RESUMO
Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondary active transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.
Assuntos
Transporte Biológico Ativo , Íons , Bicamadas Lipídicas , Lipídeos , Proteínas de Membrana Transportadoras , Transporte de Íons , Íons/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Carreadoras de Solutos/metabolismoRESUMO
The accumulation of metabolic waste is a leading cause of numerous neurological disorders, yet we still have only limited knowledge of how the brain performs self-cleansing. Here we demonstrate that neural networks synchronize individual action potentials to create large-amplitude, rhythmic and self-perpetuating ionic waves in the interstitial fluid of the brain. These waves are a plausible mechanism to explain the correlated potentiation of the glymphatic flow1,2 through the brain parenchyma. Chemogenetic flattening of these high-energy ionic waves largely impeded cerebrospinal fluid infiltration into and clearance of molecules from the brain parenchyma. Notably, synthesized waves generated through transcranial optogenetic stimulation substantially potentiated cerebrospinal fluid-to-interstitial fluid perfusion. Our study demonstrates that neurons serve as master organizers for brain clearance. This fundamental principle introduces a new theoretical framework for the functioning of macroscopic brain waves.
Assuntos
Encéfalo , Líquido Cefalorraquidiano , Líquido Extracelular , Neurônios , Potenciais de Ação , Encéfalo/citologia , Encéfalo/metabolismo , Ondas Encefálicas/fisiologia , Líquido Cefalorraquidiano/metabolismo , Líquido Extracelular/metabolismo , Sistema Glinfático/metabolismo , Cinética , Rede Nervosa/fisiologia , Neurônios/metabolismo , Optogenética , Tecido Parenquimatoso/metabolismo , Íons/metabolismoRESUMO
The introduction of AlphaFold 21 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design2-6. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein-ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein-nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody-antigen prediction accuracy compared with AlphaFold-Multimer v.2.37,8. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.
Assuntos
Aprendizado Profundo , Ligantes , Modelos Moleculares , Proteínas , Software , Humanos , Anticorpos/química , Anticorpos/metabolismo , Antígenos/metabolismo , Antígenos/química , Aprendizado Profundo/normas , Íons/química , Íons/metabolismo , Simulação de Acoplamento Molecular , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas/química , Proteínas/metabolismo , Reprodutibilidade dos Testes , Software/normasRESUMO
A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.
Assuntos
Bacteriófagos , Capsídeo , DNA Viral , Genoma Viral , Vírion , Montagem de Vírus , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Difusão , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Íons/análise , Íons/química , Íons/metabolismo , Eletricidade Estática , Vírion/química , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/genética , Água/análise , Água/química , Água/metabolismoRESUMO
Bio-integrated devices need power sources to operate1,2. Despite widely used technologies that can provide power to large-scale targets, such as wired energy supplies from batteries or wireless energy transduction3, a need to efficiently stimulate cells and tissues on the microscale is still pressing. The ideal miniaturized power source should be biocompatible, mechanically flexible and able to generate an ionic current for biological stimulation, instead of using electron flow as in conventional electronic devices4-6. One approach is to use soft power sources inspired by the electrical eel7,8; however, power sources that combine the required capabilities have not yet been produced, because it is challenging to obtain miniaturized units that both conserve contained energy before usage and are easily triggered to produce an energy output. Here we develop a miniaturized soft power source by depositing lipid-supported networks of nanolitre hydrogel droplets that use internal ion gradients to generate energy. Compared to the original eel-inspired design7, our approach can shrink the volume of a power unit by more than 105-fold and it can store energy for longer than 24 h, enabling operation on-demand with a 680-fold greater power density of about 1,300 W m-3. Our droplet device can serve as a biocompatible and biological ionic current source to modulate neuronal network activity in three-dimensional neural microtissues and in ex vivo mouse brain slices. Ultimately, our soft microscale ionotronic device might be integrated into living organisms.
