RESUMO
Highly resolved spectra of the 16O35Cl16O isotopologue of chlorine dioxide were recorded with a Bruker IFS 125HR Fourier transform infrared spectrometer in the region of the ν3 band. The analysis was made in the frame of the spin-rotational effective Hamiltonian (in A-reduction and Ir-representation) taking into account spin-rotational coupling operators up to the sixth order and the corresponding reduction of the Hamiltonian. The mathematical description of the ro-vibrational spectra was implemented to the specially created computer program ROVDES. Under the present experimental conditions, we were able to assign more than 5200 spin-rotational transitions to the ν3 band. This number is 2.4 times higher compared to the previous studies available from the literature. The vibrational ground state parameters were improved, and the 2220 upper spin-rotation-vibration energy levels were determined and used as initial data in the inverse spectroscopic problem with the derived effective spin-rotational Hamiltonian. A total of 37 fitted parameters were determined (22 rotational and centrifugal parameters and 15 parameters of spin-rotation coupling). The appearance of strong Coriolis resonance interactions between the (001) and (100) vibrational states in the sets of (001)[N,Ka = 9], (001)[N,Ka = 17], (001)[N,Ka = 18] and (001)[N,Ka = 19] spin-rotation-vibration levels was experimentally observed for the first time and explained. The drms = 1.4 × 10-4 cm-1 reproduction of the initial "experimental" upper ro-vibrational energy values was achieved which is considerably better compared to the use of parameters from a previous study ([J. Ortigoso et al., J. Mol. Spectrosc., 1992, 155, 25-43]).
RESUMO
The aims of this study were to develop co-delivery systems of paclitaxel (PTX) and etoposide prodrug (4'-O-benzyloxycarbonyl-etoposide, ETP-cbz) based on non-cross-linked human serum albumin (HSA) and poly(lactide-co-glycolide) nanoparticles and to evaluate the synergistic potential of these drugs in vitro. The nanoformulations were prepared by the high-pressure homogenisation technique and characterised using DLS, TEM, SEM, AFM, HPLC, CZE, in-vitro release, and cytotoxicity in human and murine glioma cells. All nanoparticles had 90-150 nm in size and negative ζ-potentials. The Neuro2A cells were the most sensitive to both HSA- and PLGA-based co-delivery systems (IC50 0.024 µM and 0.053 µM, respectively). The drugs' synergistic effect (combination index < 0.9) was observed in the GL261 cells for both types of co-delivery formulations and in the Neuro2A cells for the HSA-based system. These nanodelivery systems may be useful to improve combination chemotherapy for brain tumour treatment. To our knowledge, this is the first report describing the non-cross-linked HSA-based co-delivery nanosuspension which was prepared using nab™ technology.
Assuntos
Neoplasias Encefálicas , Nanopartículas , Pró-Fármacos , Humanos , Camundongos , Animais , Paclitaxel/farmacologia , Etoposídeo/farmacologia , Pró-Fármacos/farmacologia , Albumina Sérica Humana , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológicoRESUMO
A primary function of the H+-ATPase (or V-ATPase) is to create an electrochemical proton gradient across eukaryotic cell membranes, which energizes fundamental cellular processes. Its activity allows for the acidification of intracellular vesicles and organelles, which is necessary for many essential cell biological events to occur. In addition, many specialized cell types in various organ systems such as the kidney, bone, male reproductive tract, inner ear, olfactory mucosa, and more, use plasma membrane V-ATPases to perform specific activities that depend on extracellular acidification. It is, however, increasingly apparent that V-ATPases are central players in many normal and pathophysiological processes that directly influence human health in many different and sometimes unexpected ways. These include cancer, neurodegenerative diseases, diabetes, and sensory perception, as well as energy and nutrient-sensing functions within cells. This review first covers the well-established role of the V-ATPase as a transmembrane proton pump in the plasma membrane and intracellular vesicles and outlines factors contributing to its physiological regulation in different cell types. This is followed by a discussion of the more recently emerging unconventional roles for the V-ATPase, such as its role as a protein interaction hub involved in cell signaling, and the (patho)physiological implications of these interactions. Finally, the central importance of endosomal acidification and V-ATPase activity on viral infection will be discussed in the context of the current COVID-19 pandemic.
