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1.
Genes (Basel) ; 11(9)2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32899439

RESUMO

The importance of host genetics and demography in coronavirus disease 2019 (COVID-19) is a crucial aspect of infection, prognosis and associated case fatality rate. Individual genetic landscapes can contribute to understand Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) burden and can give information on how to fight virus spreading and the associated severe acute respiratory distress syndrome (ARDS). The spread and pathogenicity of the virus have become pandemic on specific geographic areas and ethnicities. Interestingly, SARS-CoV-2 firstly emerged in East Asia and next in Europe, where it has caused higher morbidity and mortality. This is a peculiar feature of SARS-CoV-2, different from past global viral infections (i.e., SARS-1 or MERS); it shares with the previous pandemics strong age- and sex-dependent gaps in the disease outcome. The observation that the severest COVID-19 patients are more likely to have a history of hypertension, diabetes and/or cardiovascular disease and receive Renin-Angiotensin-System (RAS) inhibitor treatment raised the hypothesis that RAS-unbalancing may have a crucial role. Accordingly, we recently published a genetic hypothesis on the role of RAS-pathway genes (ACE1, rs4646994, rs1799752, rs4340, rs13447447; and ACE2, rs2285666, rs1978124, rs714205) and ABO-locus (rs495828, rs8176746) in COVID-19 prognosis, suspecting inherited genetic predispositions to be predictive of COVID-19 severity. In addition, recently, Genome-Wide Association Studies (GWAS) found COVID-19-association signals at locus 3p21.31 (rs11385942) comprising the solute carrier SLC6A20 (Na+ and Cl- coupled transporter family) and at locus 9q34.2 (rs657152) coincident with ABO-blood group (rs8176747, rs41302905, rs8176719), and interestingly, both loci are associated to RAS-pathway. Finally, ACE1 and ACE2 haplotypes seem to provide plausible explanations for why SARS-CoV-2 have affected more heavily some ethnic groups, namely people with European ancestry, than Asians.


Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Sistema Renina-Angiotensina/genética , Sistema ABO de Grupos Sanguíneos/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Haplótipos , Humanos , Proteínas de Membrana Transportadoras/genética , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Polimorfismo Genético , Prognóstico , Caracteres Sexuais
2.
Nat Commun ; 11(1): 3825, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732874

RESUMO

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.


Assuntos
Lipídeos , Malária Falciparum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/parasitologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Transporte Proteico , Vacúolos/metabolismo , Vacúolos/parasitologia
3.
Nat Commun ; 11(1): 3802, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732903

RESUMO

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Here, we kinetically dissect Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Desdobramento de Proteína , Canais de Translocação SEC/metabolismo , Proteínas SecA/metabolismo , Estresse Mecânico , Membrana Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/metabolismo , NADP/metabolismo , Transporte Proteico , Proteínas SecA/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
4.
PLoS Biol ; 18(8): e3000790, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776918

RESUMO

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.


Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Uridina/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Uridina/metabolismo
5.
Ecotoxicol Environ Saf ; 202: 110885, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32650140

RESUMO

Silicon (Si) is a metalloid which is gaining worldwide attention of plant scientists due to its ameliorating impact on plants' growth and development. The beneficial response of Si is observed predominantly under numerous abiotic and biotic stress conditions. However, under favorable conditions, most of the plant can grow without it. Therefore, Si has yet not been fully accepted as essential element rather it is being considered as quasi-essential for plants' growth. Si is also known to enhance resilience in plants by reducing the plant's stress. Besides its second most abundance on the earth crust, most of the soils lack plant available form of Si i.e. silicic acid. In this regard, understanding the role of Si in plant metabolism, its uptake from roots and transport to aerial tissues along with its ionomics and proteomics under different circumstances is of great concern. Plants have evolved a well-optimized Si-transport system including various transporter proteins like Low silicon1 (Lsi1), Low silicon2 (Lsi2), Low silicon3 (Lsi3) and Low silicon6 (Lsi6) at specific sub-cellular locations along with the expression profiling that creates precisely coordinated network among these transporters, which also facilitate uptake and accumulation of Si. Though, an ample amount of information is available pertinent to the solute specificity, active sites, transcriptional and post-transcriptional regulation of these transporter genes. Similarly, the information regarding transporters involved in Si accumulation in different organelles is also available particularly in silica cells occurred in poales. But in this review, we have attempted to compile studies related to plants vis à vis Si, its role in abiotic and biotic stress, its uptake in various parts of plants via different types of Si-transporters, expression pattern, localization and the solute specificity. Besides these, this review will also provide the compiled knowledge about the genetic variation among crop plants vis à vis enhanced Si uptake and related benefits.


