RESUMO
Thiamine (vitamin B1) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane, which is mainly mediated by the two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters lead to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report seven cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enable us to capture different states of SLC19A3's transport cycle, revealing the molecular details of thiamine recognition and transport. We identify seven previously unknown drug interactions of SLC19A3 and present structures of the transporter in complex with the inhibitors fedratinib, amprolium and hydroxychloroquine. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3.
Assuntos
Microscopia Crioeletrônica , Proteínas de Membrana Transportadoras , Tiamina , Tiamina/metabolismo , Tiamina/química , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Transporte Biológico , Modelos Moleculares , Ligantes , Células HEK293 , Interações MedicamentosasRESUMO
Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance.
Assuntos
Apresentação de Antígeno , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Antígenos de Histocompatibilidade Classe I , Proteínas de Membrana Transportadoras , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Here we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.
Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Transporte Biológico , Microscopia Crioeletrônica , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Conformação ProteicaRESUMO
Over 50 billion cells undergo apoptosis each day in an adult human to maintain tissue homeostasis by eliminating damaged or unwanted cells. Apoptotic deficiency can lead to age-related diseases with reduced apoptotic metabolites. However, whether apoptotic metabolism regulates aging is unclear. Here, we show that aging mice and apoptosis-deficient MRL/lpr (B6.MRL-Faslpr/J) mice exhibit decreased apoptotic levels along with increased aging phenotypes in the skeletal bones, which can be rescued by the treatment with apoptosis inducer staurosporine (STS) and stem cell-derived apoptotic vesicles (apoVs). Moreover, embryonic stem cells (ESC)-apoVs can significantly reduce senescent hallmarks and mtDNA leakage to rejuvenate aging bone marrow mesenchymal stem cells (MSCs) and ameliorate senile osteoporosis when compared to MSC-apoVs. Mechanistically, ESC-apoVs use TCOF1 to upregulate mitochondrial protein transcription, resulting in FLVCR1-mediated mitochondrial functional homeostasis. Taken together, this study reveals a previously unknown role of apoptotic metabolites in ameliorating bone aging phenotypes and the unique role of TCOF1/FLVCR1 in maintaining mitochondrial homeostasis.
Assuntos
Envelhecimento , Apoptose , Homeostase , Células-Tronco Mesenquimais , Mitocôndrias , Animais , Humanos , Camundongos , Envelhecimento/metabolismo , Apoptose/efeitos dos fármacos , Osso e Ossos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Osteoporose/metabolismo , Fenótipo , Estaurosporina/farmacologiaRESUMO
In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1Apo), fluconazole-bound state (Cdr1Flu), and milbemycin oxime-inhibited state (Cdr1Mil). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance.
Assuntos
Antifúngicos , Azóis , Candida albicans , Microscopia Crioeletrônica , Proteínas Fúngicas , Proteínas de Membrana Transportadoras , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/antagonistas & inibidores , Antifúngicos/farmacologia , Antifúngicos/química , Azóis/farmacologia , Azóis/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Farmacorresistência Fúngica , Fluconazol/farmacologia , Sítios de LigaçãoRESUMO
The complexity of branching and curvilinear morphology of a complete mitochondrial network within each cell is challenging to analyze and quantify. To address this challenge, we developed an image analysis technique using persistent homology with a multiparameter filtration framework, combining image processing techniques in mathematical morphology. We show that such filtrations contain both topological and geometric information about complex cellular organelle structures, which allows a software program to extract meaningful features. Using this information, we also develop a connectivity index that describes the morphology of the branching patterns. As proof of concept, we utilize this approach to study how mitochondrial networks are altered by genetic changes in the Optineurin gene. Mutations in the autophagy gene Optineurin (OPTN) are associated with primary open-angle glaucoma (POAG), amyotrophic lateral sclerosis (ALS), and Paget's disease of the bone, but the pathophysiological mechanism is unclear. We utilized the proposed mathematical morphology-based multiparameter filtration and persistent homology approach to analyze and quantitatively compare how changes in the OPTN gene alter mitochondrial structures from their normal interconnected, tubular morphology into scattered, fragmented pieces.
