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1.
Nat Commun ; 12(1): 761, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536412

RESUMO

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.


Assuntos
Arginina/metabolismo , Membrana Celular/metabolismo , Fusão de Membrana , Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Animais , Arginina/química , Sítios de Ligação , Cálcio/metabolismo , Neurotransmissores/metabolismo , Ligação Proteica , Domínios Proteicos , Ratos , Proteínas SNARE/química , Sinaptotagmina I/química
2.
Nat Commun ; 12(1): 796, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542236

RESUMO

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Desoxirribose/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
3.
Nat Commun ; 12(1): 1055, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594058

RESUMO

mTORC1, a central controller of cell proliferation in response to growth factors and nutrients, is dysregulated in cancer. Whereas arginine activates mTORC1, it is overridden by high expression of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1). Because cancer cells often encounter low levels of nutrients, an alternative mechanism might exist to regulate CASTOR1 expression. Here we show K29-linked polyubiquitination and degradation of CASTOR1 by E3 ubiquitin ligase RNF167. Furthermore, AKT phosphorylates CASTOR1 at S14, significantly increasing its binding to RNF167, and hence its ubiquitination and degradation, while simultaneously decreasing its affinity to MIOS, leading to mTORC1 activation. Therefore, AKT activates mTORC1 through both TSC2- and CASTOR1-dependent pathways. Several cell types with high CASTOR1 expression are insensitive to arginine regulation. Significantly, AKT and RNF167-mediated CASTOR1 degradation activates mTORC1 independent of arginine and promotes breast cancer progression. These results illustrate a mTORC1 regulating mechanism and identify RNF167 as a therapeutic target for mTORC1-dysregulated diseases.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Arginina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Cinética , Lisina/metabolismo , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação/efeitos dos fármacos
4.
Nat Commun ; 12(1): 872, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558506

RESUMO

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases-homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Sequência de Bases , Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Ligação a RNA/química , Tensão Superficial
5.
Nat Commun ; 12(1): 807, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547325

RESUMO

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.


Assuntos
Apoproteínas/química , Cálcio/química , Calmodulina/química , Hipertermia Maligna/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Substituição de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Arginina/química , Arginina/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Cisteína/metabolismo , Expressão Gênica , Humanos , Transporte de Íons , Hipertermia Maligna/genética , Hipertermia Maligna/patologia , Modelos Moleculares , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Mutação , 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 , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Suínos
6.
Monogr Oral Sci ; 29: 80-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33427222

RESUMO

Untreated dental caries is the most prevalent disease worldwide. Development of caries is associated with the intake of sugar. The microorganisms utilize the sugar and create an acidic environment, which results in mineral loss. The appropriate use of fluoride is associated with a decline of caries. Another approach in preventing caries might be the increase of pH in dental plaque. During recent years, arginine has increasingly become the focus of interest. This is based on the fact that certain streptococci possess an arginine deiminase system (ADS) which metabolizes free arginine. In vivo, the incidence of caries was inversely correlated with ADS activity in saliva and dental plaque. ADS is highly active in Streptococcus gordonii and S. sanguinis, but is absent in S. sobrinus and S. mutans. In the presence of 1.5% L-arginine, S. gordonii and S. sanguinis, but not S. mutans and S. sobrinus, synthesize the metabolite citrulline and increase the pH of the environment.In defined multispecies biofilms consisting of ADS-positive and ADS-negative streptococci, supplementation with 1.5% arginine suppressed the growth of ADS-negative by favoring ADS-positive streptococci together with an increase in the pH of the environment. Evaluating the influence of daily manual removal of the biofilm in vitro by brushing with a commercial toothpaste containing fluoride and arginine resulted in less surface microhardness even when compared with a toothpaste with fluoride only. Recent studies clinically investigated the effect of using an arginine-containing dentifrice and reported a decrease of DMFS by about 10-20%.


