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1.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198853

RESUMO

In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipase/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Hepatopatia Gordurosa não Alcoólica/genética , Adulto , Idoso , Feminino , Regulação da Expressão Gênica/genética , Hepacivirus/genética , Hepatócitos/virologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Gotículas Lipídicas/virologia , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/virologia , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos
2.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205228

RESUMO

BACKGROUND: Artemether-lumefantrine is a highly effective artemisinin-based combination therapy that was adopted in Mali as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study was designed to measure the efficacy of artemether-lumefantrine and to assess the selection of the P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genotypes that have been associated with drug resistance. METHODS: A 28-day follow-up efficacy trial of artemether-lumefantrine was conducted in patients aged 6 months and older suffering from uncomplicated falciparum malaria in four different Malian areas during the 2009 malaria transmission season. The polymorphic genetic markers MSP2, MSP1, and Ca1 were used to distinguish between recrudescence and reinfection. Reinfection and recrudescence were then grouped as recurrent infections and analyzed together by PCR-restriction fragment length polymorphism (RFLP) to identify candidate markers for artemether-lumefantrine tolerance in the P. falciparum chloroquine resistance transporter (pfcrt) gene and the P. falciparum multi-drug resistance 1 (pfmdr1) gene. RESULTS: Clinical outcomes in 326 patients (96.7%) were analyzed and the 28-day uncorrected adequate clinical and parasitological response (ACPR) rate was 73.9%. The total PCR-corrected 28-day ACPR was 97.2%. The pfcrt 76T and pfmdr1 86Y population prevalence decreased from 49.3% and 11.0% at baseline (n = 337) to 38.8% and 0% in patients with recurrent infection (n = 85); p = 0.001), respectively. CONCLUSION: Parasite populations exposed to artemether-lumefantrine in this study were selected toward chloroquine-sensitivity and showed a promising trend that may warrant future targeted reintroduction of chloroquine or/and amodiaquine.


Assuntos
Combinação Arteméter e Lumefantrina/administração & dosagem , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Protozoários/genética , Alelos , Combinação Arteméter e Lumefantrina/efeitos adversos , Artemisininas/administração & dosagem , Artemisininas/efeitos adversos , Criança , Pré-Escolar , Cloroquina/administração & dosagem , Cloroquina/efeitos adversos , Resistência a Medicamentos/genética , Feminino , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Malária Falciparum/patologia , Masculino , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade
3.
Nat Commun ; 12(1): 4236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244493

RESUMO

The repertoire of peptides presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface is tailored by the ER-resident peptide loading complex (PLC), which contains the exchange catalyst tapasin. Tapasin stabilizes MHC-I molecules and promotes the formation of stable peptide-MHC-I (pMHC-I) complexes that serve as T cell antigens. Exchange of suboptimal by high-affinity ligands is catalyzed by tapasin, but the underlying mechanism is still elusive. Here we analyze the tapasin-induced changes in MHC-I dynamics, and find the catalyst to exploit two essential features of MHC-I. First, tapasin recognizes a conserved allosteric site underneath the α2-1-helix of MHC-I, 'loosening' the MHC-I F-pocket region that accomodates the C-terminus of the peptide. Second, the scoop loop11-20 of tapasin relies on residue L18 to target the MHC-I F-pocket, enabling peptide exchange. Meanwhile, tapasin residue K16 plays an accessory role in catalysis of MHC-I allotypes bearing an acidic F-pocket. Thus, our results provide an explanation for the observed allele-specificity of catalyzed peptide exchange.