Assuntos
Plantas/metabolismo , Silício/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Raízes de Plantas/metabolismo , Solo
6.
BMC Infect Dis ; 20(1): 513, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677899

RESUMO

BACKGROUND: Imported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China. METHOD: Fifty-five imported falciparum malaria cases in Guizhou Province during 2012-2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software. RESULTS: The prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%. CONCLUSIONS: The high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.


Assuntos
Doenças Transmissíveis Importadas/parasitologia , Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Adulto , África/epidemiologia , Substituição de Aminoácidos/genética , Antimaláricos/uso terapêutico , China/epidemiologia , Estudos de Coortes , Feminino , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/isolamento & purificação , Doença Relacionada a Viagens
7.
PLoS Comput Biol ; 16(7): e1008024, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609716

RESUMO

Vitamin B12 (or cobalamin) is an enzymatic cofactor essential both for mammals and bacteria. However, cobalamin can be synthesized only by few microorganisms so most bacteria need to take it up from the environment through the TonB-dependent transport system. The first stage of cobalamin import to E. coli cells occurs through the outer-membrane receptor called BtuB. Vitamin B12 binds with high affinity to the extracellular side of the BtuB protein. BtuB forms a ß-barrel with inner luminal domain and extracellular loops. To mechanically allow for cobalamin passage, the luminal domain needs to partially unfold with the help of the inner-membrane TonB protein. However, the mechanism of cobalamin permeation is unknown. Using all-atom molecular dynamics, we simulated the transport of cobalamin through the BtuB receptor embedded in an asymmetric and heterogeneous E. coli outer-membrane. To enhance conformational sampling of the BtuB loops, we developed the Gaussian force-simulated annealing method (GF-SA) and coupled it with umbrella sampling. We found that cobalamin needs to rotate in order to permeate through BtuB. We showed that the mobility of BtuB extracellular loops is crucial for cobalamin binding and transport and resembles an induced-fit mechanism. Loop mobility depends not only on the position of cobalamin but also on the extension of luminal domain. We provided atomistic details of cobalamin transport through the BtuB receptor showing the essential role of the mobility of BtuB extracellular loops. A similar TonB-dependent transport system is used also by many other compounds, such as haem and siderophores, and importantly, can be hijacked by natural antibiotics. Our work could have implications for future delivery of antibiotics to bacteria using this transport system.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Vitamina B 12/metabolismo , Algoritmos , Antibacterianos/química , Sítios de Ligação , Biologia Computacional , Cristalografia por Raios X , Heme/química , Íons , Bicamadas Lipídicas/química , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Distribuição Normal , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Sacarose/química , Água/química
8.
Nat Commun ; 11(1): 3479, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661250

RESUMO

Genetic factors contribute to the risk of thrombotic diseases. Recent genome wide association studies have identified genetic loci including SLC44A2 which may regulate thrombosis. Here we show that Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial energetics. We find that Slc44a2 null mice (Slc44a2(KO)) have increased bleeding times and delayed thrombosis compared to wild-type (Slc44a2(WT)) controls. Platelets from Slc44a2(KO) mice have impaired activation in response to thrombin. We discover that Slc44a2 mediates choline transport into mitochondria, where choline metabolism leads to an increase in mitochondrial oxygen consumption and ATP production. Platelets lacking Slc44a2 contain less ATP at rest, release less ATP when activated, and have an activation defect that can be rescued by exogenous ADP. Taken together, our data suggest that mitochondria require choline for maximum function, demonstrate the importance of mitochondrial metabolism to platelet activation, and reveal a mechanism by which Slc44a2 influences thrombosis.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Ativação Plaquetária/fisiologia , Trombose/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Masculino , Espectrometria de Massas , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Ativação Plaquetária/genética , Agregação Plaquetária/genética , Agregação Plaquetária/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Trombose/genética
9.
PLoS Comput Biol ; 16(7): e1007884, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32614821

RESUMO

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a "competitive" environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest.


