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1.
J Agric Food Chem ; 69(35): 10114-10120, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428895

RESUMO

Our previous study showed that lycopene reduced the absorption of cholesterol in Caco-2 cells through inhibiting Niemann-Pick C1-Like 1 (NPC1L1) expression. Herein, we aimed to explore whether lycopene supplementation can decrease cholesterol absorption in the intestine and prevent atherosclerosis progression in high-fat diet (HFD)-fed apolipoprotein E knockout (ApoE-/-) mice. Male ApoE-/- mice were fed a high-fat diet with or without lycopene for 19 weeks. Supplementation of lycopene markedly lowered serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels. Additionally, serum high-density lipoprotein cholesterol (HDL-C) levels were increased after lycopene administration. Lycopene also downregulated the expression of NPC1L1 and hepatocyte nuclear factor-1α (HNF-1α) in the small intestine. Furthermore, the Oil Red O staining of the aorta and aortic sinus showed that lycopene supplementation remarkably reduced atherosclerotic lesions. These results indicated that lycopene inhibited intestinal cholesterol absorption and protected against HFD-induced atherosclerosis through inhibiting HNF-1α and NPC1L1 expression. Lycopene exhibits a potential antiatherosclerotic effect through suppressing intestinal cholesterol absorption.


Assuntos
Apolipoproteínas E , Aterosclerose , Animais , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/prevenção & controle , Células CACO-2 , Colesterol , Fator 1-alfa Nuclear de Hepatócito , Humanos , Absorção Intestinal , Licopeno , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
2.
Structure ; 29(8): 781-782, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34358463

RESUMO

The bacterial Sec translocase transports unfolded proteins across membranes. In this issue of Structure, Krishnamurthy et al. (2021) report a nexus of conformational dynamics in the translocase motor protein, SecA. Their findings shed light on the Sec activation mechanism and suggest a general role for multi-level dynamics in protein functions.


Assuntos
Proteínas de Bactérias , Proteínas de Membrana Transportadoras , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico , Canais de Translocação SEC/genética , Proteínas SecA
3.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361043

RESUMO

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.


Assuntos
Antígenos de Neoplasias/química , ATPases Transportadoras de Cobre/química , Melanossomas/metabolismo , Proteínas de Membrana Transportadoras/química , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/química , Prótons , Antígenos de Neoplasias/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Melanossomas/química , Proteínas de Membrana Transportadoras/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Estabilidade Proteica
4.
Molecules ; 26(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34279427

RESUMO

The functioning of mitochondria and their biogenesis are largely based on the proper function of the mitochondrial outer membrane channels, which selectively recognise and import proteins but also transport a wide range of other molecules, including metabolites, inorganic ions and nucleic acids. To date, nine channels have been identified in the mitochondrial outer membrane of which at least half represent the mitochondrial protein import apparatus. When compared to the mitochondrial inner membrane, the presented channels are mostly constitutively open and consequently may participate in transport of different molecules and contribute to relevant changes in the outer membrane permeability based on the channel conductance. In this review, we focus on the channel structure, properties and transported molecules as well as aspects important to their modulation. This information could be used for future studies of the cellular processes mediated by these channels, mitochondrial functioning and therapies for mitochondria-linked diseases.


Assuntos
Canais Iônicos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Membranas Mitocondriais/metabolismo , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Transporte Proteico
5.
Am J Physiol Regul Integr Comp Physiol ; 321(3): R377-R384, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34318705