Assuntos
Alelos , Apresentação do Antígeno/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Regulação Alostérica , Biocatálise , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Imunoglobulinas/metabolismo , Imunoglobulinas/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
4.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070225

RESUMO

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Peptidoglicano/química , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética
5.
Artigo em Russo | MEDLINE | ID: mdl-34184482

RESUMO

OBJECTIVE: To estimate the proportion and spectrum of infrequent autosomal dominant spastic paraplegias in a group of families with DNA-confirmed diagnosis and to investigate their molecular and clinical characteristics. MATERIAL AND METHODS: Ten families with 6 AD-SPG: SPG6 (n=1), SPG8 (n=2), SPG9A (n=1), SPG12 (n=1), SPG17 (n=3), SPG31 (n=2) were studied using clinical, genealogical, molecular-genetic (massive parallel sequencing, spastic paraplegia panel, whole-exome sequencing, multiplex ligation-dependent amplification, Sanger sequencing) and bioinformatic methods. RESULTS AND CONCLUSION: Nine heterozygous mutations were detected in 6 genes, including the common de novo mutation p.Gly106Arg in NIPA1 (SPG6), the earlier reported mutation p.Val626Phe in WASHC5 (SPG8) in isolated case and the novel p.Val695Ala in WASHC5 (SPG8) in a family with 4 patients, the novel mutation p.Thr301Arg in RTN2 (SPG12) in a family with 2 patients, the novel mutation c.105+4A>G in REEP1 (SPG31) in a family with 4 patients and the reported earlier p.Lys101Lys in REEP1 (SPG31) in a family with 3 patients, the known de novo mutation p.Arg252Gln in ALDH18A1 (SPG9A) in two monozygous twins; the common mutation p.Ser90Leu in BSCL2 (SPG17) in a family with 3 patients and in isolated case, reported mutation p.Leu363Pro in a family with 2 patients. SPG6, SPG8, SPG12 and SPG31 presented 'pure' phenotypes, SPG31 had most benign course. Age of onset varied in SPG31 family and was atypically early in SPG6 case. Patients with SPG9A and SPG17 had 'complicated' paraplegias; amyotrophy of hands typical for SPG17 was absent in a child and in an adolescent from 2 families, but may develop later.


Assuntos
Subunidades gama da Proteína de Ligação ao GTP , Paraplegia Espástica Hereditária , Adolescente , Criança , Heterozigoto , Humanos , Proteínas de Membrana Transportadoras/genética , Mutação , Fenótipo , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/genética
6.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069257

RESUMO

Candida albicans is an opportunistic pathogen that induces vulvovaginal candidiasis (VVC), among other diseases. In the vaginal environment, the source of carbon for C. albicans can be either lactic acid or its dissociated form, lactate. It has been shown that lactate, similar to the popular antifungal drug fluconazole (FLC), reduces the expression of the ERG11 gene and hence the amount of ergosterol in the plasma membrane. The Cdr1 transporter that effluxes xenobiotics from C. albicans cells, including FLC, is delocalized from the plasma membrane to a vacuole under the influence of lactate. Despite the overexpression of the CDR1 gene and the increased activity of Cdr1p, C. albicans is fourfold more sensitive to FLC in the presence of lactate than when glucose is the source of carbon. We propose synergistic effects of lactate and FLC in that they block Cdr1 activity by delocalization due to changes in the ergosterol content of the plasma membrane.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Fluconazol/farmacologia , Ácido Láctico/farmacologia , Candida albicans/genética , Candida albicans/metabolismo , Membrana Celular/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Ácido Láctico/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Transporte Proteico/efeitos dos fármacos
7.
J Stroke Cerebrovasc Dis ; 30(8): 105932, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34148020

RESUMO

OBJECTIVES: Bone marrow mononuclear cells (BM-MNC) show a significant therapeutic effect in combination with training even in the chronic phase of stroke. However, the mechanism of this combination therapy has not been investigated. Here, we examined its effects on brain metabolism in chronic stroke mice. MATERIALS AND METHODS: BM-MNC (1x105 cells in 100 µL of phosphate-buffered saline) were intravenously transplanted at 4 weeks (chronic stage) after the middle cerebral artery occlusion. At 3 h and 10 weeks after the administration of BM-MNC, we evaluated transcription changes of the metabolism-related genes, hypoxia inducible factor 1-α (Hif-1α), prolyl hydroxylase 3 (Phd3), pyruvate dehydrogenase kinase 1 (Pdk1), Na+/K+-ATPase (Atp1α1‒3), connexins, glucose transporters, and monocarboxylate transporters, in the brain during chronic phase of stroke using quantitative polymerase chain reaction. RESULTS: The results showed transcriptional activation of the metabolism-related genes in the contralateral cortex at 3 h after BM-MNC transplantation. Behavioral tests were performed after cell therapy, and the brain metabolism of mice with improved motor function was examined at 10 weeks after cell therapy. The therapeutic efficacy of the combination therapy with BM-MNC transplantation and training was evident in the form of transcriptional activation of ipsilateral anterior cerebral artery (ACA) cortex. CONCLUSIONS: BM-MNC transplantation combined with training for chronic stroke activated gene expression in both the ipsilateral and the contralateral side.


Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Encéfalo/metabolismo , Metabolismo Energético , Infarto da Artéria Cerebral Média/terapia , Condicionamento Físico Animal , Animais , Comportamento Animal , Encéfalo/fisiopatologia , Doença Crônica , Terapia Combinada , Conexinas/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Metabolismo Energético/genética , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Camundongos SCID , Atividade Motora , Recuperação de Função Fisiológica , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Transcrição Genética
8.
J Clin Neurosci ; 89: 243-248, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34119275

RESUMO

The sortilin-related receptor 1 (SORL1) gene has been the subject of many studies focusing on frequent polymorphisms, which is associated with increased risk for Alzheimer's Disease (AD). By whole-exome sequencing (WES), we identified two pathogenic missense mutations c.579C > G (p.F193L) and c.1397A > G (p.N466S) in SORL1. The two mutations were located in the same protein domain, and the two unrelated probands both had an onset of memory problems at less than 65 years of age, but their clinical manifestations and cranial imaging are different. The protein structure and function affected by these mutations were predicted using bioinformatics analysis, which suggested they were pathogenic. 3D protein structural analysis revealed that these amino acid substitutions might result in instability of protein structure and adverse intramolecular interactions. These findings suggest that both F193L and N466S should be thought as potential causative mutations in early-onset Alzheimer's disease (EOAD) patients. Further functional studies are warranted to evaluate their roles in the pathogenesis of AD.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Mutação/genética , Adulto , Sequência de Aminoácidos , Feminino , Predisposição Genética para Doença/genética , Humanos , Proteínas Relacionadas a Receptor de LDL/química , Masculino , Proteínas de Membrana Transportadoras/química , Pessoa de Meia-Idade , Linhagem , Estrutura Secundária de Proteína , Fatores de Risco , Sequenciamento Completo do Exoma/métodos
9.
Res Vet Sci ; 138: 1-10, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34087563

RESUMO

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.


Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Feminino , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Virulência/genética , Fatores de Virulência/metabolismo
10.
Nat Commun ; 12(1): 2879, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001879

RESUMO

As whole-genome sequencing capacity becomes increasingly decentralized, there is a growing opportunity for collaboration and the sharing of surveillance data within and between countries to inform typhoid control policies. This vision requires free, community-driven tools that facilitate access to genomic data for public health on a global scale. Here we present the Pathogenwatch scheme for Salmonella enterica serovar Typhi (S. Typhi), a web application enabling the rapid identification of genomic markers of antimicrobial resistance (AMR) and contextualization with public genomic data. We show that the clustering of S. Typhi genomes in Pathogenwatch is comparable to established bioinformatics methods, and that genomic predictions of AMR are highly concordant with phenotypic susceptibility data. We demonstrate the public health utility of Pathogenwatch with examples selected from >4,300 public genomes available in the application. Pathogenwatch provides an intuitive entry point to monitor of the emergence and spread of S. Typhi high risk clones.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Salmonella typhi/efeitos dos fármacos , Febre Tifoide/prevenção & controle , Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Genômica/métodos , Genótipo , Geografia , Humanos , Malaui , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana/métodos , Mutação , Salmonella typhi/genética , Salmonella typhi/fisiologia , Tanzânia , Febre Tifoide/microbiologia
11.
BMC Infect Dis ; 21(1): 421, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952197