Assuntos
COVID-19/virologia , ATPases Translocadoras de Prótons/metabolismo , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/metabolismo , Humanos , ATPases Translocadoras de Prótons/genética , Transdução de Sinais , Internalização do VírusRESUMO
Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.
Assuntos
Aquaporina 2/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Coletores/metabolismo , Rim/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Sequência de Bases , Biomarcadores/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Proteína Jagged-1/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais , Transportadores de Sulfato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
KEY POINTS: In the epididymis, elaborate communication networks between epithelial cells are important with respect to establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa, which is essential for male fertility. Proton secretion by epididymal clear cells is achieved via the proton pumping V-ATPase located in their apical membrane. In the present study, we dissect the molecular mechanisms by which clear cells respond to luminal ATP and adenosine to modulate their acidifying activity via the adenosine receptor ADORA2B and the pH-sensitive ATP receptor P2X4. We demonstrate that the hydrolysis of ATP to produce adenosine by ectonucleotidases plays a key role in V-ATPase-dependent proton secretion, and is part of a feedback loop that ensures acidification of the luminal compartment These results help us better understand how professional proton-secreting cells respond to extracellular cues to modulate their functions, and how they communicate with neighbouring cells. ABSTRACT: Cell-cell cross-talk is crucial for the dynamic function of epithelia, although how epithelial cells detect and respond to variations in extracellular stimuli to modulate their environment remains incompletely understood. In the present study, we used the epididymis as a model system to investigate epithelial cell regulation by luminal factors. In the epididymis, elaborate communication networks between the different epithelial cell types are important for establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa. In particular, clear cells (CCs) secrete protons into the lumen via the proton pumping V-ATPase located in their apical membrane, a process that is activated by luminal alkalinization. However, how CCs detect luminal pH variations to modulate their function remains uncharacterized. Purinergic regulation of epithelial transport is modulated by extracellular pH in other tissues. In the present study, functional analysis of the mouse cauda epididymis perfused in vivo showed that luminal ATP and adenosine modulate the acidifying activity of CCs via the purinergic ADORA2B and P2X4 receptors, and that luminal adenosine content is itself regulated by luminal pH. Altogether, our observations illustrate mechanisms by which CCs are activated by pH sensitive P2X4 receptor and ectonucleotidases, providing a feedback mechanism for the maintenance of luminal pH. These novel mechanisms by which professional proton-secreting cells respond to extracellular cues to modulate their functions, as well as how they communicate with neighbouring cells, might be translatable to other acidifying epithelia.
Assuntos
Trifosfato de Adenosina/farmacologia , Adenosina/farmacologia , Epididimo/fisiologia , Purinérgicos , Agonistas Purinérgicos/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Epididimo/efeitos dos fármacos , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Antagonistas Purinérgicos/farmacologia , Receptor A2B de Adenosina/genética , Receptor A2B de Adenosina/metabolismo , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismo , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N1-402) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N1-402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber Icyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N1-352 fragment. Next, we combined it with the determined NMR structures of peptides a2N368-395 and a2N386-402 of the C-terminal section of a2N1-402. The complete molecular model of a2N1-402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N1-402. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N1-402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N1-17 and a2N35-49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.
Assuntos
Desenho de Fármacos , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sequência de Aminoácidos , Animais , Bactérias , Sítios de Ligação , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
We recently reported that nuclear receptor coactivator 7 (Ncoa7) is a vacuolar proton pumping ATPase (V-ATPase) interacting protein whose function has not been defined. Ncoa7 is highly expressed in the kidney and partially colocalizes with the V-ATPase in collecting duct intercalated cells (ICs). Here, we hypothesized that targeted deletion of the Ncoa7 gene could affect V-ATPase activity in ICs in vivo. We tested this by analyzing the acid-base status, major electrolytes, and kidney morphology of Ncoa7 knockout (KO) mice. We found that Ncoa7 KO mice, similar to Atp6v1b1 KOs, did not develop severe distal renal tubular acidosis (dRTA), but they exhibited a persistently high urine pH and developed hypobicarbonatemia after acid loading with ammonium chloride. Conversely, they did not develop significant hyperbicarbonatemia and alkalemia after alkali loading with sodium bicarbonate. We also found that ICs were larger and with more developed apical microvilli in Ncoa7 KO compared with wild-type mice, a phenotype previously associated with metabolic acidosis. At the molecular level, the abundance of several V-ATPase subunits, carbonic anhydrase 2, and the anion exchanger 1 was significantly reduced in medullary ICs of Ncoa7 KO mice, suggesting that Ncoa7 is important for maintaining high levels of these proteins in the kidney. We conclude that Ncoa7 is involved in IC function and urine acidification in mice in vivo, likely through modulating the abundance of V-ATPase and other key acid-base regulators in the renal medulla. Consequently, mutations in the NCOA7 gene may also be involved in dRTA pathogenesis in humans.