Assuntos
Cárie Dentária , Arginina , Biofilmes , Cárie Dentária/prevenção & controle , Humanos , Streptococcus mutans , Streptococcus sanguis
7.
Ecotoxicol Environ Saf ; 208: 111748, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396074

RESUMO

Microcystin-leucine arginine (MC-LR) is a kind of toxin produced by cyanobacterial, resulting in decrease of testosterone levels in serum and leading to impaired spermatogenesis. Gonadotropin-releasing hormone (GnRH) neurons play crucial roles in the regulation of testosterone release. Meanwhile, it has been demonstrated that MC-LR is capable of entering the GnRH neurons and inducing apoptosis. Nevertheless, the molecular mechanism of MC-LR induced apoptosis of GnRH neurons remains elusive. In present study, we found that MC-LR inhibited the cell viability of GT1-7 cells. In addition, we discovered apoptosis of GnRH neurons and GT1-7 cells treated with MC-LR. And increased intracellular ROS production and the release of intracellular Ca2+ were all observed following exposure to MC-LR. Furthermore, we also found the endoplasmic reticulum stress (ERs) and autophagy were activated by MC-LR. Additionally, pretreatment of the ERs inhibitor (4-Phenyl butyric acid) reduced the apoptotic rate of GT1-7 cells comparing with MC-LR exposure alone. Comparing with MC-LR treatment alone, apoptotic cell death was increased by pretreatment of GT1-7 cells with an autophagy inhibitor (3-methyladenine). Together, our data implicated that the treatment of MC-LR induced the apoptosis of GnRH neurons by activating the ERs resulting in a decrease of serum testosterone level in mice. Autophagy is a protective cellular process which was activated by ER stress and thus protected cells from apoptosis upon MC-LR exposure.


Assuntos
Estresse do Retículo Endoplasmático , Microcistinas/toxicidade , Testosterona/sangue , Animais , Apoptose , Arginina/metabolismo , Bioensaio , Sobrevivência Celular , Cianobactérias/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Leucina/metabolismo , Masculino , Toxinas Marinhas/metabolismo , Camundongos , Microcistinas/metabolismo , Neurônios/metabolismo , Testosterona/metabolismo
8.
Nat Commun ; 12(1): 407, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33462223

RESUMO

The exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili.


Assuntos
Nanotubos de Peptídeos/ultraestrutura , Arginina/química , Arginina/genética , Microscopia Crioeletrônica , Modelos Moleculares , Nanotubos de Peptídeos/química , Conformação Proteica em alfa-Hélice , Relação Estrutura-Atividade
9.
Phys Chem Chem Phys ; 23(2): 1706-1717, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33427255

RESUMO

The use of water as a component of deep eutectic systems (DES) has raised some questions regarding its influence on the nature of the mixture. Does it form a DES or an aqueous solution and what is the role of water? In this work, the nature of citric acid:l-arginine:water mixtures was explored through phase equilibria studies and spectroscopic analysis. In a first step, PC-SAFT was validated as a predictive tool to model the water influence on the solid liquid equilibria (SLE) of the DES reline using the individual-component approach. Hence, activity coefficients in the ternary systems citric acid:l-arginine:water and respective binary combinations were studied and compared using ePC-SAFT. It was observed that the water-free mixtures citric acid:l-arginine showed positive deviation from Raoult's law, while upon addition of water strong negative deviation from Raoult's law was found, yielding melting depressions around 100 K. Besides these strong interactions, pH was found to become acidic (pH = 3.5) upon water addition, which yields the formation of charged species ([H2Cit]- and [l-arg]+). Thus, the increased interactions between the molecules upon water addition might be caused by several mechanisms such as hydrogen bonding or ionic forces, both being induced by water. For further investigation, the liquid mixtures citric acid:l-arginine:water were studied by FTIR and NMR spectroscopy. FTIR spectra disproved a possible solubility enhancement caused by salt formation between citric acid and l-arginine, while NMR spectra supported the formation of a hydrogen bonding network different from the binary systems citric acid:water and l-arginine:water. Either being a DES or other type of non-ideal solution, the liquefaction of the studied systems is certainly caused by a water-mediator effect based on the formation of charged species and cross interactions between the mixture constituents.


