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1.
Nature ; 581(7808): 316-322, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433612

RESUMO

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus12-14.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Motivos de Aminoácidos , Animais , Feminino , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Transdução de Sinais
2.
Am J Physiol Renal Physiol ; 318(5): F1160-F1166, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174141

RESUMO

Renal fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-ß, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-ß, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson's trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO.


Assuntos
Nefropatias/metabolismo , Rim/patologia , Proteínas de Membrana Transportadoras/deficiência , Obstrução Ureteral/complicações , Actinas/metabolismo , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibrose , Rim/metabolismo , Nefropatias/etiologia , Nefropatias/patologia , Nefropatias/prevenção & controle , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/metabolismo
3.
J Biol Chem ; 294(50): 19167-19183, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31699901

RESUMO

The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli, here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG-interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD-SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely-overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is noncompetitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptidilprolil Isomerase/metabolismo , Canais de Translocação SEC/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/deficiência , Peptidilprolil Isomerase/química , Ligação Proteica , Canais de Translocação SEC/química
4.
Elife ; 82019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31682224

RESUMO

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.


Assuntos
Transtornos da Surdocegueira/fisiopatologia , Distonia/fisiopatologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Deficiência Intelectual/fisiopatologia , Proteínas de Membrana Transportadoras/metabolismo , Neurônios/metabolismo , Atrofia Óptica/fisiopatologia , Multimerização Proteica , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Proteínas de Transporte de Cobre/metabolismo , Humanos , Proteínas de Membrana Transportadoras/deficiência , Estresse Oxidativo , Mapas de Interação de Proteínas
5.
Elife ; 82019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31661432

RESUMO

Lysosomes are major sites for intracellular, acidic hydrolase-mediated proteolysis and cellular degradation. The export of low-molecular-weight catabolic end-products is facilitated by polytopic transmembrane proteins mediating secondary active or passive transport. A number of these lysosomal transporters, however, remain enigmatic. We present a detailed analysis of MFSD1, a hitherto uncharacterized lysosomal family member of the major facilitator superfamily. MFSD1 is not N-glycosylated. It contains a dileucine-based sorting motif needed for its transport to lysosomes. Mfsd1 knockout mice develop splenomegaly and severe liver disease. Proteomics of isolated lysosomes from Mfsd1 knockout mice revealed GLMP as a critical accessory subunit for MFSD1. MFSD1 and GLMP physically interact. GLMP is essential for the maintenance of normal levels of MFSD1 in lysosomes and vice versa. Glmp knockout mice mimic the phenotype of Mfsd1 knockout mice. Our data reveal a tightly linked MFSD1/GLMP lysosomal membrane protein transporter complex.


Assuntos
Fígado/fisiologia , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Homeostase , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Camundongos Knockout , Ligação Proteica
6.
J Infect Dis ; 220(11): 1729-1737, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31325363

RESUMO

BACKGROUND: Searching for new strategies to defeat Pseudomonas aeruginosa is of paramount importance. Previous works in vitro showed that peptidoglycan recycling blockade disables AmpC-dependent resistance and enhances susceptibility against cell-wall-targeting immunity. Our objective was to validate these findings in murine models.This study shows for the first time in different murine models of infection that blocking the peptidoglycan recycling in Pseudomonas aeruginosa causes an important virulence impairment and disables AmpC-mediated resistance, being hence validated as a promising therapeutic target. METHODS: Wildtype PAO1, recycling-defective AmpG and NagZ mutants, an AmpC hyperproducer dacB mutant, and their combinations were used to cause systemic/respiratory infections in mice. Their survival, bacterial burden, inflammation level, and effectiveness of ceftazidime or subtherapeutic colistin to treat the infections were assessed. RESULTS: Inactivation of AmpG or NagZ significantly attenuated the virulence in terms of mice mortality, bacterial load, and inflammation. When inactivating these genes in the dacB-defective background, the ß-lactam resistance phenotype was abolished, disabling the emergence of ceftazidime-resistant mutants, and restoring ceftazidime for treatment. Subtherapeutic colistin was shown to efficiently clear the infection caused by the recycling-defective strains, likely due to the combined effect with the mice cell-wall- targeting immunity. CONCLUSIONS: This study brings us one step closer to new therapies intended to disable P. aeruginosa AmpC-mediated resistance and dampen its virulence, and strongly support the interest in developing efficient AmpG and/or NagZ inhibitors.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Peptidoglicano/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/metabolismo , beta-Lactamas/administração & dosagem , Animais , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Carga Bacteriana , Ceftazidima/administração & dosagem , Parede Celular/imunologia , Modelos Animais de Doenças , Feminino , Proteínas de Membrana Transportadoras/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Análise de Sobrevida , Resultado do Tratamento , Virulência
7.
Nanomedicine (Lond) ; 14(12): 1579-1593, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31038003