RESUMO

BACKGROUND: Streptococcus pneumoniae serotype 8 incidence has increased in Denmark after the introduction of pneumococcal conjugated vaccines (PCV). The mechanism behind the serotype 8 replacement is not well understood. In this study, we aimed to present epidemiological data on invasive pneumococcal disease (IPD) and molecular characterization of 96 serotype 8 clinical isolates. METHODS: IPD data from 1999 to 2019 were used to calculate the incidence and age distribution. Whole-genome sequencing (WGS) analysis was performed on 96 isolates (6.8% of the total serotype 8 IPD isolates in the period) to characterize the isolates with respect to pneumococcal lineage traits, a range of genes with potential species discrimination, presence of colonization and virulence factors, and molecular resistance pattern. RESULTS: The serotype 8 IPD incidence increased significantly (P < 0.05) for the age groups above 15 years after the introduction of PCV13, primarily affecting the elderly (65+). All isolates were phenotypically susceptible to penicillin, erythromycin and clindamycin. Molecular characterization revealed seven different MLST profiles with ST53 as the most prevalent lineage (87.5%) among the analyzed serotype 8 isolates. The genes covering the cell-surface proteins: lytA, rspB, pspA, psaA & Xisco and the pneumococcal toxin pneumolysin = ply were present in all isolates, while genes for the membrane transporter proteins: piaA/piaB/piaC; the capsular genes: cpsA (wzg) & psrP; the metallo-binding proteins zmpB & zmpC; and the neuroamidase proteins: nanA/nanB were variably present. Surprisingly, the putative transcriptional regulator gene SP2020 was not present in all isolates (98%). Susceptibility to penicillin, erythromycin and clindamycin was molecularly confirmed. CONCLUSION: The observed serotype 8 replacement was not significantly reflected with a change in the MLST profile or changes in antibiotic resistance- or virulence determinants.


Assuntos
Resistência Microbiana a Medicamentos/genética , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Dinamarca/epidemiologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Genes Bacterianos , Humanos , Incidência , Lactente , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Vacinas Pneumocócicas/farmacologia , Sorogrupo , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/isolamento & purificação , Vacinação , Vacinas Conjugadas/farmacologia , Fatores de Virulência/genética , Adulto Jovem
12.
Cell Prolif ; 54(5): e13034, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33931895

RESUMO

OBJECTIVES: Dysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self-renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs. MATERIALS AND METHODS: Parkin+/+ and parkin-/- ESCs were established from E3.5 blastocysts of parkin+/- x parkin+/- mating mice. The pink1-/- , optn-/- and ndp52-/- ESCs were generated by CRISPR-Cas9. shRNAs were used for function loss assay of target genes. Mito-Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q-PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness. RESULTS: PINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1-/- ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub-S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub-binding dead OPTN mutant (D477N). CONCLUSIONS: The mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1-activated OPTN binding of PINK1-phosphorylated Ubiquitin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitofagia , Proteínas Quinases/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Reprogramação Celular , Edição de Genes , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ubiquitina/metabolismo
13.
Front Immunol ; 12: 651656, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936072

RESUMO

Although immune dysfunction is a key feature of coronavirus disease 2019 (COVID-19), the metabolism-related mechanisms remain elusive. Here, by reanalyzing single-cell RNA sequencing data, we delineated metabolic remodeling in peripheral blood mononuclear cells (PBMCs) to elucidate the metabolic mechanisms that may lead to the progression of severe COVID-19. After scoring the metabolism-related biological processes and signaling pathways, we found that mono-CD14+ cells expressed higher levels of glycolysis-related genes (PKM, LDHA and PKM) and PPP-related genes (PGD and TKT) in severe patients than in mild patients. These genes may contribute to the hyperinflammation in mono-CD14+ cells of patients with severe COVID-19. The mono-CD16+ cell population in COVID-19 patients showed reduced transcription levels of genes related to lysine degradation (NSD1, KMT2E, and SETD2) and elevated transcription levels of genes involved in OXPHOS (ATP6V1B2, ATP5A1, ATP5E, and ATP5B), which may inhibit M2-like polarization. Plasma cells also expressed higher levels of the OXPHOS gene ATP13A3 in COVID-19 patients, which was positively associated with antibody secretion and survival of PCs. Moreover, enhanced glycolysis or OXPHOS was positively associated with the differentiation of memory B cells into plasmablasts or plasma cells. This study comprehensively investigated the metabolic features of peripheral immune cells and revealed that metabolic changes exacerbated inflammation in monocytes and promoted antibody secretion and cell survival in PCs in COVID-19 patients, especially those with severe disease.