Assuntos
Transporte Biológico/fisiologia , Simulação por Computador , Computadores Moleculares , Biologia de Sistemas/métodos , Algoritmos , Cadeias de Markov , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Método de Monte Carlo , Termodinâmica
10.
PLoS Comput Biol ; 16(7): e1007789, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32614861

RESUMO

Membrane transport is generally thought to occur via an alternating access mechanism in which the transporter adopts at least two states, accessible from two different sides of the membrane to exchange substrates from the extracellular environment and the cytoplasm or from the cytoplasm and the intracellular matrix of the organelles (only in eukaryotes). In recent years, a number of high resolution structures have supported this general framework for a wide class of transport molecules, although additional states along the transport pathway are emerging as critically important. Given that substrate binding is often weak in order to enhance overall transport rates, there exists the distinct possibility that transporters may transport the incorrect substrate. This is certainly the case for many pharmaceutical compounds that are absorbed in the gut or cross the blood brain barrier through endogenous transporters. Docking studies on the bacterial sugar transporter vSGLT reveal that many highly toxic compounds are compatible with binding to the orthosteric site, further motivating the selective pressure for additional modes of selectivity. Motivated by recent work in which we observed failed substrate delivery in a molecular dynamics simulation where the energized ion still goes down its concentration gradient, we hypothesize that some transporters evolved to harness this 'slip' mechanism to increase substrate selectivity and reduce the uptake of toxic molecules. Here, we test this idea by constructing and exploring a kinetic transport model that includes a slip pathway. While slip reduces the overall productive flux, when coupled with a second toxic molecule that is more prone to slippage, the overall substrate selectivity dramatically increases, suppressing the accumulation of the incorrect compound. We show that the mathematical framework for increased substrate selectivity in our model is analogous to the classic proofreading mechanism originally proposed for tRNA synthase; however, because the transport cycle is reversible we identified conditions in which the selectivity is essentially infinite and incorrect substrates are exported from the cell in a 'detoxification' mode. The cellular consequences of proofreading and membrane slippage are discussed as well as the impact on future drug development.


Assuntos
Sítios de Ligação , Transporte Biológico/fisiologia , Proteínas de Membrana Transportadoras , Modelos Biológicos , Ligação Proteica/fisiologia , Biologia Computacional , Humanos , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Transportador 1 de Glucose-Sódio , Especificidade por Substrato
11.
BMC Infect Dis ; 20(1): 533, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32698879

RESUMO

BACKGROUND: Plasmodium falciparum parasites, which could harbour anti-malaria drug resistance genes, are commonly detected in blood donors in malaria-endemic areas. Notwithstanding, anti-malaria drug resistant biomarkers have not been characterized in blood donors with asymptomatic P. falciparum infection. METHODS: A total of 771 blood donors were selected from five districts in the Greater Accra Region, Ghana. Each donor sample was screened with malaria rapid diagnostic test (RDT) kit and parasitaemia quantified microscopically. Dried blood spots from malaria positive samples were genotyped for P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum multi-drug resistance (Pfmdr1), P. falciparum dihydropteroate-synthetase (Pfdhps), P. falciparum dihydrofolate-reductase (Pfdhfr) and Kelch 13 propeller domain on chromosome 13 (Kelch 13) genes. RESULTS: Of the 771 blood donors, 91 (11.8%) were positive by RDT. Analysis of sequence reads indicated successful genotyping of Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes in 84.6, 81.3, 86.8, 86.9 and 92.3% of the isolates respectively. Overall, 21 different mutant haplotypes were identified in 69 isolates (75.8%). In Pfcrt, CVIET haplotype was observed in 11.6% samples while in Pfmdr1, triple mutation (resulting in YFN haplotype) was detected in 8.1% of isolates. In Pfdhfr gene, triple mutation resulting in IRNI haplotype and in Pfdhps gene, quintuple mutation resulting in AGESS haplotype was identified in 17.7% parasite isolates. Finally, five non-synonymous Kelch 13 alleles were detected; C580Y (3.6%), P615L (4.8%), A578S (4.8%), I543V (2.4%) and A676S (1.2%) were detected. CONCLUSION: Results obtained in this study indicated various frequencies of mutant alleles in Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes from P. falciparum infected blood donors. These alleles could reduce the efficacy of standard malaria treatment in transfusion-transmitted malaria cases. Incorporating malaria screening into donor screening protocol to defer infected donors is therefore recommended.