RESUMO

Creatine transporter (CrT) deficiency is an X-linked intellectual disability caused by mutations of CrT. Aim: This work focus on the preclinical development of a new therapeutic approach based on a microemulsion (ME) as drug delivery system for dodecyl creatine ester (DCE). Materials & methods: DCE-ME was prepared by titration method. Novel object recognition (NOR) tests were performed before and after DCE-ME treatment on Slc6a8-/y mice. Results: Intranasal administration with DCE-ME improved NOR performance in Slc6a8-/y mice. Slc6a8-/y mice treated with DCE-ME had increased striatal ATP levels mainly in the striatum compared with vehicle-treated Slc6a8-/y mice which was associated with increased expression of synaptic markers. Conclusion: These results highlight the potential value of DCE-ME as promising therapy for creatine transporter deficiency.


Assuntos
Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Creatina/deficiência , Emulsões/química , Emulsões/uso terapêutico , Proteínas de Membrana Transportadoras/deficiência , Retardo Mental Ligado ao Cromossomo X/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Administração Intranasal , Animais , Sistemas de Liberação de Medicamentos , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação/genética
8.
J Inherit Metab Dis ; 42(4): 581-597, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31095747

RESUMO

Thiamine is a crucial cofactor involved in the maintenance of carbohydrate metabolism and participates in multiple cellular metabolic processes within the cytosol, mitochondria, and peroxisomes. Currently, four genetic defects have been described causing impairment of thiamine transport and metabolism: SLC19A2 dysfunction leads to diabetes mellitus, megaloblastic anemia and sensory-neural hearing loss, whereas SLC19A3, SLC25A19, and TPK1-related disorders result in recurrent encephalopathy, basal ganglia necrosis, generalized dystonia, severe disability, and early death. In order to achieve early diagnosis and treatment, biomarkers play an important role. SLC19A3 patients present a profound decrease of free-thiamine in cerebrospinal fluid (CSF) and fibroblasts. TPK1 patients show decreased concentrations of thiamine pyrophosphate in blood and muscle. Thiamine supplementation has been shown to improve diabetes and anemia control in Rogers' syndrome patients due to SLC19A2 deficiency. In a significant number of patients with SLC19A3, thiamine improves clinical outcome and survival, and prevents further metabolic crisis. In SLC25A19 and TPK1 defects, thiamine has also led to clinical stabilization in single cases. Moreover, thiamine supplementation leads to normal concentrations of free-thiamine in the CSF of SLC19A3 patients. Herein, we present a literature review of the current knowledge of the disease including related clinical phenotypes, treatment approaches, update of pathogenic variants, as well as in vitro and in vivo functional models that provide pathogenic evidence and propose mechanisms for thiamine deficiency in humans.