Assuntos
COVID-19/imunologia , Glicólise/genética , Lisina/metabolismo , Monócitos/metabolismo , Análise de Célula Única/métodos , Adenosina Trifosfatases/sangue , Adenosina Trifosfatases/genética , Anticorpos/metabolismo , COVID-19/metabolismo , COVID-19/fisiopatologia , Bases de Dados Genéticas , Proteínas Ligadas por GPI/metabolismo , Ontologia Genética , Hematopoese/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Receptores de Lipopolissacarídeos/metabolismo , Lisina/genética , Proteínas de Membrana Transportadoras/sangue , Proteínas de Membrana Transportadoras/genética , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Monócitos/imunologia , Monócitos/patologia , Fosforilação Oxidativa , RNA-Seq , Receptores de IgG/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/genética
14.
ACS Infect Dis ; 7(6): 1833-1847, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33980014

RESUMO

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N. gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N. gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.


Assuntos
Gonorreia , Neisseria gonorrhoeae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Neisseria gonorrhoeae/genética
15.
Nat Commun ; 12(1): 3174, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039964

RESUMO

Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high-affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and Tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Based on structural and functional data, a loop sequence of variable lengths has been proposed to stabilize empty MHC-I molecules through direct interactions with the floor of the groove. Using deep mutagenesis on two complementary expression systems, we find that important residues for the Tapasin/TAPBPR chaperoning activity are located on a large scaffolding surface, excluding the loop. Conversely, loop mutations influence TAPBPR interactions with properly conformed MHC-I molecules, relevant for peptide editing. Detailed biophysical characterization by solution NMR, ITC and FP-based assays shows that the loop hovers above the MHC-I groove to promote the capture of incoming peptides. Our results suggest that the longer loop of TAPBPR lowers the affinity requirements for peptide selection to facilitate peptide loading under conditions and subcellular compartments of reduced ligand concentration, and to prevent disassembly of high-affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.


Assuntos
Apresentação do Antígeno , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Antígenos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Imunoglobulinas/genética , Imunoglobulinas/isolamento & purificação , Imunoglobulinas/ultraestrutura , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/ultraestrutura , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Biblioteca de Peptídeos , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
16.
Medicine (Baltimore) ; 100(16): e25585, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33879716

RESUMO

RATIONALE: The sortilin-related receptor 1 gene (SORL1) encodes a key protein (SORLA) involved in the pathophysiology of Alzheimer disease (AD). SORLA also mediates a trophic pathway that acts through glial cell line-derived neurotrophic factor (GDNF), a critical survival factor for the midbrain dopaminergic (DA) neurons. PATIENT CONCERNS: Four patients presented to our hospital with complaints of progressive memory decline, who developed extrapyramidal signs (EPS) and psychiatric symptoms. DIAGNOSES: All 4 patients were diagnosed with AD based on their symptoms, findings from cranial magnetic resonance imaging, and cerebrospinal fluid analysis. INTERVENTIONS: We also performed whole-exome sequencing (WES) and found 4 novel mutations in SORL1. Donepezil, rivastigmine, memantine, madopar, quetiapine, and risperidone were administrated as therapy. OUTCOMES: The four mutations would change the thermal stability of SORLA domain. This could be associated with parkinsonian and psychiatric features in AD. These patients showed improvements in parkinsonian and psychiatric features. LESSONS: These cases suggest that SORL1 mutations might result in aggregation of a-synuclein through altered function of GDNF and further lead to appearance of core dementia with Lewy bodies features.