RESUMO

The purpose of this study was to investigate if the cardiovascular system is important for ammonia excretion in the early life stages of zebrafish. Morpholino knockdowns of cardiac troponin T (TNNT2) or vascular endothelial growth factor A (VEGFA) provided morphants with nonfunctional circulation. At the embryonic stage [30-36 h postfertilization (hpf)], ammonia excretion was not constrained by a lack of cardiovascular function. At 2 days postfertilization (dpf) and 4 dpf, morpholino knockdowns of TNNT2 or VEGFA significantly reduced ammonia excretion in all morphants. Expression of rhag, rhbg, and rhcgb showed no significant changes but the mRNA levels of the urea transporter (ut) were upregulated in the 4 dpf morphants. Taken together, rhag, rhbg, rhcgb, and ut gene expression and an unchanged tissue ammonia concentration but an increased tissue urea concentration, suggest that impaired ammonia excretion led to increased urea synthesis. However, in larvae anesthetized with tricaine or clove oil, ammonia excretion was not reduced in the 4 dpf morphants compared with controls. Furthermore, oxygen consumption was reduced in morphants regardless of anesthesia. These results suggest that cardiovascular function is not directly involved in ammonia excretion, but rather reduced activity and external convection may explain reduced ammonia excretion and compensatory urea accumulation in morphants with reduced cardiovascular function.


Assuntos
Sistema Cardiovascular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ureia/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Brânquias/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
6.
Genes (Basel) ; 12(6)2021 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-34204109

RESUMO

Increasing evidence suggests that the Translocase of Outer Mitochondria Membrane 40 (TOMM40) gene may contribute to the risk of Alzheimer's disease (AD). Currently, there is no consensus as to whether TOMM40 expression is up- or down-regulated in AD brains, hindering a clear interpretation of TOMM40's role in this disease. The aim of this study was to determine if TOMM40 RNA levels differ between AD and control brains. We applied RT-qPCR to study TOMM40 transcription in human postmortem brain (PMB) and assessed associations of these RNA levels with genetic variants in APOE and TOMM40. We also compared TOMM40 RNA levels with mitochondrial functions in human cell lines. Initially, we found that the human genome carries multiple TOMM40 pseudogenes capable of producing highly homologous RNAs that can obscure precise TOMM40 RNA measurements. To circumvent this obstacle, we developed a novel RNA expression assay targeting the primary transcript of TOMM40. Using this assay, we showed that TOMM40 RNA was upregulated in AD PMB. Additionally, elevated TOMM40 RNA levels were associated with decreases in mitochondrial DNA copy number and mitochondrial membrane potential in oxidative stress-challenged cells. Overall, differential transcription of TOMM40 RNA in the brain is associated with AD and could be an indicator of mitochondrial dysfunction.


Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Linhagem Celular , Células Cultivadas , Feminino , Dosagem de Genes , Humanos , Masculino , Potencial da Membrana Mitocondrial , Proteínas de Membrana Transportadoras/metabolismo , Estresse Oxidativo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima
7.
Methods Mol Biol ; 2342: 339-365, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272701

RESUMO

Drug transporters are universally acknowledged as important determinants of the absorption, distribution, metabolism, and excretion of both endogenous and exogenous compounds. Altered transporter function, whether due to genetic polymorphism, DDIs, disease, or environmental factors such as dietary constituents, can result in changes in drug efficacy and/or toxicity due to changes in circulating or tissue levels of either drugs or endogenous substrates.Prediction of whether and to what extent the biological fate of a drug is influenced by drug transporters, therefore, requires in vitro test systems that can accurately predict the risk and magnitude of clinical DDIs. While these in vitro assessments appear simple in theory, practitioners recognize that there are multiple factors that can influence experimental outcomes. A better understanding of these variables, including test compound characteristics, test systems, assay formats, and experimental design, will enable clear, actionable steps and translatable outcomes that may avoid unnecessary downstream clinical engagement. This chapter will delineate the role of these variables in improving in vitro assay outcomes.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Desenho de Fármacos , Interações Medicamentosas , Humanos , Cinética , Proteínas de Membrana Transportadoras/química , Projetos de Pesquisa
8.
Methods Mol Biol ; 2342: 369-417, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272702