Assuntos
Proteínas de Membrana Transportadoras/deficiência , Deficiência de Tiamina/genética , Tiamina/metabolismo , Anemia Megaloblástica , Transporte Biológico , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Diabetes Mellitus , Perda Auditiva Neurossensorial , Humanos , Doença de Leigh , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Fenótipo , Tiamina/líquido cefalorraquidiano , Tiamina/uso terapêutico , Deficiência de Tiamina/congênito , Deficiência de Tiamina/tratamento farmacológico , Tiamina Pirofosfato/metabolismo
9.
EBioMedicine ; 40: 184-197, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30686754

RESUMO

BACKGROUND: Fast growing cancer cells require greater amounts of ATP than normal cells. Although glycolysis was suggested as a source of anabolic metabolism based on lactate production, the main source of ATP to support cancer cell metabolism remains unidentified. METHODS: We have proposed that the oxoglutarate carrier SLC25A11 is important for ATP production in cancer by NADH transportation from the cytosol to mitochondria as a malate. We have examined not only changes of ATP and NADH but also changes of metabolites after SLC25A11 knock down in cancer cells. FINDINGS: The mitochondrial electron transport chain was functionally active in cancer cells. The cytosolic to mitochondrial NADH ratio was higher in non-small cell lung cancer (NSCLC) and melanoma cells than in normal cells. This was consistent with higher levels of the oxoglutarate carrier SLC25A11. Blocking malate transport by knockdown of SLC25A11 significantly impaired ATP production and inhibited the growth of cancer cells, which was not observed in normal cells. In in vivo experiments, heterozygote of SLC25A11 knock out mice suppressed KRASLA2 lung tumor formation by cross breeding. INTERPRETATION: Cancer cells critically depended on the oxoglutarate carrier SLC25A11 for transporting NADH from cytosol to mitochondria as a malate form for the purpose of ATP production. Therefore blocking SLC25A11 may have an advantage in stopping cancer growth by reducing ATP production. FUND: The Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT to SYK (NRF-2017R1A2B2003428).


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Transformação Celular Neoplásica/genética , Neoplasias Pulmonares/genética , Melanoma/genética , Proteínas de Membrana Transportadoras/deficiência , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Genes ras , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Melanoma/metabolismo , Melanoma/patologia , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Mutação , Transporte Proteico
10.
Microbiology (Reading) ; 165(2): 224-232, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30620266

RESUMO

Helicobacter pylori lacks the genes involved in the de novo synthesis of thiamin, and is therefore a thiamin auxotroph. The PnuT transporter, a member of the Pnu transporter family, mediates the uptake of thiamin across the membrane. In the genome of H. pylori, the pnuT gene is clustered with the thiamin pyrophosphokinase gene thi80. In this study, we found that [3H]thiamin is incorporated into the H. pylori SS1 strain via facilitated diffusion with a Km value of 28 µM. The incorporation of radioactive thiamin was inhibited to some extent by 2-methyl-4-amino-5-hydroxymethylpyrimidine or pyrithiamine, but was largely unaffected by thiamin phosphate or thiamin pyrophosphate. RT-PCR analysis demonstrated that the pnuT and thi80 genes are cotranscribed as a single transcript. The estimated Km value for thiamin in the thiamin pyrophosphokinase activity exerted by the recombinant Thi80 protein was 0.40 µM, which is much lower than the Km value of thiamin transport in H. pylori cells. These findings suggested that the incorporated thiamin from the environment is efficiently trapped by pyrophosphorylation to make the transport directional. In addition, the thiamin transport activity in the pnuT-deficient H. pylori strain was less than 20 % of that in the wild-type strain at extracellular thiamin concentration of 1 µM, but the incorporated scintillation signals of the pnuT-deficient strain with 100 nM [3H]thiamin were nearly at the background level. We also found that the pnuT-deficient strain required 100-times more thiamin to achieve growth equal to that of the wild-type. These findings reflect the presence of multiple routes for entry of thiamin into H. pylori, and PnuT is likely responsible for the high-affinity thiamin transport and serves as a target for antimicrobial agents against H. pylori.