Assuntos
Materiais Biocompatíveis , Fontes de Energia Bioelétrica , Materiais Biomiméticos , Condutividade Elétrica , Eletrônica , Íons , Animais , Camundongos , Elétrons , Hidrogéis/química , Íons/análise , Íons/metabolismo , Enguias , Rede Nervosa/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , MicroquímicaRESUMO
The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , Íons/metabolismo , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Linhagem Celular , Microscopia Crioeletrônica/métodos , AMP Cíclico/metabolismo , Células HEK293 , HumanosRESUMO
Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1-4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6-9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchange that is unrelated to vertebrate size and activity-perhaps at the very inception of pharyngeal pores in stem deuterostomes.
Assuntos
Brânquias , Íons , Oxigênio , Filogenia , Vertebrados , Animais , Brânquias/metabolismo , Anfioxos/metabolismo , Oxigênio/metabolismo , Vertebrados/classificação , Vertebrados/metabolismo , Íons/metabolismo , Tamanho Corporal , Lampreias/metabolismo , Pele/metabolismoRESUMO
Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore-a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.
Assuntos
Conexinas , Humanos , Conexinas/metabolismo , Conexinas/genética , Transporte de Íons , Animais , Mutação , Íons/metabolismo , Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Canais Iônicos/genéticaRESUMO
Signal transduction across biological membranes is central to life. This process generally happens through communication between different domains and hierarchical coupling of information. Here, we review structural and thermodynamic principles behind transmembrane (TM) signal transduction and discuss common themes. Communication between signaling domains can be understood in terms of thermodynamic and kinetic principles, and complex signaling patterns can arise from simple wiring of thermodynamically coupled domains. We relate this to functions of several signal transduction systems: the M2 proton channel from influenza A virus, potassium channels, integrin receptors, and bacterial kinases. We also discuss key features in the structural rearrangements responsible for signal transduction in these systems.
Assuntos
Comunicação Celular/fisiologia , Membrana Celular/fisiologia , Integrinas , Canais de Potássio , Proteínas da Matriz Viral , Integrinas/química , Integrinas/metabolismo , Íons/química , Íons/metabolismo , Ligantes , Modelos Moleculares , Canais de Potássio/química , Canais de Potássio/metabolismo , Conformação Proteica , Transdução de Sinais/fisiologia , Termodinâmica , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismoRESUMO
Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.
Assuntos
Artemia , Salinidade , Animais , Artemia/genética , Lisina , Sódio/metabolismo , Cloreto de Sódio/metabolismo , Íons/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Flower color diversity is a key taxonomic trait in Meconopsis species, enhancing their appeal as ornamental flowers. However, knowledge of the molecular mechanisms of flower color formation in Meconopsis species is still limited. M. wilsonii subsp. australis (Australis) and M. wilsonii subsp. orientalis (Orientalis) have a developmental stage presenting red-purple flowers, while Orientalis also presents blue coloration at the full-bloom period, making them an important model for exploring the mechanism of blue flower formation in M. wilsonii. In this study, we collected petals from Australis and Orientalis at different developmental stages to compare the coloration differences between the two species and detect the molecular mechanisms of blue color in Orientalis. We identified that cyanidin was the main anthocyanin in the flowers of both species, and the blue color in Orientalis primarily arises from anthocyanins (Cyanidin-3-O-sambubioside). RNA sequencing analysis was performed to detect the gene expression in the anthocyanin biosynthesis pathway, and the results suggested that gene regulation for anthocyanin biosynthesis may not be the direct reason for blue color formation in Orientalis. In addition, the growth solid of Orientalis was rich in Fe and Mg ions, and a large amount of Fe and Mg ions accumulated in the petals of Orientalis. Combined with the gene functional enrichment results, we found that the purple and red-purple colors of these two species were presented by different glycosylation levels of cyanidin, while the violet color of Orientalis might be the results of metalloanthocyanins by Fe and Mg ions, which also relieved the toxicity caused by the high content of Fe and Mg ions in its cells. The environmental adaptation-related genes were highly expressed of in both species, such as adaptation to desiccation, water deprivation, freezing, etc. Our results revealed the coloration differences between Australis and Orientalis and described the molecular mechanisms of blue coloration in Orientalis. The data in our analysis could enrich the genetic resources for M. wilsonii for further studies.
