Líquido-Líquido , RNA Viral/metabolismo , /metabolismo , Trifosfato de Adenosina/química , Sítios de Ligação , /genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Viral/química , RNA Viral/genética , Motivos de Ligação ao RNA/genética , /química
12.
Int J Biol Macromol ; 171: 28-36, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33412198

RESUMO

Mycobacterium tuberculosis HddA enzyme phosphorylates the M7P substrate and converts it to M7PP product in GDP-D-α-D-heptose biosynthetic pathway. For structural and functional studies on MtbHddA, we have purified the enzyme, which eluted as a monomer from size exclusion column. Purified MtbHddA had ATPase activity. The SAXS analysis supported globular monomeric scattering profile of MtbHddA in solution. The CD analysis showed that MtbHddA contains 45% α-helix, 18% ß-stands, and 32% random coil structures and showed unfolding temperature (TM) ~ 47.5 °C. The unfolding temperature of MtbHddA is enhanced by 1.78±0.41 °C in ATP+Mg2+ bound state, 2.12±0.41 °C in Mannose bound state and 3.07±0.41 °C in Mannose+ ATP+Mg2+ bound state. The apo and M7P +ATP + Mg2+ complexed models of MtbHddA showed that enzyme adopts a classical GHMP sugar kinase fold with conserved ATP+Mg2+ and M7P binding sites. The dynamics simulation analysis on four MtbHddA models showed that ATP+Mg2+ and M7P binding enhanced the stability of active site conformation of MtbHddA. Our study provides important insights into MtbHddA structure and activity, which can be targeted for therapeutic development against M. tuberculosis.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Magnésio/química , Mycobacterium tuberculosis/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfatos Açúcares/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Fosfatos Açúcares/metabolismo , Termodinâmica
13.
Int J Biol Macromol ; 171: 59-73, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33412199

RESUMO

Mycobacterium tuberculosis (M. tuberculosis H37Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include Vmax and Km for the hydrolysis of GTP by ftsY which were 20.25±5.16 µM/min/mg and 39.95±7.7 µM respectively. kcat and catalytic efficiency of the reaction were 0.012±0.003 s-1 and 0.00047±0.0001 µM/s-1 respectively. These values were affected upon changing the standard conditions. Cations (Mg2+ and Mn2+) play important role in FtsY enzymatic activity as increasing Mg2+ decrease the activity. Mn2+on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Mycobacterium tuberculosis/genética , Receptores Citoplasmáticos e Nucleares/química , Partícula de Reconhecimento de Sinal/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Cátions Bivalentes , Expressão Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Cinética , Magnésio/química , Magnésio/metabolismo , Manganês/química , Manganês/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium tuberculosis/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Transdução de Sinais , Especificidade por Substrato , Termodinâmica
14.
Biochemistry ; 60(4): 289-302, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33440120

RESUMO

Pseudokinases play important roles in signal transduction and cellular processes similar to those of catalytically competent kinases. However, pseudokinase pharmacological tractability and conformational space accessibility are poorly understood. Pseudokinases have only recently been suggested to adopt "inactive" conformations or interact with conformation-specific kinase inhibitors (e.g., type II compounds). In this work, the heavily substituted pseudokinase STRADα, which possesses a DFG → GLR substitution in the catalytic site that permits nucleotide binding while impairing divalent cation coordination, is used as a test case to demonstrate the potential applicability of conformation-specific, type II compounds to pseudokinase pharmacology. Integrated structural modeling is employed to generate a "GLR-out" conformational ensemble. Likely interacting type II compounds are identified through virtual screening against this ensemble model. Biophysical validation of compound binding is demonstrated through protein thermal stabilization and ATP competition. Localization of a top-performing compound through surface methylation strongly suggests that STRADα can adopt the "GLR-out" conformation and interact with compounds that comply with the standard type II pharmacophore. These results suggest that, despite a loss of catalytic function, some pseudokinases, including STRADα, may retain the conformational switching properties of conventional protein kinases.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Trifosfato de Adenosina/química , Humanos , Domínios Proteicos , Estabilidade Proteica
15.
Nat Commun ; 12(1): 121, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402676

RESUMO

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.