Assuntos
Helicobacter pylori/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Tiamina Pirofosfoquinase/metabolismo , Tiamina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Mutação , Óperon , Pirimidinas/farmacologia , Piritiamina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiamina Pirofosfoquinase/genética
11.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1182-1191, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30658162

RESUMO

Pyrimidine nucleotides are essential for a vast number of cellular processes and dysregulation of pyrimidine metabolism has been associated with a variety of clinical abnormalities. Inborn errors of pyrimidine metabolism affecting enzymes in the pyrimidine de novo and degradation pathway have been identified but no patients have been described with a deficiency in proteins affecting the cellular import of ribonucleosides. In this manuscript, we report the elucidation of the genetic basis of the observed uridine-cytidineuria in a patient presenting with fever, hepatosplenomegaly, persistent lactate acidosis, severely disturbed liver enzymes and ultimately multi-organ failure. Sequence analysis of genes encoding proteins directly involved in the metabolism of uridine and cytidine showed two variants c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in SLC28A1, encoding concentrative nucleotide transporter 1 (hCNT1). Functional analysis showed that these variants affected the three-dimensional structure of hCNT1, altered glycosylation and decreased the half-life of the mutant proteins which resulted in impaired transport activity. Co-transfection of both variants, mimicking the trans disposition of c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in the patient, significantly impaired hCNT1 biological function. Whole genome sequencing identified two pathogenic variants c.50delT; p.(Leu17Argfs*34) and c.853_855del; p.(Lys285del) in the PRF1 gene, indicating that our patient was also suffering from Familial Hemophagocytic Lymphohistiocytosis type 2. The identification of two co-existing monogenic defects might have resulted in a blended phenotype. Thus, the clinical presentation of isolated hCNT1 deficiency remains to be established.


Assuntos
Proteínas de Membrana Transportadoras/deficiência , Insuficiência de Múltiplos Órgãos/metabolismo , Perforina/deficiência , Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Pirimidinas/metabolismo , Evolução Fatal , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana Transportadoras/genética , Insuficiência de Múltiplos Órgãos/genética , Perforina/genética , Fenótipo , Erros Inatos do Metabolismo da Purina-Pirimidina/genética
12.
mBio ; 9(6)2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538184

RESUMO

While Neisseria meningitidis typically exists in an asymptomatic nasopharyngeal carriage state, it may cause potentially lethal diseases in humans, such as septicemia or meningitis, by invading deeper sites in the body. Since the nutrient compositions of human cells are not always conducive to meningococci, N. meningitidis needs to exploit nutrients from host environments. In the present study, the utilization of cysteine by the meningococcal cysteine transport system (CTS) was analyzed for the pathogenesis of meningococcal infections. A N. meningitidis strain deficient in one of the three cts genes annotated as encoding cysteine-binding protein (cbp) exhibited approximately 100-fold less internalization into human brain microvascular endothelial cells (HBMEC) than the wild-type strain. This deficiency was restored by complementation with the three cts genes together, and the infectious phenotype of HBMEC internalization correlated with cysteine uptake activity. However, efficient accumulation of ezrin was observed beneath the cbp mutant. The intracellular survival of the cbp mutant in HBMEC was markedly reduced, whereas equivalent reductions of glutathione concentrations and of resistance to reactive oxygens species in the cbp mutant were not found. The cbp mutant grew well in complete medium but not in synthetic medium supplemented with less than 300 µM cysteine. Taking cysteine concentrations in human cells and other body fluids, including blood and cerebrospinal fluid, into consideration, the present results collectively suggest that the meningococcal CTS is crucial for the acquisition of cysteine from human cells and participates in meningococcal nutrient virulence.IMPORTANCE Neisseria meningitidis colonizes at a nasopharynx of human as a unique host and has many strains that are auxotrophs for amino acids for their growth. To cause invasive meningococcal diseases (IMD) such as sepsis and meningitis, N. meningitidis passes through epithelial and endothelial barriers and infiltrates into blood and cerebrospinal fluid as well as epithelial and endothelial cells. However, meningococcal nutrients, including cysteine, become less abundant when it more deeply infiltrates the human body even during inflammation, such that N. meningitidis has to acquire nutrients in order to survive/persist, disseminate, and proliferate in humans. This was the first study to examine the relationship between meningococcal cysteine acquisition and the pathogenesis of meningococcal infections. The results of the present study provide insights into the mechanisms by which pathogens with auxotrophs acquire nutrients in hosts and may also contribute to the development of treatments and prevention strategies for IMD.