Assuntos
Doença de Alzheimer/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Transtornos Mentais/genética , Transtornos Parkinsonianos/genética , Adulto , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação
17.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917029

RESUMO

Endometrial cancer (EC) is associated with increased estrogen actions. Locally, estrogens can be formed from estrone-sulphate (E1-S) after cellular uptake by organic anion-transporting polypeptides (OATP) or organic anion transporters (OAT). Efflux of E1-S is enabled by ATP Binding Cassette transporters (ABC) and organic solute transporter (OST)αß. Currently, 19 E1-S transporters are known but their roles in EC are not yet understood. Here, we analysed levels of E1-S transporters in Ishikawa (premenopausal EC), HEC-1-A (postmenopausal EC), HIEEC (control) cell lines, in EC tissue, examined metabolism of steroid precursor E1-S, studied effects of OATPs' inhibition and gene-silencing on E1-S uptake, and assessed associations between transporters and histopathological data. Results revealed enhanced E1-S metabolism in HEC-1-A versus Ishikawa which could be explained by higher levels of OATPs in HEC-1-A versus Ishikawa, especially 6.3-fold up-regulation of OATP1B3 (SLCO1B3), as also confirmed by immunocytochemical staining and gene silencing studies, lower ABCG2 expression and higher levels of sulfatase (STS). In EC versus adjacent control tissue the highest differences were seen for ABCG2 and SLC51B (OSTß) which were 3.0-fold and 2.1-fold down-regulated, respectively. Immunohistochemistry confirmed lower levels of these two transporters in EC versus adjacent control tissue. Further analysis of histopathological data indicated that SLCO1B3 might be important for uptake of E1-S in tumours without lymphovascular invasion where it was 15.6-fold up-regulated as compared to adjacent control tissue. Our results clearly indicate the importance of E1-S transporters in EC pathophysiology and provide a base for further studies towards development of targeted treatment.


Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Estrona/análogos & derivados , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Fatores Etários , Transporte Biológico , Linhagem Celular Tumoral , Neoplasias do Endométrio/patologia , Estrona/metabolismo , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica , Gradação de Tumores , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Pós-Menopausa , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo
18.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923747

RESUMO

Placental insufficiency is a known consequence of maternal heat stress during gestation in farm animals. The molecular regulation of placentae during the stress response is little known in pigs. This study aims to identify differential gene expression in pig placentae caused by maternal heat exposure during early to mid-gestation. RNA sequencing (RNA-seq) was performed on female placental samples from pregnant pigs exposed to thermoneutral control (CON; constant 20 °C; n = 5) or cyclic heat stress (HS; cyclic 28 to 33 °C; n = 5) conditions between d40 and d60 of gestation. On d60 of gestation, placental efficiency (fetal/placental weight) was decreased (p = 0.023) by maternal HS. A total of 169 genes were differentially expressed (FDR ≤ 0.1) between CON and HS placentae of female fetuses, of which 35 genes were upregulated and 134 genes were downregulated by maternal HS. The current data revealed transport activity (FDR = 0.027), glycoprotein biosynthetic process (FDR = 0.044), and carbohydrate metabolic process (FDR = 0.049) among the terms enriched by the downregulated genes (HS vs. CON). In addition, solute carrier (SLC)-mediated transmembrane transport (FDR = 0.008) and glycosaminoglycan biosynthesis (FDR = 0.027), which modulates placental stroma synthesis, were identified among the pathways enriched by the downregulated genes. These findings provide evidence that heat-stress induced placental inefficiency may be underpinned by altered expression of genes associated with placental nutrient transport capacity and metabolism. A further understanding of the molecular mechanism contributes to the identification of placental gene signatures of summer infertility in pigs.