Assuntos
Coenzimas/química , Ditiocarb/análogos & derivados , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Nucleares/química , Compostos Organometálicos/química , Ubiquitina/química , Proteína com Valosina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Clonagem Molecular , Coenzimas/genética , Coenzimas/metabolismo , Microscopia Crioeletrônica , Dissulfiram/química , Dissulfiram/metabolismo , Ditiocarb/química , Ditiocarb/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compostos Organometálicos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Proteína com Valosina/antagonistas & inibidores , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Dedos de Zinco
16.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140544, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971286

RESUMO

Murine serine racemase (SR), the enzyme responsible for the biosynthesis of the neuromodulator d-serine, was reported to form a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in SR inhibition. In this work, we investigated the interaction between the two human orthologues. We were not able to observe neither the inhibition nor the formation of the SR-GAPDH complex. Rather, hSR is inhibited by the hGAPDH substrate glyceraldehyde 3-phosphate (G3P) in a time- and concentration-dependent fashion, likely through a covalent reaction of the aldehyde functional group. The inhibition was similar for the two G3P enantiomers but it was not observed for structurally similar aldehydes. We ruled out a mechanism of inhibition based on the competition with either pyridoxal phosphate (PLP) - described for other PLP-dependent enzymes when incubated with small aldehydes - or ATP. Nevertheless, the inhibition time course was affected by the presence of hSR allosteric and orthosteric ligands, suggesting a conformation-dependence of the reaction.


Assuntos
Trifosfato de Adenosina/química , Inibidores Enzimáticos/química , Gliceraldeído 3-Fosfato/química , Gliceraldeído-3-Fosfato Desidrogenases/química , Fosfato de Piridoxal/química , Racemases e Epimerases/química , 2,3-Difosfoglicerato/química , 2,3-Difosfoglicerato/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Domínio Catalítico , Clonagem Molecular , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Gliceraldeído/química , Gliceraldeído/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Fosfato de Piridoxal/metabolismo , Racemases e Epimerases/antagonistas & inibidores , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato
17.
Biochem J ; 478(1): 235-245, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33346350

RESUMO

Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. BpHldC is one of the essential enzymes in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei. Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of BpHldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and ßG1P. Analyses with EGCG homologs could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei.


Assuntos
Burkholderia pseudomallei/enzimologia , Flavonoides/química , Manose/química , Nucleotidiltransferases/química , Piranos/química , Trifosfato de Adenosina/química , Catequina/análogos & derivados , Catequina/química , Cristalografia por Raios X , Escherichia coli/metabolismo , Concentração Inibidora 50 , Cinética , Ligantes , Simulação de Acoplamento Molecular , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo
18.
Nat Commun ; 11(1): 6387, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318494

RESUMO

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.


Assuntos
Trifosfato de Adenosina/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleases/metabolismo , Trifosfato de Adenosina/química , Sítio Alostérico/efeitos dos fármacos , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Endorribonucleases/química , Técnicas de Inativação de Genes , Humanos , Ligantes , Modelos Moleculares , Fosforilação , Conformação Proteica , Dobramento de Proteína , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Ribonucleases/química , Resposta a Proteínas não Dobradas
19.
PLoS One ; 15(12): e0243747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315907

RESUMO

Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.


Assuntos
Luciferina de Vaga-Lumes/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Trifosfato de Adenosina/química , Alquilação , Indicadores e Reagentes , Luciferases de Vaga-Lume/química , Especificidade por Substrato , Temperatura
20.
J Phys Chem Lett ; 11(21): 9144-9151, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33052685

RESUMO

The raging COVID-19 pandemic caused by SARS-CoV-2 has infected tens of millions of people and killed several hundred thousand patients worldwide. Currently, there are no effective drugs or vaccines available for treating coronavirus infections. In this study, we have focused on the SARS-CoV-2 helicase (Nsp13), which is critical for viral replication and the most conserved nonstructural protein within the coronavirus family. Using homology modeling that couples published electron-density with molecular dynamics (MD)-based structural refinements, we generated structural models of the SARS-CoV-2 helicase in its apo- and ATP/RNA-bound conformations. We performed virtual screening of ∼970 000 chemical compounds against the ATP-binding site to identify potential inhibitors. Herein, we report docking hits of approved human drugs targeting the ATP-binding site. Importantly, two of our top drug hits have significant activity in inhibiting purified recombinant SARS-CoV-2 helicase, providing hope that these drugs can be potentially repurposed for the treatment of COVID-19.


Assuntos
Antivirais/química , Betacoronavirus/enzimologia , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Antivirais/metabolismo , Antivirais/uso terapêutico , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , RNA Helicases/química , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
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