Assuntos
Arginina/química , Ácido Cítrico/química , Água/química , Cloreto de Cálcio/química , Congelamento , Modelos Químicos , Termodinâmica , Temperatura de Transição , Ureia/química
10.
Nucleic Acids Res ; 49(2): 928-953, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406258

RESUMO

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP's function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP's carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.


Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , Replicação do DNA , Endodesoxirribonucleases/fisiologia , Processamento de Proteína Pós-Traducional , Sumoilação , Substituição de Aminoácidos , Arginina/química , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Genes Reporter , Instabilidade Genômica , Humanos , Lisina/química , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Inibidoras de STAT Ativados/fisiologia , Mapeamento de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Reparo de DNA por Recombinação/genética , Reparo de DNA por Recombinação/fisiologia
11.
Food Chem ; 338: 128055, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32950008

RESUMO

This study examined the ability of l-arginine, l-cysteine and l-methionine, to inhibit postharvest senescence of broccoli. Florets were dipped in aqueous solutions of the amino acids at concentrations from 1.0 to 100 mM and stored at 10 °C. A 5 mM dip was found to be optimal in delaying senescence as measured by retention of green colour, vitamin C and antioxidant activity, and a lower level of ethylene production, respiration, weight loss, phenylalanine ammonia lyase (PAL) activity and ion leakage with the benefits being similar for all three amino acids. Arginine, cysteine and methionine have Generally Recognised As Safe (GRAS) status and should have few impediments in obtaining regulatory approval for commercial use if similar effects were found for other leafy vegetables.


Assuntos
Arginina/farmacologia , Brassica/efeitos dos fármacos , Cisteína/farmacologia , Metionina/farmacologia , Proteínas de Plantas/metabolismo , Amônia-Liases/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Brassica/metabolismo , Etilenos/metabolismo , Fatores de Tempo
12.
Biochim Biophys Acta Gen Subj ; 1865(1): 129776, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33127433

RESUMO

BACKGROUND: Protein arginine methyltransferase 1 (PRMT1), a major type I arginine methyltransferase in mammals, methylates histone and non-histone proteins to regulate various cellular functions such as transcription, DNA damage response, and signal transduction. SCOPE OF REVIEW: This review summarizes previous and recent studies on PRMT1 functions in major cell types of the central nervous system. We also discuss the potential involvement of PRMT1 in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. Also, we raise key questions that must be addressed in the future to more precisely understand the roles of PRMT1. MAJOR CONCLUSIONS: Recent studies revealed that PRMT1 is essential for the development of neurons, astrocytes, and oligodendrocytes, although further investigation using cell type-specific PRMT1-deficient animals is required. In addition, the relevance of PRMT1 in neurodegenerative diseases will continue to be a hot topic. GENERAL SIGNIFICANCE: PRMT1 is important for neural development and neurodegenerative diseases.


Assuntos
Arginina/metabolismo , Encéfalo/crescimento & desenvolvimento , Doenças Neurodegenerativas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Arginina/análogos & derivados , Arginina/genética , Encéfalo/metabolismo , Encéfalo/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Metilação , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética
13.
Phys Chem Chem Phys ; 23(1): 415-424, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33319872

RESUMO

Proteins are the most abundant biomacromolecules in living cells, where they perform vital roles in virtually every biological process. To maintain their function, proteins need to remain in a stable (native) state. Inter- and intramolecular interactions in aqueous protein solutions govern the fate of proteins, as they can provoke their unfolding or association into aggregates. The initial steps of protein aggregation are difficult to capture experimentally, therefore we used molecular dynamics simulations in this study. We investigated the initial phase of aggregation of two different lysozymes, hen egg-white (HEWL) and T4 WT* lysozyme and also human lens γ-D crystallin by using atomistic simulations. We monitored the phase stability of their aqueous solutions by calculating time-dependent density fluctuations. We found that all proteins remained in their compact form despite aggregation. With an extensive analysis of intermolecular residue-residue interactions we discovered that arginine is of paramount importance in the initial stage of aggregation of HEWL and γ-D crystallin, meanwhile lysine was found to be the most involved amino acid in forming initial contacts between T4 WT* molecules.