RESUMO

Accurate estimation of in vivo clearance in human is pivotal to determine the dose and dosing regimen for drug development. In vitro-in vivo extrapolation (IVIVE) has been performed to predict drug clearance using empirical and physiological scalars. Multiple in vitro systems and mathematical modeling techniques have been employed to estimate in vivo clearance. The models for predicting clearance have significantly improved and have evolved to become more complex by integrating multiple processes such as drug metabolism and transport as well as passive diffusion. This chapter covers the use of conventional as well as recently developed methods to predict metabolic and transporter-mediated clearance along with the advantages and disadvantages of using these methods and the associated experimental considerations. The general approaches to improve IVIVE by use of appropriate scalars, incorporation of extrahepatic metabolism and transport and application of physiologically based pharmacokinetic (PBPK) models with proteomics data are also discussed. The chapter also provides an overview of the advantages of using such dynamic mechanistic models over static models for clearance predictions to improve IVIVE.


Assuntos
Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Cálculos da Dosagem de Medicamento , Vias de Eliminação de Fármacos , Hepatócitos/química , Humanos , Técnicas In Vitro , Cinética , Taxa de Depuração Metabólica , Modelos Teóricos , Proteômica
9.
Methods Mol Biol ; 2342: 709-735, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272714

RESUMO

Assessing the interactions of a new drug candidate with transporters, either as a substrate, inhibitor, or inducer, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to 11 for the EMA as of 2020. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the most relevant tools for the study and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter are unpublished at the time of the compilation of this book. It has been included in this chapter to provide a sense of the complexities in transporter kinetics to the reader.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Transporte Biológico , Células CACO-2 , Linhagem Celular , Cães , Interações Medicamentosas , Humanos , Células Madin Darby de Rim Canino , Projetos de Pesquisa
10.
Nat Commun ; 12(1): 4625, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330923

RESUMO

Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.


Assuntos
Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Canais Iônicos/metabolismo , Klebsiella/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Transporte Biológico , Microscopia Crioeletrônica/métodos , Canais Iônicos/química , Canais Iônicos/ultraestrutura , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica
11.
Commun Biol ; 4(1): 836, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226658

RESUMO

Transporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Anticorpos de Domínio Único/metabolismo , Staphylococcus aureus/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único/química , Infecções Estafilocócicas/microbiologia
12.
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
13.
Plant Sci ; 310: 110990, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315604

RESUMO

Transfer cells (TCs) develop extensive wall ingrowths to facilitate enhanced rates of membrane transport. In Arabidopsis, TCs trans-differentiate from phloem parenchyma (PP) cells abutting the sieve element/companion cell complex in minor veins of foliar tissues and, based on anatomy and expression of SWEET sucrose uniporters, are assumed to play pivotal roles in phloem loading. While wall ingrowth deposition in PP TCs is a dynamic process responding to abiotic stresses such as high light and cold, the transcriptional control of PP TC development, including deposition of the wall ingrowths themselves, is not understood. PP TC development is a trait of vegetative phase change, potentially linking wall ingrowth deposition with floral induction. Transcript profiling by RNA-seq identified NAC056 and NAC018 (NARS1 and NARS2) as putative regulators of wall ingrowth deposition, while recent single cell RNA-seq analysis of leaf vasculature identified PP-specific expression of NAC056. Numerous membrane transporters, particularly of the UmamiT family of amino acid efflux carriers, were also identified. Collectively, these findings, and the recent discovery that wall ingrowth deposition is regulated by sucrose-dependent loading activity of these cells, provide new insights into the biology of PP TCs and their importance to phloem loading in Arabidopsis, establishing these cells as a key transport hub for phloem loading.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Floema/metabolismo , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Folhas de Planta/metabolismo , Análise de Sequência de RNA/métodos
14.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299309

