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1.
Biomolecules ; 10(9)2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32967116

RESUMO

We report the results of our in silico study of approved drugs as potential treatments for COVID-19. The study is based on the analysis of normal modes of proteins. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). We applied the tools we developed and standard tools used in the structural biology community. Our results indicate that small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic sites over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Pneumonia Viral/tratamento farmacológico , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Antivirais/química , Antivirais/uso terapêutico , Betacoronavirus , Sítios de Ligação , Cloroquina/química , Cloroquina/farmacologia , Infecções por Coronavirus/prevenção & controle , Reposicionamento de Medicamentos , Eflornitina/química , Eflornitina/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ivermectina/química , Ivermectina/farmacologia , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias/prevenção & controle , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/efeitos dos fármacos , Pneumonia Viral/prevenção & controle , Prolina/análogos & derivados , Prolina/química , Prolina/farmacologia , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Receptores da Glicina/química , Receptores da Glicina/efeitos dos fármacos , Saposinas/química , Saposinas/efeitos dos fármacos , Sofosbuvir/química , Sofosbuvir/farmacologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , Relação Estrutura-Atividade
2.
Science ; 369(6510): 1501-1505, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32703906

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).


Assuntos
Substituição de Aminoácidos , Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Humanos , Prolina/química , Domínios Proteicos , Estabilidade Proteica , Vacinas Virais/química
3.
Proc Natl Acad Sci U S A ; 117(25): 14158-14167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513738

RESUMO

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 µM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 µM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.


Assuntos
Prolina/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte Vesicular/química , Humanos , Modelos Moleculares , Prolina/metabolismo , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
4.
Food Chem ; 329: 126775, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512387

RESUMO

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.


Assuntos
Colágeno Tipo I/química , Hidroxiprolina/química , Prolina/química , Serina/química , Animais , Dicroísmo Circular , Peixes , Desnaturação Proteica , Temperatura
5.
Food Chem ; 327: 127057, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32464461

RESUMO

Chilling injury (CI) restricts the quality and shelf life of bell pepper fruits; reducing these CI-induced detrimental effects is therefore of high economic and agricultural relevance. Here, we investigated the effects of trisodium phosphate (TSP), salicylic acid (SA), and TSP + SA treatments on pepper fruits under cold stress at 4 °C for 25 d. Combined TSP + SA treatment performed an optimal effect. Specifically, TSP + SA treatment enhanced fatty-acid desaturation efficiency, as indicated by the increased expression of key fatty acid desaturase genes, and higher content of unsaturated fatty acids. Meanwhile, TSP + SA treatment inhibited the CI-induced membrane damage, manifested as lower electrolyte leakage and malondialdehyde content. Furthermore, low field-nuclear magnetic resonance and proline content also revealed that TSP + SA treatment mitigated CI through enhancing water retention in pepper fruits. Collectively, our results may shed new light on optimizing the low-temperature storage conditions of post-harvest peppers.


Assuntos
Capsicum/química , Ácidos Graxos/química , Fosfatos/química , Ácido Salicílico/química , Água/química , Capsicum/efeitos dos fármacos , Capsicum/metabolismo , Parede Celular/efeitos dos fármacos , Temperatura Baixa , Ácidos Graxos/metabolismo , Frutas/química , Frutas/efeitos dos fármacos , Frutas/metabolismo , Malondialdeído/química , Malondialdeído/metabolismo , Permeabilidade/efeitos dos fármacos , Fosfatos/farmacologia , Prolina/química , Ácido Salicílico/farmacologia
6.
Chem Biol Interact ; 324: 109092, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32278739

RESUMO

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70 µM, compared to 13 µM for HuBChE. The kcat was about 100,000 min-1 for MaBChE and 30,000 min-1 for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05 µM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5 × 107 M-1 min-1 for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4 M-1 min-1, but was 380 fold faster for DFP-inhibited HuBChE (kr 531 M-1 min-1). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8-fold (kr 92 M-1 min-1) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2 M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.