Assuntos
Muramidase/metabolismo , Multimerização Proteica , gama-Cristalinas/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Bacteriófago T4/química , Galinhas , Humanos , Lisina/química , Simulação de Dinâmica Molecular , Muramidase/química , Ligação Proteica , Temperatura , gama-Cristalinas/química
14.
Arch Biochem Biophys ; 698: 108716, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33309545

RESUMO

The effects of phosphorylation of histone H3 at serine 10 have been studied in the context of other posttranslational modifications such as lysine methylation. We set out to investigate the impact of phosphoserine-10 on arginine-8 methylation. We performed methylation reactions using peptides based on histone H3 that contain a phosphorylated serine and compared the extent of arginine methylation with unmodified peptides. Results obtained via fluorography indicate that peptides containing a phosphorylated serine-10 inhibit deposition of methyl groups to arginine-8 residues. To further explore the effects of phosphoserine on neighboring arginine residues, we physically characterized the non-covalent interactions between histone H3 phosphoserine-10 and arginine-8 using 31P NMR spectroscopy. A salt bridge was detected between the negatively charged phosphoserine-10 and the positively charged unmodified arginine-8 residue. This salt bridge was not detected when arginine-8 was symmetrically dimethylated. Finally, molecular simulations not only confirm the presence of a salt bridge but also identify a subset of electrostatic interactions present when arginine is replaced with alanine. Taken together, our work suggests that the negatively charged phosphoserine maximizes its interactions. By limiting its exposure and creating new contacts with neighboring residues, it will inhibit deposition of neighboring methyl groups, not through steric hindrance, but by forming intrapeptide interactions that may mask substrate recognition. Our work provides a mechanistic framework for understanding the role of phosphoserine on nearby amino acid residues and arginine methylation.


Assuntos
Arginina/metabolismo , Histonas/metabolismo , Fosfosserina/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina/química , Histonas/química , Humanos , Metilação , Simulação de Dinâmica Molecular , Fosfosserina/química , Eletricidade Estática , Xenopus laevis
15.
Nucleic Acids Res ; 49(2): 1033-1045, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367793

RESUMO

Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.


Assuntos
Adenina , Bacteriófagos/genética , Mutagênese , Retroelementos/genética , Adenina/química , Arginina/química , Sequência de Bases , Bordetella/virologia , Catálise , Sistema Livre de Células , Simulação por Computador , DNA Complementar/genética , Glicina/química , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Moleculares , Conformação Proteica , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/metabolismo
16.
Nucleic Acids Res ; 49(2): 1163-1172, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367820

RESUMO

Transcription factor decoy binding sites are short DNA sequences that can titrate a transcription factor away from its natural binding site, therefore regulating gene expression. In this study, we harness synthetic transcription factor decoy systems to regulate gene expression for metabolic pathways in Escherichia coli. We show that transcription factor decoys can effectively regulate expression of native and heterologous genes. Tunability of the decoy can be engineered via changes in copy number or modifications to the DNA decoy site sequence. Using arginine biosynthesis as a showcase, we observed a 16-fold increase in arginine production when we introduced the decoy system to steer metabolic flux towards increased arginine biosynthesis, with negligible growth differences compared to the wild type strain. The decoy-based production strain retains high genetic integrity; in contrast to a gene knock-out approach where mutations were common, we detected no mutations in the production system using the decoy-based strain. We further show that transcription factor decoys are amenable to multiplexed library screening by demonstrating enhanced tolerance to pinene with a combinatorial decoy library. Our study shows that transcription factor decoy binding sites are a powerful and compact tool for metabolic engineering.


Assuntos
Sítios de Ligação , Regulação da Expressão Gênica/efeitos dos fármacos , Engenharia Metabólica/métodos , Mimetismo Molecular , Fatores de Transcrição/metabolismo , Arginina/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Monoterpenos Bicíclicos , Ligação Competitiva , Desenho de Fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dosagem de Genes , Genes Sintéticos , Repressores Lac/genética , Repressores Lac/metabolismo , Mutagênese , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética
17.
BMC Nephrol ; 21(1): 486, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198670