Assuntos
Antocianinas , Papaveraceae , Antocianinas/metabolismo , Papaveraceae/metabolismo , Fenótipo , Íons/metabolismo , Flores , Pigmentação/genética , Cor , Regulação da Expressão Gênica de PlantasRESUMO
The MerR family of transcriptional regulators includes a variety of bacterial cytoplasmic proteins that respond to a wide range of signals, including toxins, metal ions, and endogenous metabolites. Its best-characterized members share similar structural and functional features with the family founder, the mercury sensor MerR, although most of them do not respond to metal ions. The group of "canonical" MerR homologs displays common molecular mechanisms for controlling the transcriptional activation of their target genes in response to inducer signals. This includes the recognition of distinctive operator sequences located at suboptimal σ70 -dependent promoters. Interestingly, an increasing number of proteins assigned to the MerR family based on their DNA-binding domain do not match in structure, sequence, or mode of action with any of the canonical MerR-like regulators. Here, we analyzed several members of the family, including this last group. Based on a phylogenetic analysis, and similarities in structural/functional features and position of their target operators relative to the promoter elements, we propose to assign these "atypical/divergent" MerR regulators to a phylogenetically separated group. These atypical/divergent homologs represent a new class of transcriptional regulators with novel regulatory mechanisms.
Assuntos
Proteínas de Ligação a DNA , Metais , Proteínas de Ligação a DNA/metabolismo , Sequência de Bases , Filogenia , Regiões Promotoras Genéticas/genética , Metais/metabolismo , Proteínas de Bactérias/metabolismo , Íons/metabolismo , Regulação Bacteriana da Expressão Gênica/genéticaRESUMO
The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.
Assuntos
Proteínas Associadas a CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas do Segundo Mensageiro , Transdução de Sinais , Endonucleases/metabolismo , Íons/metabolismo , Sistemas CRISPR-Cas , Proteínas Associadas a CRISPR/metabolismoRESUMO
DNA double-strand breaks (DSBs) are functionally linked to genomic instability in spermatocytes and to male infertility. The heavy metal cadmium (Cd) is known to induce DNA damage in spermatocytes by unknown mechanisms. Here, we showed that Cd ions impaired the canonical non-homologous end-joining (NHEJ) repair pathway, but not the homologous recombination (HR) repair pathway, through stimulation of Ser2056 and Thr2609 phosphorylation of DNA-PKcs at DSB sites. Hyper-phosphorylation of DNA-PKcs led to its premature dissociation from DNA ends and the Ku complex, preventing recruitment of processing enzymes and further ligation of DNA ends. Specifically, this cascade was initiated by the loss of PP5 phosphatase activity, which results from the dissociation of PP5 from its activating ions (Mn), that is antagonized by Cd ions through a competitive mechanism. In accordance, in a mouse model Cd-induced genomic instability and consequential male reproductive dysfunction were effectively reversed by a high dosage of Mn ions. Together, our findings corroborate a protein phosphorylation-mediated genomic instability pathway in spermatocytes that is triggered by exchange of heavy metal ions.
Assuntos
Cádmio , Instabilidade Genômica , Infertilidade Masculina , Espermatócitos , Animais , Humanos , Masculino , Camundongos , Cádmio/toxicidade , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Instabilidade Genômica/efeitos dos fármacos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Íons/metabolismo , Fosforilação , Reparo de DNA por Recombinação , Espermatócitos/efeitos dos fármacosRESUMO
Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to GltPh. The binding sites for three Na+ ions (Na1-3) have been identified in GltPh, but the specific roles of the individual Na+ sites in the binding process have not been elucidated. In this study, we developed assays to probe Na+ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na+ binding to the Na3 site is required for the NMDGT conformational switch while Na+ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na+ binding to both the Na1 and the Na3 sites. Based on our studies, we also propose an alternate pathway for the coupled binding of Na+ and Asp.