Assuntos
Butirilcolinesterase/química , Reativadores da Colinesterase/química , Compostos de Pralidoxima/química , Prolina/química , Sequência de Aminoácidos , Animais , Inibidores da Colinesterase/farmacologia , Humanos , Cinética , Macaca , Macaca mulatta , Fenotiazinas/farmacologia , Alinhamento de Sequência
7.
Food Chem ; 319: 126598, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32182540

RESUMO

Collagens are large structural proteins that are prevalent in mammalian connective tissue. Peptides designed to include a glycine-proline-hydroxyproline (GPO) amino acid triad are biomimetic analogs of the collagen triple helix, a fold that is a hallmark of collagen-like sequences. To inform the rational engineering of collagen-like peptides and proteins for food systems, we report the crystal structure of the (GPO)10 peptide at 0.89-Å resolution, solved using direct methods. We determined that a single chain in the asymmetric unit forms a pseudo-hexagonal network of triple helices that have a pitch variation consistent with the model 7/2 helix (3.5 residues per turn). The proline rings occupied one of two states, while the helix was found to have a well-defined hydration shell involved in the stabilization of the inter-helix crystal network. This structure offers a new high-resolution basis for understanding the hierarchical assembly of native collagens, which will aid the food industry in engineering new sustainable food systems.


Assuntos
Colágeno/química , Prolina/química , Cristalografia por Raios X , Glicina , Hidroxiprolina/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica
8.
Food Chem ; 320: 126665, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32220705

RESUMO

The effects of using proline to solubilise fish myosin under low ionic strength conditions were studied. After solubilising myosin in 0.1 M NaCl containing 5, 10, 15, and 20 mM proline, respectively, it was observed that more than 80% of the myosin was effectively solubilised using 10 mM proline. The addition of 10 mM proline lowered the surface hydrophobicity of myosin from 18.25 to 8.22 mg/g, increased the amount of ß-sheet structure from 33.87% to 46.88%, both of which facilitated solubilisation. As revealed by transfer free energy measurements, the interactions between proline and tyrosine and tryptophan residues were more favourable. Furthermore, the ability of proline to shield hydrophobic sites of myosin and to partially break disulphide bonds helped to form myosin oligomer aggregates. Transmission electron microscopy images verified the effects of proline on myosin proteins. A solubilisation mechanism based mainly on chemical interactions between myosin and proline was proposed.


Assuntos
Proteínas de Peixes da Dieta/química , Miosinas/química , Prolina/química , Tilápia , Animais , Dissulfetos/química , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Concentração Osmolar , Solubilidade , Triptofano , Tirosina
9.
Chemistry ; 26(22): 5070-5074, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32043659

RESUMO

Cγ -substituted proline derivatives are valuable tools for developing functionalized collagen peptides for biological and materials investigations, yet the stereochemistry at Cγ can produce undesired steric or stereoelectronic constraints. Alkylated γ-azaproline (γ-azPro) derivatives are proline mimetics that lack a stereogenic center at the γ-position of the ring and can thus utilize the invertibility of nitrogen to adapt their conformation. NMR spectroscopic analyses and DFT calculations highlighted how alkylated γ-azPro derivatives are conformationally dynamic and adopt conformational preferences through ring pucker flip along with nitrogen inversion. Lastly, incorporation of alkylated γ-azPro into collagen peptides produced functionalized pH-responsive triple helices with similar thermal stabilities, regardless of their placement in the Xaa or Yaa position within the characteristic Xaa-Yaa-Gly repeating unit of collagen peptides.


Assuntos
Compostos Aza/química , Colágeno/química , Peptídeos/química , Prolina/química , Alquilação , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética
10.
Org Biomol Chem ; 18(10): 1881-1885, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32100807

RESUMO

A convenient two-step method is reported for the ligation of alkoxyamine- or hydrazine-bearing cargo to proline N-termini. Using this approach, bifunctional proline N-terminal bioconjugates are constructed and proline N-terminal proteins are immobilized.