RESUMO

BACKGROUND: Critically ill coronavirus disease 2019 (COVID-19) patients have a high risk of acute kidney injury (AKI) that requires renal replacement therapy (RRT). A state of hypercoagulability reduces circuit life spans. To maintain circuit patency and therapeutic efficiency, an optimized anticoagulation strategy is needed. This study investigates whether alternative anticoagulation strategies for RRT during COVID-19 are superior to administration of unfractionated heparin (UFH). METHODS: Retrospective cohort study on 71 critically ill COVID-19 patients (≥18 years), admitted to intensive care units at a tertiary health care facility in the southwestern part of Germany between February 26 and May 21, 2020. We collected data on the disease course, AKI, RRT, and thromboembolic events. Four different anticoagulatory regimens were administered. Anticoagulation during continuous veno-venous hemodialysis (CVVHD) was performed with UFH or citrate. Anticoagulation during sustained low-efficiency daily dialysis (SLEDD) was performed with UFH, argatroban, or low molecular weight heparin (LMWH). Primary outcome is the effect of the anticoagulation regimen on mean treatment times of RRT. RESULTS: In patients receiving CVVHD, mean treatment time in the UFH group was 21.3 h (SEM: ±5.6 h), in the citrate group 45.6 h (SEM: ±2.7 h). Citrate anticoagulation significantly prolonged treatment times by 24.4 h (P = .001). In patients receiving SLEDD, mean treatment time with UFH was 8.1 h (SEM: ±1.3 h), with argatroban 8.0 h (SEM: ±0.9 h), and with LMWH 11.8 h (SEM: ±0.5 h). LMWH significantly prolonged treatment times by 3.7 h (P = .008) and 3.8 h (P = .002), respectively. CONCLUSIONS: UFH fails to prevent early clotting events in the dialysis circuit during COVID-19. For patients, who do not require effective systemic anticoagulation, regional citrate dialysis is the most effective strategy. For patients, who require effective systemic anticoagulation, the usage of LMWH results in the longest circuit life spans. The proposed anticoagulatory strategies are safe, can easily be monitored, and allow an individualized treatment.


Assuntos
Lesão Renal Aguda/terapia , Anticoagulantes/administração & dosagem , Betacoronavirus , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Terapia de Substituição Renal/métodos , Lesão Renal Aguda/sangue , Lesão Renal Aguda/epidemiologia , Adulto , Idoso , Arginina/análogos & derivados , Coagulação Sanguínea , Ácido Cítrico/administração & dosagem , Comorbidade , Infecções por Coronavirus/sangue , Cuidados Críticos , Estado Terminal , Falha de Equipamento , Feminino , Alemanha/epidemiologia , Heparina/administração & dosagem , Heparina de Baixo Peso Molecular/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Ácidos Pipecólicos/administração & dosagem , Pneumonia Viral/sangue , Terapia de Substituição Renal/instrumentação , Estudos Retrospectivos , Sulfonamidas , Centros de Atenção Terciária
18.
J Clin Pediatr Dent ; 44(5): 332-341, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33181840

RESUMO

OBJECTIVE(S): To: 1) examine the fluoride concentrations in commercial child formula dentifrices (CFD)s; and 2) investigate the effect of arginine incorporation in CFDs on fluoride bioavailability. STUDY DESIGN: Five commercial CFDs were examined for fluoride concentrations. Total, total soluble, and insoluble fluorides in CFDs were determined by the modified Taves acid-diffusion method (TAD). Ionic F and MFP were estimated by modified direct method with standard addition technique. L-arginine (L-Arg)/L-arginine monohydrochloride (L-Arg.HCl) were incorporated at 2% w/w in the commercial CFDs. The pH of the toothpaste slurries, buffer capacity of the added Arg, potentially available fluorides (PAF) and 1-min PAF by TAD were determined. RESULTS: The CFDs had 4 to 32% of insoluble fluorides. Addition of L-Arg/L-Arg.HCl significantly improved the fluoride bioavailability in CFDs (p<0.05). Incorporation of L-Arg significantly increased the pH of toothpaste slurries (p<0.05); while L-Arg.HCl decreased the pH. Principal component analysis showed that L-Arg.HCl decreased the pH of toothpaste slurries due to the presence of Cl in the form of HCl; whereas the inherent elements/molecules (Na/P/Pi/F) remain distinct with unidentified influence on the variables. CONCLUSION(S): The CFDs containing NaF only have higher concentrations of bioavailable fluoride. Incorporating arginine (L-arginine or L-arginine monohydrochloride) at 2% w/w improves fluoride bioavailability of the child formula dentifrices.