Assuntos
Cisteína/metabolismo , Células Endoteliais/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Viabilidade Microbiana , Neisseria meningitidis/crescimento & desenvolvimento , Neisseria meningitidis/metabolismo , Fatores de Virulência/metabolismo , Células Cultivadas , Meios de Cultura/química , Endocitose , Deleção de Genes , Teste de Complementação Genética , Humanos , Proteínas de Membrana Transportadoras/deficiência , Neisseria meningitidis/genética , Virulência , Fatores de Virulência/deficiência
13.
J Craniomaxillofac Surg ; 46(12): 2058-2062, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30446326

RESUMO

Clinical and experimental studies show a clear positive effect of B-vitamins in the prevention of oromaxillofacial clefts, especially cleft lip and palate (CL/P). Hereby the local effect of thiamin (B1) in the amniotic fluid is very important for the embryonic facial development as seen in palatal organ models stimulated by topical B-vitamin application (Scheller et al., 2013a). Moreover a low B1 concentration in the serum and amniotic fluid was found in pregnant mice with clefts in their offspring (Scheller et al., 2013b). Immunochemical analyses of midface sections (ThTr-1 transporter) and the placenta (ThTr-2 transporter) of cleft fetuses with orofacial clefts showed an atypical cytoplasmatic localization (Scheller et al., 2017). mRNA nalyses of different B-vitamin transporters (B1, B2, B5, B7, B9) were performed and showed ThTr2 transporter in a short splice variant in all cleft fetuses. This splice variant may cause a functional loss of the transport capacity through the placenta barrier and result in a low amniotic fluid concentration of vitamin B1. All other analyzed transport proteins showed no functional change. These findings confirm the hypothesis that cleft prevention by high vitamin B1 substitution fails in genetically determined cleft mice, caused by an insufficient B1 uptake and missing local effect.


Assuntos
Fenda Labial/prevenção & controle , Fissura Palatina/prevenção & controle , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Tiamina/metabolismo , Complexo Vitamínico B/farmacologia , Animais , Transporte Biológico , Modelos Animais de Doenças , Eletroforese em Gel de Ágar , Feminino , Camundongos , Reação em Cadeia da Polimerase , Gravidez , RNA Mensageiro/análise , Análise de Sequência de DNA
14.
Cell Death Dis ; 9(11): 1122, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30405116

RESUMO

Mitochondria are the prime energy source in most eukaryotic cells, but these highly dynamic organelles are also involved in a multitude of cellular events. Disruption of mitochondrial homeostasis and the subsequent mitochondrial dysfunction plays a key role in the pathophysiology of Parkinson's disease (PD). Therefore, maintenance of mitochondrial integrity through different surveillance mechanisms is critical for neuronal survival. Here, we have studied the mitochondrial protein import system in in vitro and in vivo models of PD. Complex I inhibition, a characteristic pathological hallmark in PD, impaired mitochondrial protein import, which was associated with a downregulation of two key components of the system: translocase of the outer membrane 20 (TOM20) and translocase of the inner membrane 23 (TIM23), both in vitro and in vivo. In vitro, those changes were associated with OXPHOS protein downregulation, accumulation of aggregated proteins inside mitochondria and downregulation of mitochondrial chaperones. Most of these pathogenic changes, including mitochondrial dysfunction and dopaminergic cell death, were abrogated by TOM20 or TIM23 overexpression, in vitro. However, in vivo, while TOM20 overexpression exacerbated neurodegeneration in both substantia nigra (SN) pars compacta (pc) and striatum, overexpression of TIM23 partially protected dopaminergic neurons in the SNpc. These results highlight mitochondrial protein import dysfunction and the distinct role of two of their components in the pathogenesis of PD and suggest the need for future studies to further characterize mitochondrial protein import deficit in the context of PD.