Assuntos
Aminas/química , Hidrazinas/química , Prolina/química , Proteínas/síntese química , Hidrazonas/síntese química , Cetonas/síntese química , Oxirredução , Oximas/síntese química , Pyrococcus furiosus/química , Vírus do Mosaico do Tabaco/química
11.
Biochemistry ; 59(4): 582-593, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31895557

RESUMO

Gid4, a subunit of the ubiquitin ligase GID, is the recognition component of the Pro/N-degron pathway. Gid4 targets proteins in particular through their N-terminal (Nt) proline (Pro) residue. In Saccharomyces cerevisiae and other Saccharomyces yeasts, the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 bear Nt-Pro and are conditionally destroyed by the Pro/N-degron pathway. However, in mammals and in many non-Saccharomyces yeasts, for example, in Kluyveromyces lactis, these enzymes lack Nt-Pro. We used K. lactis to explore evolution of the Pro/N-degron pathway. One question to be addressed was whether the presence of non-Pro Nt residues in K. lactis Fbp1, Icl1, and Mdh2 was accompanied, on evolutionary time scales (S. cerevisiae and K. lactis diverged ∼150 million years ago), by a changed specificity of the Gid4 N-recognin. We used yeast-based two-hybrid binding assays and protein-degradation assays to show that the non-Pro (Ala) Nt residue of K. lactis Fbp1 makes this enzyme long-lived in K. lactis. We also found that the replacement, through mutagenesis, of Nt-Ala and the next three residues of K. lactis Fbp1 with the four-residue Nt-PTLV sequence of S. cerevisiae Fbp1 sufficed to make the resulting "hybrid" Fbp1 a short-lived substrate of Gid4 in K. lactis. We consider a blend of quasi-neutral genetic drift and natural selection that can account for these and related results. To the best of our knowledge, this work is the first study of the ubiquitin system in K. lactis, including development of the first protein-degradation assay (based on the antibiotic blasticidin) suitable for use with this organism.


Assuntos
Kluyveromyces/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Evolução Molecular , Frutose-Bifosfatase/química , Frutose-Bifosfatase/metabolismo , Gluconeogênese/genética , Kluyveromyces/enzimologia , Kluyveromyces/genética , Malato Desidrogenase/metabolismo , Mutagênese , Prolina/química , Proteólise , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia
12.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941785

RESUMO

Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV's transcription factors.IMPORTANCE Protein condensates can be assembled via liquid-liquid phase separation (LLPS), a process involving the concentration of molecules in a confined liquid-like compartment. LLPS allows for the compartmentalization and sequestration of materials and can be harnessed as a sensitive strategy for responding to small changes in the environment. This study identified the Epstein-Barr virus (EBV) proteins EBNA2 and EBNALP, which mediate virus and cellular gene transcription, as transcription factors that can form liquid-like condensates at superenhancer sites of MYC and Runx3. This study discovered the first identified LLPS of EBV proteins and emphasized the importance of LLPS in controlling host gene expression.


Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/química , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Virais/química , Linhagem Celular Tumoral , Nucléolo Celular/química , Núcleo Celular , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Genes myc , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Leucócitos Mononucleares , Microscopia de Fluorescência , Prolina/química , Regiões Promotoras Genéticas , Domínios Proteicos
13.
Chemistry ; 26(14): 3049-3053, 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-31961029

RESUMO

A general and powerful method for the stereo-controlled Pd-catalyzed N-allylation of amino acid esters is reported, as a previously largely unsolved synthetic challenge. Employing a new class of tartaric acid-derived C2 -symmetric chiral diphosphane ligands the developed asymmetric amination protocol allows the conversion of various amino acid esters to the N-allylated products with highest levels of enantio- or diastereoselectivity in a fully catalyst-controlled fashion and predictable configuration. Remarkably, the in situ generated catalysts also exhibit outstanding levels of activity (ligand acceleration). The usefulness of the method was demonstrated in the stereo-divergent synthesis of a set of new conformationally defined dipeptide mimetics, which represent new modular building blocks for the development of peptide-inspired bioactive compounds.