RESUMO

Rab GTPases are major coordinators of intracellular membrane trafficking, including vesicle transport, membrane fission, tethering, docking, and fusion events. Rab GTPases are roughly divided into two groups: conventional "small" Rab GTPases and atypical "large" Rab GTPases that have been recently reported. Some members of large Rab GTPases in mammals include Rab44, Rab45/RASEF, and Rab46. The genes of these large Rab GTPases commonly encode an amino-terminal EF-hand domain, coiled-coil domain, and the carboxyl-terminal Rab GTPase domain. A common feature of large Rab GTPases is that they express several isoforms in cells. For instance, Rab44's two isoforms have similar functions, but exhibit differential localization. The long form of Rab45 (Rab45-L) is abundantly distributed in epithelial cells. The short form of Rab45 (Rab45-S) is predominantly present in the testes. Both Rab46 (CRACR2A-L) and the short isoform lacking the Rab domain (CRACR2A-S) are expressed in T cells, whereas Rab46 is only distributed in endothelial cells. Although evidence regarding the function of large Rab GTPases has been accumulating recently, there are only a limited number of studies. Here, we report the recent findings on the large Rab GTPase family concerning their function in membrane trafficking, cell differentiation, related diseases, and knockout mouse phenotypes.


Assuntos
Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/metabolismo , Masculino , Mastócitos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Fenótipo , Domínios Proteicos , Linfócitos T/metabolismo , Proteínas rab de Ligação ao GTP/genética
15.
Nat Commun ; 12(1): 4554, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315891

RESUMO

The planktonic synthesis of reduced organophosphorus molecules, such as alkylphosphonates and aminophosphonates, represents one half of a vast global oceanic phosphorus redox cycle. Whilst alkylphosphonates tend to accumulate in recalcitrant dissolved organic matter, aminophosphonates do not. Here, we identify three bacterial 2-aminoethylphosphonate (2AEP) transporters, named AepXVW, AepP and AepSTU, whose synthesis is independent of phosphate concentrations (phosphate-insensitive). AepXVW is found in diverse marine heterotrophs and is ubiquitously distributed in mesopelagic and epipelagic waters. Unlike the archetypal phosphonate binding protein, PhnD, AepX has high affinity and high specificity for 2AEP (Stappia stellulata AepX Kd 23 ± 4 nM; methylphosphonate Kd 3.4 ± 0.3 mM). In the global ocean, aepX is heavily transcribed (~100-fold>phnD) independently of phosphate and nitrogen concentrations. Collectively, our data identifies a mechanism responsible for a major oxidation process in the marine phosphorus redox cycle and suggests 2AEP may be an important source of regenerated phosphate and ammonium, which are required for oceanic primary production.


Assuntos
Ácido Aminoetilfosfônico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Minerais/metabolismo , Fósforo/metabolismo , Rhodobacteraceae/metabolismo , Água do Mar/microbiologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Cinética , Oceanos e Mares , Oxirredução , Filogenia , Proteômica , Pseudomonas putida/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rhodobacteraceae/genética
16.
Methods Mol Biol ; 2315: 31-41, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302668

RESUMO

Droplet interface bilayers (DIBs) are an emerging tool within synthetic biology that aims to recreate biological processes in artificial cells. A critical component for the utility of these bilayers is controlled flow between compartments and, notably, uphill transport against a substrate concentration gradient. A versatile method to achieve the desired flow is to exploit the specificity of membrane proteins that regulate the movement of ions and transport of specific metabolic compounds. Methods have been in existence for some time to synthesize proteins within a droplet as well as incorporate membrane proteins into DIBS; however, there have been few reports combining synthesis and DIB incorporation for membrane transporters that demonstrate specific, uphill transport. This chapter presents two methods for the incorporation of a membrane transporter into a simple two-droplet DIB system, with the downhill and uphill transport reaction readily monitored by fluorescence microscopy.