Assuntos
Processamento de Imagem Assistida por Computador , Proteínas de Membrana Transportadoras , Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Mutação , SoftwareRESUMO
Mycobacterial membrane protein Large 3 (MmpL3) of Mycobacterium tuberculosis (Mtb) is crucial for the translocation of trehalose monomycolate (TMM) across the inner bacterial cell membrane, making it a promising target for anti-tuberculosis (TB) drug development. While several structural, microbiological, and in vitro studies have provided significant insights, the precise mechanisms underlying TMM transport by MmpL3 and its inhibition remain incompletely understood at the atomic level. In this study, molecular dynamic (MD) simulations for the apo form and seven inhibitor-bound forms of Mtb MmpL3 were carried out to obtain a thorough comprehension of the protein's dynamics and function. MD simulations revealed that the seven inhibitors in this work stably bind to the central channel of the transmembrane domain and primarily forming hydrogen bonds with ASP251, ASP640, or both residues. Through dynamical cross-correlation matrix and principal component analysis analyses, several types of coupled motions between different domains were observed in the apo state, and distinct conformational states were identified using Markov state model analysis. These coupled motions and varied conformational states likely contribute to the transport of TMM. However, simulations of inhibitor-bound MmpL3 showed an enlargement of the proton channel, potentially disrupting coupled motions. This indicates that inhibitors may impair MmpL3's transport function by directly blocking the proton channel, thereby hindering coordinated domain movements and indirectly affecting TMM translocation.
Assuntos
Proteínas de Bactérias , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Trealose/química , Trealose/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Ligação Proteica , Fatores CordaRESUMO
Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.
Assuntos
Doença de Alzheimer , Endossomos , Proteínas Relacionadas a Receptor de LDL , Proteínas de Membrana Transportadoras , Linhagem , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Endossomos/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Feminino , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação de Sentido Incorreto , Transporte Proteico , Multimerização Proteica , Idoso , Pessoa de Meia-Idade , Células HEK293RESUMO
Visceral cestodiases, like cysticercoses and echinococcoses, are caused by cystic larvae from parasites of the Cestoda class and are endemic or hyperendemic in many areas of the world. Current therapeutic approaches for these diseases are complex and present limitations and risks. Therefore, new safer and more effective treatments are urgently needed. The Niemann-Pick C1 (NPC1) protein is a cholesterol transporter that, based on genomic data, would be the solely responsible for cholesterol uptake in cestodes. Considering that human NPC1L1 is a known target of ezetimibe, used in the treatment of hypercholesterolemia, it has the potential for repurposing for the treatment of visceral cestodiases. Here, phylogenetic, selective pressure and structural in silico analyses were carried out to assess NPC1 evolutive and structural conservation, especially between cestode and human orthologs. Two NPC1 orthologs were identified in cestode species (NPC1A and NPC1B), which likely underwent functional divergence, leading to the loss of cholesterol transport capacity in NPC1A. Comparative interaction analyses performed by molecular docking of ezetimibe with human NPC1L1 and cestode NPC1B pointed out to similarities that consolidate the idea of cestode NPC1B as a target for the repurposing of ezetimibe as a drug for the treatment of visceral cestodiases.
Assuntos
Cestoides , Ezetimiba , Proteína C1 de Niemann-Pick , Ezetimiba/farmacologia , Ezetimiba/uso terapêutico , Humanos , Animais , Proteína C1 de Niemann-Pick/metabolismo , Cestoides/metabolismo , Cestoides/efeitos dos fármacos , Cestoides/genética , Filogenia , Simulação de Acoplamento Molecular , Reposicionamento de Medicamentos/métodos , Simulação por Computador , Colesterol/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêuticoRESUMO
SUMMARYThe discovery of bacterial efflux pumps significantly advanced our understanding of how bacteria can resist cytotoxic compounds that they encounter. Within the structurally and functionally distinct families of efflux pumps, those of the Resistance-Nodulation-Division (RND) superfamily are noteworthy for their ability to reduce the intracellular concentration of structurally diverse antimicrobials. RND systems are possessed by many Gram-negative bacteria, including those causing serious human disease, and frequently contribute to resistance to multiple antibiotics. Herein, we review the current literature on the structure-function relationships of representative transporter proteins of tripartite RND efflux pumps of clinically important pathogens. We emphasize their contribution to bacterial resistance to clinically used antibiotics, host defense antimicrobials and other biocides, as well as highlighting structural similarities and differences among efflux transporters that help bacteria survive in the face of antimicrobials. Furthermore, we discuss technical advances that have facilitated and advanced efflux pump research and suggest future areas of investigation that will advance antimicrobial development efforts.