Assuntos
Equilíbrio Ácido-Base , Acidose Tubular Renal/genética , Deleção de Genes , Túbulos Renais/metabolismo , Coativadores de Receptor Nuclear/genética , Acidose Tubular Renal/patologia , Acidose Tubular Renal/fisiopatologia , Acidose Tubular Renal/urina , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anidrase Carbônica II/genética , Anidrase Carbônica II/metabolismo , Predisposição Genética para Doença , Concentração de Íons de Hidrogênio , Túbulos Renais/patologia , Túbulos Renais/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativadores de Receptor Nuclear/deficiência , Fenótipo , Urina/química , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The vacuolar H+ ATPase (V-ATPase) is a complex multisubunit machine that regulates important cellular processes through controlling acidity of intracellular compartments in eukaryotes. Existing small-molecule modulators of V-ATPase either are restricted to targeting one membranous subunit of V-ATPase or have poorly understood mechanisms of action. Small molecules with novel and defined mechanisms of inhibition are thus needed to functionally characterize V-ATPase and to fully evaluate the therapeutic relevance of V-ATPase in human diseases. We have discovered electrophilic quinazolines that covalently modify a soluble catalytic subunit of V-ATPase with high potency and exquisite proteomic selectivity as revealed by fluorescence imaging and chemical proteomic activity-based profiling. The site of covalent modification was mapped to a cysteine residue located in a region of V-ATPase subunit A that is thought to regulate the dissociation of V-ATPase. We further demonstrate that a previously reported V-ATPase inhibitor, 3-bromopyruvate, also targets the same cysteine residue and that our electrophilic quinazolines modulate the function of V-ATPase in cells. With their well-defined mechanism of action and high proteomic specificity, the described quinazolines offer a powerful set of chemical probes to investigate the physiological and pathological roles of V-ATPase.
Assuntos
Modelos Biológicos , Bibliotecas de Moléculas Pequenas/química , ATPases Vacuolares Próton-Translocadoras/química , Estrutura Molecular , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H(+)-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1-17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1-17) and its amino acids Phe(5), Met(10), and Gln(14) involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1-17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1-a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , DNA Complementar/metabolismo , Epitopos/química , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Microscopia Confocal/métodos , Dados de Sequência Molecular , Peptídeos/química , Isoformas de Proteínas , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/química , Transdução de Sinais , Triptofano/químicaRESUMO
The proton pumping V-ATPase drives essential biological processes, such as acidification of intracellular organelles. Critically, the V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting kidney intercalated cells (ICs). However, little is known about the molecular regulation of V-ATPase in mammals. We identified a novel interactor of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), aka Rabconnectin-3A. The yeast homologue of Dmxl1, Rav1p, is part of a complex that catalyzes the reversible assembly of the domains. We, therefore,hypothesized that Dmxl1 is a mammalian V-ATPase assembly factor. Here, we generated kidney IC-specific Dmxl1 knockout (KO) mice, which had high urine pH, like B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting showed decreased B1 expression and B1 (V1) and a4 (VO) subunits were more intracellular and less colocalized in Dmxl1 KO ICs. In parallel, subcellular fractionation revealed less V1 associated B1 in the membrane fraction of KO cells relative to the cytosol. Furthermore, a proximity ligation assay performed using probes against B1 and a4 V-ATPase subunits also revealed decreased association. We propose that loss of Dmxl1 reduces V-ATPase holoenzyme assembly, thereby inhibiting proton pumping function. Dmxl1 may recruit the V1 domain to the membrane and facilitate assembly with the VO domain and in its absence V1 may be targeted for degradation. We conclude that Dmxl1 is a bona fide mammalian V-ATPase assembly factor.