Assuntos
Sistema X-AG de Transporte de Aminoácidos , Sódio , Sistema X-AG de Transporte de Aminoácidos/química , Sítios de Ligação , Ácido Glutâmico/metabolismo , Íons/metabolismo , Sódio/metabolismoRESUMO
Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Biofilmes/crescimento & desenvolvimento , Proteínas Amiloidogênicas/metabolismo , Proteínas de Bactérias/metabolismo , Matriz Extracelular/fisiologia , Íons/metabolismo , Espalhamento de Radiação , Espalhamento a Baixo Ângulo , Raios XRESUMO
ATB[Formula: see text] (SLC6A14) is a member of the amino acid transporter branch of the SLC6 family along with GlyT1 (SLC6A9) and GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na[Formula: see text]:Cl[Formula: see text], respectively. In contrast, ATB[Formula: see text] exhibits broad substrate specificity for all neutral and cationic amino acids, and its ionic coupling remains unsettled. Using the reversal potential slope method, we demonstrate a 3:1:1 Na[Formula: see text]:Cl[Formula: see text]:Gly stoichiometry for ATB[Formula: see text] that is consistent with its 2.1 e/Gly charge coupling. Like GlyT2, ATB[Formula: see text] behaves as a unidirectional transporter with virtually no glycine efflux at negative potentials after uptake, except by heteroexchange as remarkably shown by leucine activation of NMDARs in Xenopus oocytes coexpressing both membrane proteins. Analysis and computational modeling of the charge movement of ATB[Formula: see text] reveal a higher affinity for sodium in the absence of substrate than GlyT2 and a gating mechanism that locks Na[Formula: see text] into the apo-transporter at depolarized potentials. A 3:1 Na[Formula: see text]:Cl[Formula: see text] stoichiometry justifies the concentrative transport properties of ATB[Formula: see text] and explains its trophic role in tumor growth, while rationalizing its phylogenetic proximity to GlyT2 despite their extreme divergence in specificity.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina , Sódio , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos , Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/química , Íons/metabolismo , Leucina , Filogenia , Sódio/metabolismoRESUMO
The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.
Assuntos
Proteínas de Transporte de Cobre , Cobre , Peptídeos e Proteínas de Sinalização Intracelular , Metalochaperonas , Chaperonas Moleculares , Linhagem Celular Tumoral , Cobre/metabolismo , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Íons/metabolismo , Metalochaperonas/genética , Metalochaperonas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxirredução , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
The resistance of gram-negative bacteria to silver ions is mediated by a silver efflux pump, which mainly relies on a tripartite efflux complex SilCBA, a metallochaperone SilF and an intrinsically disordered protein SilE. However, the precise mechanism by which silver ions are extruded from the cell and the different roles of SilB, SilF, and SilE remain poorly understood. To address these questions, we employed nuclear magnetic resonance and mass spectrometry to investigate the interplay between these proteins. We first solved the solution structures of SilF in its free and Ag+-bound forms, and we demonstrated that SilB exhibits two silver binding sites in its N and C termini. Conversely to the homologous Cus system, we determined that SilF and SilB interact without the presence of silver ions and that the rate of silver dissociation is eight times faster when SilF is bound to SilB, indicating the formation of a SilF-Ag-SilB intermediate complex. Finally, we have shown that SilE does not bind to either SilF or SilB, regardless of the presence or absence of silver ions, further corroborating that it merely acts as a regulator that prevents the cell from being overloaded with silver. Collectively, we have provided further insights into protein interactions within the sil system that contribute to bacterial resistance to silver ions.