Assuntos
Doadores de Sangue , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Plasmodium falciparum/genética , Adolescente , Adulto , Alelos , Antimaláricos/uso terapêutico , Doenças Assintomáticas , Biomarcadores , Cloroquina/uso terapêutico , Estudos Transversais , Di-Hidropteroato Sintase/genética , Feminino , Frequência do Gene , Gana/epidemiologia , Haplótipos , Humanos , Repetição Kelch/genética , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/isolamento & purificação , Prevalência , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Adulto Jovem
12.
Proc Natl Acad Sci U S A ; 117(29): 17011-17018, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636271

RESUMO

Few antibiotics are effective against Acinetobacter baumannii, one of the most successful pathogens responsible for hospital-acquired infections. Resistance to chlorhexidine, an antiseptic widely used to combat A. baumannii, is effected through the proteobacterial antimicrobial compound efflux (PACE) family. The prototype membrane protein of this family, AceI (Acinetobacter chlorhexidine efflux protein I), is encoded for by the aceI gene and is under the transcriptional control of AceR (Acinetobacter chlorhexidine efflux protein regulator), a LysR-type transcriptional regulator (LTTR) protein. Here we use native mass spectrometry to probe the response of AceI and AceR to chlorhexidine assault. Specifically, we show that AceI forms dimers at high pH, and that binding to chlorhexidine facilitates the functional form of the protein. Changes in the oligomerization of AceR to enable interaction between RNA polymerase and promoter DNA were also observed following chlorhexidine assault. Taken together, these results provide insight into the assembly of PACE family transporters and their regulation via LTTR proteins on drug recognition and suggest potential routes for intervention.


Assuntos
Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Clorexidina , Proteínas de Membrana Transportadoras , Acinetobacter baumannii/química , Acinetobacter baumannii/enzimologia , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clorexidina/química , Clorexidina/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Resistência Microbiana a Medicamentos , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
13.
PLoS Negl Trop Dis ; 14(6): e0008255, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32530913

RESUMO

BACKGROUND: Vivax malaria is an important public health problem in the Greater Mekong Subregion (GMS), including the China-Myanmar border. Previous studies have found that Plasmodium vivax has decreased sensitivity to antimalarial drugs in some areas of the GMS, but the sensitivity of P. vivax to antimalarial drugs is unclear in the China-Myanmar border. Here, we investigate the drug sensitivity profile and genetic variations for two drug resistance related genes in P. vivax isolates to provide baseline information for future drug studies in the China-Myanmar border. METHODOLOGY/PRINCIPAL FINDINGS: A total of 64 P. vivax clinical isolates collected from the China-Myanmar border area were assessed for ex vivo susceptibility to eight antimalarial drugs by the schizont maturation assay. The medians of IC50 (half-maximum inhibitory concentrations) for chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate, artemether, dihydroartemisinin were 84.2 nM, 34.9 nM, 4.0 nM, 22.3 nM, 41.4 nM, 2.8 nM, 2.1 nM and 2.0 nM, respectively. Twelve P. vivax clinical isolates were found over the cut-off IC50 value (220 nM) for chloroquine resistance. In addition, sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvcrt-o (P. vivax chloroquine resistance transporter-o), and difference in pvmdr1 copy number were studied. Sequencing of the pvmdr1 gene in 52 samples identified 12 amino acid substitutions, among which two (G698S and T958M) were fixed, M908L were present in 98.1% of the isolates, while Y976F and F1076L were present in 3.8% and 78.8% of the isolates, respectively. Amplification of the pvmdr1 gene was only detected in 4.8% of the samples. Sequencing of the pvcrt-o in 59 parasite isolates identified a single lysine insertion at position 10 in 32.2% of the isolates. The pvmdr1 M908L substitutions in pvmdr1 in our samples was associated with reduced sensitivity to chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate and dihydroartemisinin. CONCLUSIONS: Our findings depict a drug sensitivity profile and genetic variations of the P. vivax isolates from the China-Myanmar border area, and suggest possible emergence of chloroquine resistant P. vivax isolates in the region, which demands further efforts for resistance monitoring and mechanism studies.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Malária Vivax/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium vivax/efeitos dos fármacos , Polimorfismo Genético , Proteínas de Protozoários/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , China , Feminino , Genótipo , Humanos , Lactente , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Mianmar , Testes de Sensibilidade Parasitária , Plasmodium vivax/isolamento & purificação , Análise de Sequência de DNA , Adulto Jovem
14.
Int J Nanomedicine ; 15: 3877-3886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32581535