Assuntos
Complexo I de Transporte de Elétrons/genética , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Doença de Parkinson/genética , Transtornos Parkinsonianos/genética , Receptores de Superfície Celular/genética , Animais , Linhagem Celular Tumoral , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Complexo I de Transporte de Elétrons/deficiência , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana Transportadoras/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Neurônios/metabolismo , Neurônios/patologia , Fosforilação Oxidativa , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Agregados Proteicos , Transporte Proteico , Receptores de Superfície Celular/deficiência , Transdução de Sinais
15.
Food Chem ; 266: 292-298, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30381188

RESUMO

Epigallocatechin-3-O-gallate (EGCG), the main green tea component, is intensively studied for its anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer effects. In the present study, a screen on a Saccharomyces cerevisiae gene deletion library was performed to identify conditions under which EGCG had deleterious rather than beneficial effects. Two genes were identified whose deletion resulted in sensitivity to EGCG: FET3 and FTR1, encoding the components of the Fet3/Ftr1 high-affinity iron uptake system, also involved in Cu(I)/Cu(II) balance on the surface of yeast cells. The presence of EGCG in the growth medium induced the production of Cu(I), with deleterious effects on fet3Δ and ftr1Δ cells. Additionally, when combined, physiological surpluses of Cu(II) and EGCG acted in synergy not only against fet3Δ and ftr1Δ, but also against wild type cells, by generating surplus Cu(I) in the growth medium. The results imply that caution should be taken when combining EGCG-rich beverages/nutraceuticals with copper-rich foods.


Assuntos
Catequina/análogos & derivados , Ceruloplasmina/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Chá/química , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Ceruloplasmina/deficiência , Cobre/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Saccharomyces cerevisiae/genética , Chá/metabolismo
16.
Semin Pediatr Neurol ; 26: 10-14, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29961494

RESUMO

This report describes the first case of a child with genetically confirmed Brown-Vialetto-van Laere syndrome in sub-Saharan Africa. This is an extremely rare clinical condition that presents with an auditory neuropathy, bulbar palsy, stridor, muscle weakness, and respiratory compromise that manifests with diaphragmatic and vocal cord paralysis. It is an autosomal recessive condition for which the genetic mutation has only recently been linked to a riboflavin transporter deficiency. We describe an 11-month-old affected male infant. He has required long-term respiratory support and a gastrostomy tube to support feeding. With high-dose riboflavin supplementation, he had limited recovery of motor function. His respiratory chain enzyme studies were abnormal suggestive of mitochondrial (mt) dysfunction. In the setting of limited resources, recognition of this striking clinical phenotype is important to highlight, specifically regarding the genetic implications of the condition and the potentially remedial response to vitamin supplementation.


Assuntos
Paralisia Bulbar Progressiva/terapia , Perda Auditiva Neurossensorial/terapia , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , África ao Sul do Saara , Paralisia Bulbar Progressiva/genética , Paralisia Bulbar Progressiva/patologia , Paralisia Bulbar Progressiva/fisiopatologia , Suplementos Nutricionais , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Lactente , Masculino , Fenótipo , Riboflavina/administração & dosagem
17.
Artigo em Inglês | MEDLINE | ID: mdl-29988395

RESUMO

Our previous study showed that the inactivation of the efflux pump TolC could abolish biofilm formation and curli production of extraintestinal pathogenic Escherichia coli (ExPEC) strain PPECC42 under hyper-osmotic conditions. In this study we investigated the role of OmpX in biofilm formation and curli production of ExPEC PPECC42. Our data showed that OmpX disruption or overexpression didn't significantly affect the biofilm formation and curli production of the wild-type strain. However, in the tolC-deleted mutant, overexpressing OmpX suppressed the effect of TolC inactivation on ExPEC biofilm formation and curli production under hyper-osmotic growth conditions. Real-time qRT-PCR confirmed that OmpX overexpression affected curli production by regulating the transcription of the curli biosynthesis-related genes in the ΔtolC strain. Our findings suggest that OmpX is involved in biofilm formation and curli production.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Escherichia coli Extraintestinal Patogênica/crescimento & desenvolvimento , Expressão Gênica , Hidrolases/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli Extraintestinal Patogênica/genética , Deleção de Genes , Perfilação da Expressão Gênica , Hidrolases/genética , Reação em Cadeia da Polimerase em Tempo Real
18.
Int J Mol Sci ; 19(7)2018 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-29937503