Assuntos
Aminoácidos/química , Dipeptídeos/síntese química , Ésteres/química , Paládio/química , Alanina/química , Catálise , Cristalografia por Raios X , Reação de Cicloadição , Ligantes , Modelos Moleculares , Estrutura Molecular , Oxirredução , Prolina/química , Estereoisomerismo
14.
Biochim Biophys Acta Biomembr ; 1862(2): 183149, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816324

RESUMO

Viral protein R (Vpr) is a small accessory protein of 96 amino acids that is present in Human and simian immunodeficiency viruses. Among the very different properties that Vpr possesses we can find cell penetrating capabilities. Based on this and on its capacity to interact with nucleic acids we previously investigated the DNA transfection properties of Vpr and subfragments thereof. We found that fragments of the C-terminal helical domain of Vpr are able to deliver efficiently plasmid DNA into different cell lines. As the amphipathic helix may play a role in the interactions with membranes, we investigated whether insertion of a proline residue in the α-helix modifies the transfection properties of Vpr. Unexpectedly, we found that the resulting Vpr55-82 Pro70 peptide was even more efficient than wild type Vpr55-82 in the gene delivery assays. Using circular dichroism, light scattering and solid-state NMR techniques, we characterized the secondary structure and interactions of Vpr and several mutants with model membranes. A model is proposed where the proline shifts the dissociation equilibrium of the peptide-cargo complex and thereby its endosomal release.


Assuntos
Peptídeos Penetradores de Células/química , Técnicas de Transferência de Genes , Bicamadas Lipídicas/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Substituição de Aminoácidos , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Células HEK293 , HIV-1/química , Humanos , Isoleucina/química , Isoleucina/genética , Prolina/química , Prolina/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo
15.
Phytochemistry ; 169: 112165, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31610323

RESUMO

Cyclin dependent kinase A; 1 (CDKA; 1) is essential in G1/S transition of cell cycle and its oxidation has been implicated in cell cycle arrest during plant abiotic stress. In the present study, an evaluation at the molecular level was performed to find possible sites of protein oxidative modifications. In vivo studies demonstrated that carbonylation of maize CDKA,1 is associated with a decrease in complex formation with maize cyclin D (CycD). Control and in vitro oxidized recombinant CDKA; 1 were sequenced by mass spectrometry. Proline at the PSTAIRE cyclin-binding motif was identified as the most susceptible oxidation site by comparative analysis of the resulted peptides. The specific interaction between CDKA; 1 and CycD6; 1, measured by surface plasmon resonance (SPR), demonstrated that the affinity and the kinetic of the interaction depended on the reduced-oxidized state of the CDKA; 1. CDKA; 1 protein oxidative modification would be in part responsible for affecting cell cycle progression, and thus producing plant growth inhibition under oxidative stress.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Prolina/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclinas/química , Modelos Moleculares , Oxirredução , Prolina/química , Alinhamento de Sequência
16.
Biosci Biotechnol Biochem ; 84(1): 1-16, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31538538

RESUMO

Recent investigations suggest that soluble oligomeric amyloid ß (Aß) species may be involved in early onset of Alzheimer's disease (AD). Using systematic proline replacement, solid-state NMR, and ESR, we identified a toxic turn at position 22 and 23 of Aß42, the most potent neurotoxic Aß species. Through radicalization, the toxic turn can induce formation of the C-terminal hydrophobic core to obtain putative Aß42 dimers and trimers. Synthesized dimer and trimer models showed that the C-terminal hydrophobic core plays a critical role in the formation of high molecular weight oligomers with neurotoxicity. Accordingly, an anti-toxic turn antibody (24B3) that selectively recognizes a toxic dimer model of E22P-Aß42 was developed. Sandwich enzyme-linked immunosorbent assay with 24B3 and 82E1 detected a significantly higher ratio of Aß42 with a toxic turn to total Aß42 in cerebrospinal fluid of AD patients compared with controls, suggesting that 24B3 could be useful for early onset of AD diagnosis.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/uso terapêutico , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/imunologia , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/química , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Modelos Moleculares , Emaranhados Neurofibrilares/química , Fragmentos de Peptídeos/química , Placa Amiloide/química , Prolina/química , Agregados Proteicos , Agregação Patológica de Proteínas , Estrutura Terciária de Proteína
17.
Crit Rev Biotechnol ; 40(1): 83-98, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31690132

RESUMO

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit-subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted.