Assuntos
Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Íons/metabolismo , Microscopia de Fluorescência/métodos , Movimento/fisiologia
17.
Development ; 148(14)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34137447

RESUMO

Arabidopsis VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC) is a plant-specific transmembrane protein that controls the development of veins in cotyledons. Here, we show that the expression and localization of the auxin efflux carrier PIN-FORMED1 (PIN1) is altered in vcc developing cotyledons and that overexpression of PIN1-GFP partially rescues vascular defects of vcc in a dosage-dependent manner. Genetic analyses suggest that VCC and PINOID (PID), a kinase that regulates PIN1 polarity, are both required for PIN1-mediated control of vasculature development. VCC expression is upregulated by auxin, likely as part of a positive feedback loop for the progression of vascular development. VCC and PIN1 localized to the plasma membrane in pre-procambial cells but were actively redirected to vacuoles in procambial cells for degradation. In the vcc mutant, PIN1 failed to properly polarize in pre-procambial cells during the formation of basal strands, and instead, it was prematurely degraded in vacuoles. VCC plays a role in the localization and stability of PIN1, which is crucial for the transition of pre-procambial cells into procambial cells that are involved in the formation of basal lateral strands in embryonic cotyledons.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/genética , Desenvolvimento Embrionário , Regulação da Expressão Gênica de Plantas , Proteínas de Arabidopsis/genética , Transporte Biológico , Cotilédone/citologia , Cotilédone/genética , Cotilédone/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Folhas de Planta/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
18.
Plant Cell Rep ; 40(8): 1565-1583, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34132878

RESUMO

Stress arising due to abiotic factors affects the plant's growth and productivity. Among several existing abiotic stressors like cold, drought, heat, salinity, heavy metal, etc., drought condition tends to affect the plant's growth by inducing two-point effect, i.e., it disturbs the water balance as well as induces toxicity by disturbing the ion homeostasis, thus hindering the growth and productivity of plants, and to survive under this condition, plants have evolved several transportation systems that are involved in regulating the drought stress. The role of membrane transporters has gained interest since genetic engineering came into existence, and they were found to be the important modulators for tolerance, avoidance, ion movements, stomatal movements, etc. Here in this comprehensive review, we have discussed the role of transporters (ABA, protein, carbohydrates, etc.) and channels that aids in withstanding the drought stress as well as the regulatory role of transporters involved in osmotic adjustments arising due to drought stress. This review also provides a gist of hydraulic conductivity by roots that are involved in regulating the drought stress.


Assuntos
Secas , Canais Iônicos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Desidratação , Engenharia Genética , Canais Iônicos/genética , Proteínas de Membrana Transportadoras/genética , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/fisiologia
19.
New Phytol ; 231(5): 1814-1831, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34086995

RESUMO

Auxin homeostasis and signaling affect a broad range of developmental processes in plants. The interplay between HSP90 and auxin signaling is channeled through the chaperoning capacity of the HSP90 on the TIR1 auxin receptor. The sophisticated buffering capacity of the HSP90 system through the interaction with diverse signaling protein components drastically shapes genetic circuitries regulating various developmental aspects. However, the elegant networking capacity of HSP90 in the global regulation of auxin response and homeostasis has not been appreciated. Arabidopsis hsp90 mutants were screened for gravity response. Phenotypic analysis of root meristems and cotyledon veins was performed. PIN1 localization in hsp90 mutants was determined. Our results showed that HSP90 affected the asymmetrical distribution of PIN1 in plasma membranes and influenced its expression in prompt cell niches. Depletion of HSP90 distorted polar distribution of auxin, as the acropetal auxin transport was highly affected, leading to impaired root gravitropism and lateral root formation. The essential role of the HSP90 in auxin homeostasis was profoundly evident from early development, as HSP90 depletion affected embryo development and the pattern formation of veins in cotyledons. Our data suggest that the HSP90-mediated distribution of PIN1 modulates auxin distribution and thereby auxin signaling to properly promote plant development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Choque Térmico HSP90 , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Ácidos Indolacéticos , Proteínas de Membrana Transportadoras/metabolismo
20.
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
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