Assuntos
Antibacterianos , Proteínas de Bactérias , Bactérias Gram-Negativas , Proteínas de Membrana Transportadoras , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Humanos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Relação Estrutura-Atividade , Farmacorresistência Bacteriana , Bactérias/metabolismo , Bactérias/efeitos dos fármacosRESUMO
The original UT-1 transporter gene was initially identified in the spiny dogfish (Squalus acanthias), but localization of the UT-1 protein was not determined. Subsequent UT-1 expression was shown to localize to the collecting tubule (CT) of the shark nephron in other shark species, with expression in a closely related chimaera species also located additionally at a lower level in the intermediate-I segment (IS-I) of the nephron. In spiny dogfish, two UT-1 splice variants are known (UT-1 long and short), and there was also a second UT-1 gene described (here termed Brain UT). In this study, a second splice variant of the second Brain UT gene was discovered. Expression profiles (mRNA) of UT-1 long and short and Brain UT were determined in a number of spiny dogfish tissues. Quantitative PCR in kidney samples showed that the level of the short variant of UT-1 was around 100 times higher than the long variant, which was itself expressed around 10 times higher than Brain UT cDNA/mRNA (in kidney). For the long variant, there was a significantly higher level of mRNA abundance in fish acclimatized to 75% seawater. Ultimately, three UT-1 antibodies were made that could bind to both the UT-1 short and long variant proteins. The first two of these showed bands of appropriate sizes on Western blots of around 52.5 and 46 kDa. The second antibody had some additional lower molecular weight bands. The third antibody was mainly bound to the 46 kDa band with faint 52.5 kDa staining. Both the 52.5 and 46 kDa bands were absent when the antibodies were pre-blocked with the peptide antigens used to make them. Across the three antibodies, there were many similarities in localization but differences in subcellular localization. Predominantly, antibody staining was greatest in the intermediate segment 1 (IS-I) and proximal (PIb) segments of the first sinus zone loop of the nephron, with reasonably strong expression also found at the start and middle of the late distal tubule (LDT; second sinus zone loop). While some expression in the collecting tubule (CT) could not be ruled out, the level of staining seemed to be low or non-existent in convoluted bundle zone nephron segments such as the CT. Hence, this suggests that spiny dogfish have a fundamentally different mode of urea absorption in comparison to that found in other shark species, potentially focused more on the nephron sinus zone loops than the CT.
Assuntos
Proteínas de Membrana Transportadoras , Squalus acanthias , Animais , Squalus acanthias/metabolismo , Squalus acanthias/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Tubarões/metabolismo , Tubarões/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Transportadores de Ureia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Sequência de Aminoácidos , Encéfalo/metabolismoRESUMO
Biofilms in nature often exist as communities. In this study, an experimental mixed-species community consisting of Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was used to investigate how AI-2 transporters affect interspecies interactions and composition. The K. pneumoniae lsrB/lsrD deletion mutants had a 10-25-fold higher concentration of extracellular AI-2 compared to the wild-type. Although these deletion mutants produced monospecies biofilms of similar biomass, the substitution of these mutants for the parental strain significantly altered composition. Dual-species biofilm assays demonstrated that the changes in composition were due to the cumulative effect of pairwise interactions. It was further revealed that K. pneumoniae being present physically in the consortium was important in AI-2 mediating composition in the consortium, and that AI-2 transporters were crucial in achieving maximum biomass in the community. In conclusion, these findings demonstrate that AI-2 transporters mediate interspecies interactions and is important in maintaining the compositional equilibrium of the community.
Assuntos
Proteínas de Bactérias , Biofilmes , Klebsiella pneumoniae , Pseudomonas aeruginosa , Biofilmes/crescimento & desenvolvimento , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações Microbianas , Homosserina/análogos & derivados , Homosserina/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Deleção de Genes , Biomassa , LactonasRESUMO
BACKGROUND: SQ109 is a promising candidate drug for the treatment of patients with drug-resistant tuberculosis (DR-TB). The purpose of this study was to investigate the activity of SQ109 against clinical isolates of Mycobacterium tuberculosis (MTB) from patients with multidrug-resistant TB (MDR-TB) and pre-extensively drug-resistant TB (pre-XDR-TB), and to explore new drug-resistant mechanisms of SQ109. METHODS: We evaluated the in vitro activity of SQ109 against clinical isolates from patients with MDR-TB and pre-XDR-TB using minimal inhibitory concentration (MIC) assay. The drug-resistant gene, mmpL3 of SQ109-resistant strains was sequenced, and a quantitative real-time PCR assay was used to analyze 28 efflux pump genes in SQ109-resistant strains without mmpL3 mutations. The role of candidate efflux pumps mmpL5 and mmpL7 on the MIC of SQ109 was evaluated using recombinantly cloned MmpL5 and MmpL7 expressed in Mycobacterium smegmatis. RESULTS: The MIC90, MIC95 and MIC99 values of SQ109 for 225 clinical isolates of MTB were 0.25 mg/L, 0.5 mg/L and 1.0 mg/L, respectively. Among the pre-XDR strains, six showed resistance to SQ109 despite the absence of gene mutations in mmpL3. In six resistant pre-XDR strains, the MIC of SQ109 decreased with the use of an efflux pump inhibitor, and there was significant upregulation of mmpL5 and mmpL7 in two strains after exposure to SQ109. The presence of MmpL7 in Mycobacterium smegmatis resulted in decreased susceptibility to SQ109, with the MIC increasing from 16 mg/L to 32 mg/L. CONCLUSIONS: Our data demonstrated that SQ109 exhibited excellent levels of in vitro activity against MTB. MmpL7 may be a potential gene for MTB resistance to SQ109, providing a useful target for detecting SQ109 resistance in MTB.