RESUMO

Introduction: Vaccine formulation with appropriate adjuvants is an attractive approach to develop protective immunity against pathogens. Calcium phosphate nanoparticles (CaPNs) are considered as ideal adjuvants and delivery systems because of their great potential for enhancing immune responses. In the current study, we have designed nanoparticle-based vaccine candidates to induce immune responses and protection against B. melitensis and B. abortus. Materials and Methods: For this purpose, we used three Brucella antigens (FliC, 7α-HSDH, BhuA) and two multi-epitopes (poly B and poly T) absorbed by CaPNs. The efficacy of each formulation was evaluated by measuring humoral, cellular and protective responses in immunized mice. Results: The CaPNs showed an average size of about 90 nm with spherical shape and smooth surface. The CaPNs-adsorbed proteins displayed significant increase in cellular and humoral immune responses compared to the control groups. In addition, our results showed increased ratio of specific IgG2a (associated with Th1) to specific IgG1 (associated with Th2). Also, immunized mice with different vaccine candidate formulations were protected against B. melitensis 16M and B. abortus 544, and showed same levels of protection as commercial vaccines (B. melitensis Rev.1 and B. abortus RB51) except for BhuA-CaPNs. Discussion: Our data support the hypothesis that these antigens absorbed with CaPNs could be effective vaccine candidates against B. melitensis and B. abortus.


Assuntos
Antígenos de Bactérias/química , Vacina contra Brucelose/química , Vacina contra Brucelose/imunologia , Nanopartículas/química , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Proteínas de Bactérias/imunologia , Brucella abortus/imunologia , Brucella melitensis/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Fosfatos de Cálcio/química , Sistemas de Liberação de Medicamentos , Feminino , Imunidade Humoral , Imunização , Imunoglobulina G/imunologia , Proteínas de Membrana Transportadoras/imunologia , Camundongos Endogâmicos BALB C
15.
Medicine (Baltimore) ; 99(20): e20192, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32443340