RESUMO

The chloroplast relies on proteins encoded in the nucleus, synthesized in the cytosol and subsequently transported into chloroplast through the protein complexes Toc and Tic (Translocon at the outer/inner membrane of chloroplasts). A Tic complex member, Tic55, contains a redox-related motif essential for protein import into chloroplasts in peas. However, Tic55 is not crucial for protein import in Arabidopsis. Here, a tic55-II-knockout mutant of Arabidopsis thaliana was characterized for Tic55 localization, its relationship with other translocon proteins, and its association with plant leaf senescence when compared to the wild type. Individually darkened leaves (IDLs) obtained through dark-induced leaf senescence were used to demonstrate chlorophyll breakdown and its relationship with plant senescence in the tic55-II-knockout mutant. The IDLs of the tic55-II-knockout mutant contained higher chlorophyll concentrations than those of the wild type. Our microarray analysis of IDLs during leaf senescence identified seven senescence-associated genes (SAGs) that were downregulated in the tic55-II-knockout mutant: ASP3, APG7, DIN2, DIN11, SAG12, SAG13, and YLS9. Real-time quantitative PCR confirmed the reliability of microarray analysis by showing the same expression patterns with those of the microarray data. Thus, Tic55 functions in dark-induced aging in A. thaliana by indirectly regulating downstream SAGs expression. In addition, the expression of four NAC genes, including ANAC003, ANAC010, ANAC042, and ANAC075 of IDL treated tic55-II-knockout mutant appeared to be downregulated. Yeast one hybrid assay revealed that only ANAC003 promoter region can be bound by MYB108, suggesting that a MYB-NAC regulatory network is involved in dark-stressed senescence.


Assuntos
Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Arabidopsis/classificação , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Senescência Celular , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Escuridão , Técnicas de Inativação de Genes , Proteínas de Membrana Transportadoras/deficiência , Filogenia , Células Vegetais/metabolismo , Células Vegetais/efeitos da radiação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Regiões Promotoras Genéticas , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
19.
mBio ; 9(1)2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29487231

RESUMO

Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (CuB) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the CuB atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the CuB site of cbb3-type cytochrome c oxidase (cbb3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides, a close relative of R. capsulatus that contains both cbb3- and aa3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb3-Cox and not that of aa3-Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb3-Cox biogenesis, establishing that distinct copper uptake systems provide the CuB atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions.IMPORTANCE The cbb3- and aa3-type cytochrome c oxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochrome c oxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient.


Assuntos
Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/metabolismo , Transporte Biológico , Proteínas de Membrana Transportadoras/deficiência
20.
Vet Microbiol ; 216: 38-44, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29519523

RESUMO

Over the last few years, polyamines have been described as key-signal of virulence in pathogenic bacteria. In the current study, we investigated whether the knockout of genes related to polyamine biosynthesis and putrescine transport affected the virulence of an avian pathogenic E. coli (APEC) strain. One-week-old White Leghorn chickens were infected intratracheally with mutants in polyamine biosynthesis (ΔspeB/C and ΔspeD/E) and transport genes (ΔpotE) of a well-characterized APEC strain of ST117 (O83: H4). All polyamine mutants and the wild-type strain were able to infect chicken; however, we observed significantly fewer lesions in the lungs of the chickens infected with the polyamine mutants in comparison with chicken infected with the wild-type. Results derived from histology of infected lungs detected significantly fewer lesions in the lung of birds infected within particular the putrescine transport mutant (ΔpotE). A decrease in colonization levels was observed in the liver and spleen of birds infected with the putrescine biosynthesis mutant ΔspeB/C, and likewise, a decrease of the colonization levels of all organs from birds infected with the ΔpotE was detected. Together, our data demonstrate that the deletion of polyamine genes, and in particular the PotE membrane protein, attenuates the virulence of APEC during infection of chickens.


Assuntos
Antiporters/genética , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/patogenicidade , Doenças das Aves Domésticas/microbiologia , Animais , Antiporters/metabolismo , Vias Biossintéticas/genética , Galinhas/microbiologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Pulmão/microbiologia , Pulmão/patologia , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Mutação , Poliaminas/metabolismo , Doenças das Aves Domésticas/fisiopatologia , Putrescina/metabolismo , Virulência , Fatores de Virulência
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