Assuntos
Antituberculosos , Proteínas de Bactérias , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Humanos , Antituberculosos/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Adamantano/farmacologia , Adamantano/análogos & derivados , Mutação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/metabolismo , EtilenodiaminasRESUMO
Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.
Assuntos
Antibacterianos , Proteínas de Bactérias , Farmacorresistência Bacteriana Múltipla , Klebsiella pneumoniae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cloranfenicol/farmacologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Tetraciclina/farmacologia , Sequenciamento Completo do GenomaRESUMO
KEY MESSAGE: Promoters of moso bamboo silicon transporter genes PeLsi1-1 and PeLsi1-2 contain elements in response to hormone, silicon, and abiotic stresses, and can drive the expression of PeLsi1-1 and PeLsi1-2 in transgene Arabidopsis. Low silicon 1 (Lsi1) transporters from different species have been shown to play an important role in influxing silicon from soil. In previous study, we cloned PeLsi1-1 and PeLsi1-2 from Phyllostachys edulis and verified that PeLsi1-1 and PeLsi1-2 have silicon uptake ability. Furthermore, in this study, the promoters of PeLsi1-1(1910 bp) and PeLsi1-2(1922 bp) were cloned. Deletion analysis identified the key regions of the PeLsi1-1 and PeLsi1-2 promoters in response to hormone, silicon, and abiotic stresses. RT-qPCR analysis indicated that PeLsi1-1 and PeLsi1-2 were regulated by hormones, salt stress and osmotic stress. In addition, we found that the driving activity of the PeLsi1-1 and PeLsi1-2 promoters was regulated by 2 mM K2SiO3 and PeLsi1-1-P3 ~ P4 and PeLsi1-2-P4 ~ 5 were the regions regulated by silicon. Overexpression of PeLsi1-1 or PeLsi1-2 driven by 35S promoter in Arabidopsis resulted in a threefold increase of Si accumulation, whereas transgenic plants showed deleterious symptoms and dwarf seedlings and shorter roots under 2 mM Si treatment. When the 35S promoter was replaced by PeLsi1-1 or PeLsi1-2 promoter, a similar Si absorption was achieved and the transgene plants grew normally. This study, therefore, demonstrates that the promoters of PeLsi1-1 and PeLsi1-2 are indeed effective in driving the expression of moso bamboo Lsi1 genes and leading to silicon uptake.
Assuntos
Arabidopsis , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Plantas Geneticamente Modificadas , Poaceae , Regiões Promotoras Genéticas , Silício , Silício/farmacologia , Silício/metabolismo , Regiões Promotoras Genéticas/genética , Poaceae/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Estresse Fisiológico/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/genéticaRESUMO
Proinflammatory cytokines, elevated during inflammation caused by infection and/or autoimmune disorders, result in reduced clearance of drugs eliminated primarily by cytochrome P450 enzymes (CYPs). However, the effect of cytokines on hepatic drug transporter expression or activity has not been well-studied. Here, using plated human hepatocytes (PHHs; n = 3 lots), we investigated the effect of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), on the mRNA expression and activity of hepatic drug transporters. PHHs were incubated for 72 hours at their pathophysiologically relevant plasma concentrations, both individually (0.01, 0.1, 1, 10 ng/ml) or as a cocktail (i.e., when each was combined at 0.1 or 1 ng/ml). Following cytokine cocktail exposure (1 ng/ml), significant downregulation of mRNA expression of organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, sodium/taurocholate cotransporting polypeptide (NTCP), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1, multidrug resistance proteins (MRP) 2, 3, and 4 was observed. While the mRNA expression of organic anion transporter (OAT) 2 and organic cation transporter (OCT) 1 was downregulated in two lots, it was upregulated in one lot. In agreement (mostly), the 1 ng/ml cytokine cocktail reduced OATP1B1/3, OATP2B1, OAT2, OCT1, and NTCP activity by 75%, 44%, 82%, 47%, and 80%, respectively. Interestingly, upregulation of OAT2 and OCT1 mRNA in one donor did not translate into the same directional change in activity. Although significant interlot variability was observed, in general, the above effects, using individual cytokines, could be attributed to IL-1ß, TNF-α, and IFN-γ. SIGNIFICANCE STATEMENT: To date, this is the first comprehensive study to investigate the effect of four major proinflammatory cytokines, both individually and as a cocktail, on the mRNA expression and activity of human hepatic drug transporters. The data obtained can be used in the future to predict transporter-mediated drug clearance changes during inflammation through physiologically based pharmacokinetic modeling and simulation.