RESUMO

The aims of this study were to compare diagnostic value of anti-ribosomal P protein antibody (anti-P), anti-Smith antibody (anti-Sm), anti-double-stranded DNA antibody (anti-dsDNA), anti-nucleosome antibody (ANuA), and anti-histone antibody (AHA) for systemic lupus erythematosus (SLE) as well as explore the correlation between anti-P and SLE.A retrospective study was performed with 487 SLE patients, 235 non-SLE rheumatic diseases, and 124 healthy subjects from January 2015 to December 2018. Clinical manifestations, laboratory results and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2000 scores were analyzed between anti-P/+/ and anti-P/-/ patients. SPSS19.0 statistical software was used for data analysis.The sensitivities of anti-P, anti-Sm, anti-dsDNA, ANuA, and AHA in SLE were 31.6%, 20.7%, 45.0%, 27.9%, and 14.6%, and the specificities were 99.2%, 99.4%, 98.9%, 98.3%, and 96.7%, respectively. Only 27.9% of SLE had a single positive anti-P while the other 4 antibodies were all negative. There were significant differences in the age of onset, skin erythema, urinary protein, creatinine and serum IgG, IgM, C3, C4 between anti-P/+/ and anti-P/-/ patients (P < .05). When anti-Sjogren syndrome A antibody, anti-P were positive and anti-dsDNA was negative, the incidence of skin erythema was the highest (35.1%). Compared with anti-P/-/ patients, anti-P/+/ patients had higher SLEDAI scores (P < .001).Anti-P, anti-Sm, anti-dsDNA, ANuA, and AHA have high specificity but poor sensitivity in the diagnosis of SLE; combined detection can greatly improve the detection rate. Anti-P is more valuable in the diagnosis of SLE when other specific autoantibodies are negative. SLE patients with positive anti-P have an earlier onset age and are more prone to skin erythema, lupus nephritis as well as higher disease activity.


Assuntos
Autoanticorpos/sangue , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Membrana Transportadoras/imunologia , Proteínas Ribossômicas/imunologia , Adulto , Anticorpos Antinucleares/imunologia , DNA/antagonistas & inibidores , DNA/metabolismo , Eritema/imunologia , Eritema/patologia , Feminino , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/imunologia , Masculino , Pessoa de Meia-Idade , Nucleossomos/metabolismo , Estudos Retrospectivos , Doenças Reumáticas/imunologia , Sensibilidade e Especificidade , Dermatopatias/epidemiologia
16.
Nat Commun ; 11(1): 2291, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385243

RESUMO

PIK3R2 encodes the p85ß regulatory subunit of phosphatidylinositol 3-kinase and is frequently amplified in cancers. The signaling mechanism and therapeutic implication of p85ß are poorly understood. Here we report that p85ß upregulates the protein level of the receptor tyrosine kinase AXL to induce oncogenic signaling in ovarian cancer. p85ß activates p110 activity and AKT-independent PDK1/SGK3 signaling to promote tumorigenic phenotypes, which are all abolished upon inhibition of AXL. At the molecular level, p85ß alters the phosphorylation of TRIM2 (an E3 ligase) and optineurin (an autophagy receptor), which mediate the selective regulation of AXL by p85ß, thereby disrupting the autophagic degradation of the AXL protein. Therapeutically, p85ß expression renders ovarian cancer cells vulnerable to inhibitors of AXL, p110, or PDK1. Conversely, p85ß-depleted cells are less sensitive to these inhibitors. Together, our findings provide a rationale for pharmacological blockade of the AXL signaling axis in PIK3R2-amplified ovarian cancer.


Assuntos
Autofagia , Carcinogênese/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Intervalo Livre de Doença , Ativação Enzimática , Feminino , Ontologia Genética , Humanos , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Nucleares , Neoplasias Ovarianas/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação , Regulação para Cima/genética
17.
PLoS One ; 15(5): e0226453, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379825

RESUMO

Membrane transporters carry key metabolites across the cell membrane and, from a resource standpoint, are hypothesized to be produced when necessary. The expression of membrane transporters in metabolic pathways is often upregulated by the transporter substrate. In E. coli, such systems include for example the lacY, araFGH, and xylFGH genes, which encode for lactose, arabinose, and xylose transporters, respectively. As a case study of a minimal system, we build a generalizable physical model of the xapABR genetic circuit, which features a regulatory feedback loop via membrane transport (positive feedback) and enzymatic degradation (negative feedback) of an inducer. Dynamical systems analysis and stochastic simulations show that the membrane transport makes the model system bistable in certain parameter regimes. Thus, it serves as a genetic "on-off" switch, enabling the cell to only produce a set of metabolic enzymes when the corresponding metabolite is present in large amounts. We find that the negative feedback from the degradation enzyme does not significantly disturb the positive feedback from the membrane transporter. We investigate hysteresis in the switching and discuss the role of cooperativity and multiple binding sites in the model circuit. Fundamentally, this work explores how a stable genetic switch for a set of enzymes is obtained from transcriptional auto-activation of a membrane transporter through its substrate.