Assuntos
Estabilidade Enzimática , Engenharia de Proteínas , Ciclização , Glicina/química , Glicosilação , Prolina/química , Conformação Proteica , Subunidades Proteicas , Temperatura
18.
J Colloid Interface Sci ; 559: 76-87, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610307

RESUMO

Exploring and controlling chemical reactions in compartments opens new platforms for designing bioinspired catalysts and energy-autonomous systems. Aqueous polymer networks or hydrogels serve as a perfect model for biological tissues, allowing systematic investigations of chemical transformations in compartments. Herein, we report the synthesis of a versatile, colloidal microgel catalyst containing covalently bound l-proline as an organocatalyst. The key finding of our work is that the catalytic activity can be tuned by adjusting the distribution of the organocatalyst in the microgel network as well as the properties of the solvent. We demonstrate that l-proline groups integrated into microgels enable the reaction of 4-nitrobenzaldehyde and cyclohexanone in a heterogeneous reaction mixture in which free l-proline is not active. By controlling the localization of the l-proline groups within the microgel network (core or corona), the rate of the aldol reaction in homogenous and heterogeneous reaction mixtures can be modulated. Furthermore, microgels with covalently attached catalysts can be recycled and reused in sequential catalytic runs without deterioration of the catalyst performance in terms of activity and selectivity. The internal structure of the microgel in heterogeneous reaction mixtures was studied by computer simulations.


Assuntos
Materiais Biomiméticos/química , Coloides/química , Hidrogéis/química , Prolina/química , Aldeídos/química , Benzaldeídos/química , Catálise , Simulação por Computador , Reagentes para Ligações Cruzadas/química , Cicloexanonas/química , Cinética , Polimerização , Solventes/química , Água
19.
Toxicol In Vitro ; 62: 104679, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31676337

RESUMO

Ruthenium complexes are being considered as novel chemotherapeutic alternatives for cancer treatment. In our study, we assessed the antitumoral activities of novel ruthenium complexes coupled to the amino acids proline (RuPro) and threonine (RuThr) in prostate tumor cell lines (DU145) and breast (MCF7), and normal cell lines of the lung fibroblast (GM07492A). Our results revealed that the EC50 of the complexes for DU145 and MCF7 was two times lower than that GM07492A. Moreover, RuPro and RuThr were not able to induce significant genomic instability, cell cycle arrest or cell death in GM07492A, but could induce DNA damage, arrest in G2/M and apoptosis in DU145 and MCF7. Furthermore, BAX, TP53 and ATM were found to be upregulated in DU145 and MCF7 treated with RuPro and RuThr, in which, a higher ASCT2 gene expression was also observed. Using molecular docking, RuPro and RuThr interact with ASCT2, suggesting that this transporter might have a pivotal role in the execution of their activities. Hence, our results with RuPro and RuThr are capable of selectively inducing genetic damage, cell cycle arrest and apoptosis in DU145 and MCF7. We suggest that the selective action of the RuPro and RuThr complexes is related to the higher expression of ASCT2 in the tumor cells.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quelantes/farmacologia , Instabilidade Genômica/efeitos dos fármacos , Prolina/química , Neoplasias da Próstata/tratamento farmacológico , Compostos de Rutênio/farmacologia , Treonina/química , Sistema ASC de Transporte de Aminoácidos/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Ligantes , Masculino , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Simulação de Acoplamento Molecular , Neoplasias da Próstata/patologia
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