Assuntos
Citocinas , Hepatócitos , Humanos , Citocinas/metabolismo , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fígado/metabolismo , Fígado/efeitos dos fármacos , Células Cultivadas , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genéticaRESUMO
This research cloned and expressed the sugar transporter gene KM_SUT5 from Kluyveromyces marxianus GX-UN120, which displayed remarkable sugar transportation capabilities, including pentose sugars. To investigate the impact of point mutations on xylose transport capacity, we selected four sites, predicted the suitable amino acid sites by molecular docking, and altered their codons to construct the corresponding mutants, Q74D, Y195K, S460H, and Q464F, respectively. Furthermore, we conducted site-directed truncation on six sites of KM_SUT5p. The molecular modification resulted in significant changes in mutant growth and the D-xylose transport rate. Specifically, the S460H mutant exhibited a higher growth rate and demonstrated excellent performance across 20 g L-1 xylose, achieving the highest xylose accumulation under xylose conditions (49.94 µmol h-1 gDCW-1, DCW mean dry cell weight). Notably, mutant delA554-, in which the transporter protein SUT5 is truncated at position delA554-, significantly increased growth rates in both D-xylose and D-glucose substrates. These findings offer valuable insights into potential modifications of other sugar transporters and contribute to a deeper understanding of the C-terminal function of sugar transporters.
Assuntos
Proteínas Fúngicas , Kluyveromyces , Xilose , Xilose/metabolismo , Kluyveromyces/metabolismo , Kluyveromyces/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/química , Simulação de Acoplamento Molecular , Mutação , Glucose/metabolismoRESUMO
The objectives were to determine the effects of dietary crude protein (CP) content and corn grain processing on whole-body urea kinetics and the functional roles of urea transporter-B (UT-B) and aquaporins (AQP) in serosal-to-mucosal urea flux (Jsm-urea) in ovine ruminal epithelia. Thirty-two Rideau-Arcott ram lambs were blocked by bodyweight into groups of 4 and then randomly allocated within blocks to 1 of 4 diets (nâ =â 8) in a 2â ×â 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn grain processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). Whole-body urea kinetics and N balance were determined using 4-d continuous intrajugular infusions of [15N15N]-urea with concurrent collections of urine and feces with four blocks of lambs (nâ =â 4). After 23 d on diets, lambs were killed to collect ruminal epithelia for mounting in Ussing chambers to determine Jsm-urea and the measurement of mRNA abundance of UT-B and AQP. Serosal and mucosal additions of phloretin and NiCl2 were used to inhibit UT-B- and AQP-mediated urea transport, respectively. Lambs fed HP had a greater (Pâ <â 0.01) N intake (29.4 vs. 19.1 g/d) than those fed LP; however, retained N (g/d or % of N intake) was not different. As a % of N intake, lambs fed SFC tended (Pâ =â 0.09) to have a lower N excretion (72.2 vs. 83.5%) and a greater N retention (27.8 vs. 16.6%) compared to those fed WSC. Endogenous urea-N production (UER) was greater in lambs fed HP compared to those fed LP (29.9 vs. 20.6 g/d; Pâ =â 0.02), whereas urea-N secreted into the gut (GER; g/d) and urea-N used for anabolic purposes (UUA; g/d) were similar. Lambs fed LP tended (Pâ =â 0.05) to have greater GER:UER (0.78 vs. 0.66) and UUA:GER (0.23 vs. 0.13) ratios, and a greater Jsm-urea (144.7 vs. 116.1 nmol/[cm2â ×â h]; Pâ =â 0.07) compared to those fed HP. Lambs fed SFC tended to have a lower NiCl2-insensitive Jsm-urea (117.4 vs. 178.4 nmol/[cm2â ×â h]; Pâ =â 0.09) and had a lower phloretin-insensitive Jsm-urea (87.1 vs. 143.1 nmol/[cm2â ×â h]; Pâ =â 0.02) compared to those fed WSC. The mRNA abundance of UT-B (0.89 vs. 1.07; Pâ =â 0.08) and AQP-3 (0.90 vs. 1.05; Pâ =â 0.07) tended to be lower in lambs fed SFC compared to those fed WSC. Overall, reducing CP content tended to increase the GER:UER ratio with no changes in the expression or function of UT-B and AQP. Although corn grain processing had no effects on GER, feeding SFC increased the portion of urea secretion into the rumen that was mediated via UT-B and AQP.