Assuntos
Adaptação Fisiológica/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Genes de Troca , Modelos Biológicos , Sítios de Ligação , Transporte Biológico/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ribonucleosídeos/metabolismo , Processos Estocásticos , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética
19.
Plant Mol Biol ; 103(4-5): 561-580, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32405802

RESUMO

KEY MESSAGE: CmHKT1;1 selectively exports Na+ from plant cells. Upon NaCl stress, its expression increased in a salt-tolerant melon cultivar. Overexpression of CmHKT1;1 increased transgenic Arabidopsis salt tolerance through improved K+/Na+ balance. High-affinity K+ transporters (HKTs) are thought to be involved in reducing Na+ in plant shoots under salt stress and modulating salt tolerance, but their function in a moderately salt-tolerant species of melon (Cucumis melo L.) remains unclear. In this study, a Na+ transporter gene, CmHKT1;1 (GenBank accession number: MK986658), was isolated from melons based on genome data. The transcript of CmHKT1;1 was relatively more abundant in roots than in stems or leaves from melon seedlings. The tobacco transient expression system showed that CmHKT1;1 was plasma-membrane localized. Upon salt stress, CmHKT1;1 expression was more strongly upregulated in a salt-tolerant melon cultivar, 'Bingxuecui' (BXC) compared with a salt-sensitive cultivar, 'Yulu' (YL). Electrophysiological evidence demonstrated that CmHKT1;1 only transported Na+, rather than K+, when expressed in Xenopus laevis oocytes. Overexpression of CmHKT1;1 increased salt sensitivity in Saccharomyces cerevisiae and salt tolerance in Arabidopsis thaliana. Under NaCl treatments, transgenic Arabidopsis plants accumulated significantly lower concentrations of Na+ in shoots than wild type plants and showed a better K+/Na+ balance, leading to better Fv/Fm, root length, biomass, and enhanced plant growth. The CmHKT1;1 gene may serve as a useful candidate for improving crop salt tolerance.


Assuntos
Arabidopsis/metabolismo , Cucumis melo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Clorofila/análise , Clonagem Molecular , Cucumis melo/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Saccharomyces cerevisiae/genética , Tolerância ao Sal , Plântula/genética , Plântula/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Simportadores/genética , Simportadores/metabolismo , Tabaco/genética , Tabaco/metabolismo
20.
Life Sci ; 254: 117751, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32387413

RESUMO

AIMS: Urea transporter B (UTB) is encoded by the SLC14α1 gene, and exerts its activity in the choroid plexus (CP) by regulating [Na+] in the cerebrospinal fluid (CSF) and maintaining normal blood pressure in mice fed on high salt diet. The aim of this study is to investigate the effect of high salt diet on the mean arterial pressure (MAP) in SLC14α1 depletion mice and its possible molecular mechanism. MAIN METHODS: Adult male mice were divided into four groups: 1) UTB+/+(wild type) mice + normal salt diet (0.3% NaCl, NS); 2) UTB+/+ mice + high salt diet (8% NaCl, HS); 3) UTB-/- (SLC14α1 knockout) mice + NS; 4) UTB-/- mice + HS, each group consisted of 6 mice. The MAP of mice was measured by non-invasive detection method after HS diet for 4 weeks, followed by euthanization for brain and blood collection. KEY FINDINGS: HS significantly elevated the MAP and CSF [Na+] in UTB-/- mice in comparison with wild type mice; however, NS didn't alter the MAP and CSF [Na+] in either wild type mice or UTB-/- mice. HS also induced the expression of ENaC-α and α1-Na+-K+-ATPase in UTB-/- mice as confirmed by RT-PCR and Western blot. SIGNIFICANCE: These results suggest that the depletion of SLC14α1 gene in mice may contribute to the HS-induced abnormality of sodium transportation in the CSF, and lead to the elevation of MAP, which eventually promote the development of salt-sensitive hypertension.


Assuntos
Pressão Sanguínea , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Cloreto de Sódio na Dieta/administração & dosagem , Animais , Camundongos , Camundongos Transgênicos
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