Assuntos
Resposta ao Choque Térmico , Nutrientes/metabolismo , Placenta/metabolismo , Transcriptoma , Animais , Metabolismo dos Carboidratos , Feminino , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Nutrientes/genética , Gravidez , Suínos
19.
Clin Drug Investig ; 41(6): 513-527, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33886098

RESUMO

Riboflavin is classified as one of the water-soluble B vitamins. It is part of the functional group of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors and is required for numerous flavoprotein-catalysed reactions. Riboflavin has important antioxidant properties, essential for correct cell functioning. It is required for the conversion of oxidised glutathione to the reduced form and for the mitochondrial respiratory chain as complexes I and II contain flavoprotein reductases and electron transferring flavoproteins. Riboflavin deficiency has been demonstrated to impair the oxidative state of the body, especially in relation to lipid peroxidation status, in both animal and human studies. In the nervous system, riboflavin is essential for the synthesis of myelin and its deficiency can determine the disruption of myelin lamellae. The inherited condition of restricted riboflavin absorption and utilisation, reported in about 10-15% of world population, warrants further investigation in relation to its association with the main neurodegenerative diseases. Several successful trials testing riboflavin for migraine prevention were performed, and this drug is currently classified as a Level B medication for migraine according to the American Academy of Neurology evidence-based rating, with evidence supporting its efficacy. Brown-Vialetto-Van Laere syndrome and Fazio-Londe diseases are now renamed as "riboflavin transporter deficiency" because these are autosomal recessive diseases caused by mutations of SLC52A2 and SLC52A3 genes that encode riboflavin transporters. High doses of riboflavin represent the mainstay of the therapy of these diseases and high doses of riboflavin should be rapidly started as soon as the diagnosis is suspected and continued lifelong. Remarkably, some mitochondrial diseases respond to supplementation with riboflavin. These include multiple acyl-CoA-dehydrogenase deficiency (which is caused by ETFDH gene mutations in the majority of the cases, or mutations in the ETFA and ETFB genes in a minority), mutations of ACAD9 gene, mutations of AIFM1 gene, mutations of the NDUFV1 and NDUFV2 genes. Therapeutic riboflavin administration has been tried in other neurological diseases, including stroke, multiple sclerosis, Friedreich's ataxia and Parkinson's disease. Unfortunately, the design of these clinical trials was not uniform, not allowing to accurately assess the real effects of this molecule on the disease course. In this review we analyse the properties of riboflavin and its possible effects on the pathogenesis of different neurological diseases, and we will review the current indications of this vitamin as a therapeutic intervention in neurology.


Assuntos
Doenças do Sistema Nervoso/tratamento farmacológico , Riboflavina/uso terapêutico , Animais , Paralisia Bulbar Progressiva/tratamento farmacológico , Paralisia Bulbar Progressiva/genética , Flavoproteínas Transferidoras de Elétrons/genética , Perda Auditiva Neurossensorial/tratamento farmacológico , Humanos , Proteínas de Membrana Transportadoras/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/tratamento farmacológico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Mutação , Doenças do Sistema Nervoso/fisiopatologia , Riboflavina/metabolismo
20.
Nat Commun ; 12(1): 2460, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33911082

RESUMO

It is well established that antibiotic treatment selects for resistance, but the dynamics of this process during infections are poorly understood. Here we map the responses of Pseudomonas aeruginosa to treatment in high definition during a lung infection of a single ICU patient. Host immunity and antibiotic therapy with meropenem suppressed P. aeruginosa, but a second wave of infection emerged due to the growth of oprD and wbpM meropenem resistant mutants that evolved in situ. Selection then led to a loss of resistance by decreasing the prevalence of low fitness oprD mutants, increasing the frequency of high fitness mutants lacking the MexAB-OprM efflux pump, and decreasing the copy number of a multidrug resistance plasmid. Ultimately, host immunity suppressed wbpM mutants with high meropenem resistance and fitness. Our study highlights how natural selection and host immunity interact to drive both the rapid rise, and fall, of resistance during infection.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Meropeném/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Seleção Genética/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Humanos , Hidroliases/genética , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Plasmídeos/genética , Porinas/genética , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Análise de Sequência de DNA , Choque Hemorrágico/microbiologia
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