In ruminants, urea produced in the liver as a nitrogenous waste can be secreted into the rumen where it can be used by rumen microorganisms as a source of nitrogen (N) for their growth. Therefore, urea secretion into the rumen is nutritionally important for ruminants particularly when dietary N intake is deficient. Urea secretion into the rumen occurs via transporter proteins in rumen tissue referred to as urea transporters (UT-B) and aquaporins (AQP). The purpose of this research was to investigate the effects of dietary crude protein (CP) content and corn grain processing on urea secretion into the rumen and the function of UT-B and AQP. Thirty-two Rideau-Arcott lambs were assigned to 1 of 4 diets in a 2â ×â 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). When compared to feeding HP, feeding LP tended to increase urea secretion into the rumen, but there were no corresponding changes in UT-B and AQP function. Corn processing did not influence urea secretion into the rumen; however, the portion of urea secretion that was facilitated via UT-B and AQP was greater in lambs fed SFC compared to those fed WSC.
Assuntos
Ração Animal , Aquaporinas , Dieta , Proteínas de Membrana Transportadoras , Rúmen , Transportadores de Ureia , Ureia , Zea mays , Animais , Ureia/metabolismo , Rúmen/metabolismo , Aquaporinas/metabolismo , Aquaporinas/genética , Zea mays/metabolismo , Ração Animal/análise , Dieta/veterinária , Ovinos/fisiologia , Ovinos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Masculino , Proteínas Alimentares/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , CinéticaRESUMO
Plants synthesize hundreds of small secretory peptides, which are perceived by the receptor-like kinase (RLK) family at the cell surface. Various signaling peptide-RLK pairs ensure plant adaptation to distinct environmental conditions. Here, we report that SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) immune peptides modulate root growth and development by regulating PIN-FORMED (PIN)-regulated polar auxin transport in Arabidopsis. The SCOOP4 and SCOOP12 treatments impaired root gravitropic growth, auxin redistribution in response to gravistimulation, and PIN abundance in the PM. Furthermore, genetic and cell biological analyses revealed that these physiological and cellular effects of SCOOP4 and SCOOP12 peptides are mediated by the receptor MALE DISCOVERER1-INTERACTING RECEPTOR LIKE KINASE2 (MIK2) and the downstream mitogen-activated kinase MPK6. Biochemical evidence indicates that MPK6 directly phosphorylates the cytosolic loop of PIN proteins. Our work established a link between the immune signaling peptide SCOOPs and root growth pathways, providing insights into the molecular mechanisms underlying plant root adaptive growth in the defense response.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Proteínas Quinases Ativadas por Mitógeno , Raízes de Plantas , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Transporte Biológico , Transdução de Sinais , Regulação da Expressão Gênica de Plantas , Fosforilação , Gravitropismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genéticaRESUMO
The phenotypic switch of vascular smooth cells (VSMCs) from a contractile to a synthetic state is associated with the development and progression of aortic aneurysm (AA). However, the mechanism underlying this process remains unclear. In this issue of the JCI, Song et al. identified SLC44A2 as a regulator of the phenotypic switch in VSMCs. Inhibition of SLC44A2 facilitated the switch to the synthetic state, contributing to the development of AA. Mechanistically, SLC44A2 interacted with NRP1 and ITGB3 to activate the TGF-ß/SMAD signaling pathway, resulting in VSMCs with a contractile phenotype. Furthermore, VSMC-specific SLC44A2 overexpression by genetic or pharmacological manipulation reduced AA in mouse models. These findings suggest the potential of targeting the SLC44A2 signaling pathway for AA prevention and treatment.