Assuntos
Dentifrícios , Fluoretos , Arginina , Disponibilidade Biológica , Cariostáticos , Criança , Humanos , Fosfatos , Fluoreto de Sódio , Cremes Dentais
19.
Yakugaku Zasshi ; 140(11): 1373-1380, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33132273

RESUMO

The treatment of acute ischemic stroke usually involves argatroban administration by continuous infusion for 2 d and by intravenous infusion twice a day for 5 d after that. However, the appropriate dose of argatroban to be administered is not clear. Therefore, no studies have been reported a comparison of intravenous and continuous argatroban infusion after day 3 for acute ischemic stroke patients. We aimed to identify the connection between differences in argatroban administration and worsening of symptoms after day 3 in ischemic stroke patients. We retrospectively evaluated the data of 107 ischemic stroke patients who received treatment with argatroban. The study endpoint was defined as the worsening of symptoms from days 3 to 7. Logistic regression analysis was used to determine the risk factors that were significantly associated with worsening of symptoms. Patients were administered argatroban, with rates of 72.0%, and 28.0% for continuous, and intravenous infusion, respectively. A total of 10 (9.3%) patients experienced worsening of symptoms. In the single logistic regression analysis, carotid stenosis [non-adjusted odds ratio (OR) 5.775, 95% confidence interval (CI) 1.486-22.442, p=0.011] was only significantly associated with worsening of symptoms. Worsening of symptoms was not related to either intravenous or continuous infusion group (16.7% vs. 6.5%, p=0.104). Bleeding was also not associated with either group (6.7% vs. 3.9%, p=0.618). We suggest that the differences in the mode of argatroban administration were not related to the worsening of symptoms in ischemic stroke patients. We also found that safety was equivalent regardless of the administration route.


Assuntos
Administração Intravenosa/métodos , Ácidos Pipecólicos/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Exacerbação dos Sintomas , Idoso , Idoso de 80 Anos ou mais , Arginina/análogos & derivados , Vias de Administração de Medicamentos , Feminino , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados da Assistência ao Paciente , Ácidos Pipecólicos/efeitos adversos , Estudos Retrospectivos , Segurança , Sulfonamidas
20.
Niger J Clin Pract ; 23(11): 1542-1547, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33221779

RESUMO

Background: The relationships among serum Apelin, Asymmetric- dimethylarginine (ADMA), N-terminal probrain natriureticpeptide (NT-proBNP) levels, and blood pressures in dialysis patients are not well known. Materials and Methods: Age and sex matched 30 hemodialysis (HD), 30 peritoneal dialysis (PD) patients, and 20 healthy controls were recruited. Serum apelin-36, ADMA, NT-proBNP levels, and blood pressures of both patients and healthy controls were measured and compared. Results: Serum ADMA and Apelin levels in HD patients were significantly higher than in PD patients. In multiple regression analyses the predictors of higher serum apelin levels were higher BMI, higher ADMA and lower systolic blood pressure. The predictors of serum ADMA levels were being on HD. The predictors of serum NT-proBNP levels were lower serum albumin and higher systolic blood pressure. Conclusion: Being on HD is a predictor of high ADMA levels. HD might be less effective on ADMA removal than PD. It seems that higher serum apelin levels related with lower sytolic blood pressure levels, whereas higher NT-proBNP levels related with higher sytolic blood pressure levels indicating potential roles as independent prognostic factors for systolic hypertension in dialysis patients.


Assuntos
Apelina/sangue , Arginina/análogos & derivados , Pressão Sanguínea/fisiologia , Falência Renal Crônica/sangue , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Adulto , Idoso , Arginina/sangue , Biomarcadores/sangue , Determinação da Pressão Arterial , Feminino , Humanos , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal , Diálise Renal
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