Assuntos
Camundongos Knockout , ATPases Vacuolares Próton-Translocadoras , Animais , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Camundongos , Rim/metabolismo , Genes Essenciais/genéticaRESUMO
Previously, we demonstrated that the vacuolar-type H(+)-ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1-a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: K(D) = 2.84 × 10(-10) M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms.
Assuntos
Forma Celular , Vesículas Citoplasmáticas/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/ultraestrutura , Endossomos/metabolismo , Frutose-Bifosfato Aldolase/genética , Proteínas Ativadoras de GTPase/genética , Gelsolina/genética , Gelsolina/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant KD=3.44x10(-7) M, similar to the KD=3.13x10(-7) M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser392. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser392 phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.
Assuntos
Proteínas Ativadoras de GTPase/química , ATPases Vacuolares Próton-Translocadoras/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Isoenzimas/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Homologia Estrutural de ProteínaRESUMO
Induction of the iron regulatory hormone hepcidin contributes to the anemia of inflammation. Bone morphogenetic protein 6 (BMP6) signaling is a central regulator of hepcidin expression in the liver. Recently, the TGF-ß/BMP superfamily member activin B was implicated in hepcidin induction by inflammation via noncanonical SMAD1/5/8 signaling, but its mechanism of action and functional significance in vivo remain uncertain. Here, we show that low concentrations of activin B, but not activin A, stimulate prolonged SMAD1/5/8 signaling and hepcidin expression in liver cells to a similar degree as canonical SMAD2/3 signaling, and with similar or modestly reduced potency compared with BMP6. Activin B stimulates hepcidin via classical activin type II receptors ACVR2A and ACVR2B, noncanonical BMP type I receptors activin receptor-like kinase 2 and activin receptor-like kinase 3, and SMAD5. The coreceptor hemojuvelin binds to activin B and facilitates activin B-SMAD1/5/8 signaling. Activin B-SMAD1/5/8 signaling has some selectivity for hepatocyte-derived cells and is not enabled by hemojuvelin in other cell types. Liver activin B mRNA expression is up-regulated in multiple mouse models of inflammation associated with increased hepcidin and hypoferremia, including lipopolysaccharide, turpentine, and heat-killed Brucella abortus models. Finally, the activin inhibitor follistatin-315 blunts hepcidin induction by lipopolysaccharide or B. abortus in mice. Our data elucidate a novel mechanism for noncanonical SMAD activation and support a likely functional role for activin B in hepcidin stimulation during inflammation in vivo.
Assuntos
Ativinas/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepcidinas/efeitos dos fármacos , Inflamação , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Linhagem Celular Tumoral , Hepatócitos/metabolismo , Hepcidinas/genética , Hepcidinas/metabolismo , Humanos , Immunoblotting , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/efeitos dos fármacos , Proteína Smad1/metabolismo , Proteína Smad5/efeitos dos fármacos , Proteína Smad5/metabolismo , Proteína Smad8/efeitos dos fármacos , Proteína Smad8/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Protein-lipid interactions are important for protein targeting, signal transduction, lipid transport, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. We recently cloned a 45-kDa protein from rat olfactory epithelium, which is homologous to the yeast Sec14p phosphatidylinositol (PtdIns) transfer protein and we report here that this protein binds to PtdIns(3,4,5)P3 and far weaker to less phosphorylated derivatives of PtdIns. Expression of the p45 protein in COS-1 cells resulted in accumulation of the protein in secretory vesicles and in the extracellular space. The secreted material contained PtdIns(3,4,5)P3. Our findings are the first report of a Sec14p-like protein involved in transport out of a cell and, to the best of our knowledge, inositol-containing phospholipids have not previously been detected in the extracellular space. Our findings suggest that p45 and phosphoinositides may participate in the formation of the protective mucus on nasal epithelium.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Animais , Carboxipeptidase H/metabolismo , Peso Molecular , RatosRESUMO
V-ATPases (H(+) ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology.
Assuntos
Mapas de Interação de Proteínas , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Imunoprecipitação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Dobramento de Proteína , Mapeamento de Interação de Proteínas/métodos , Transporte Proteico , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N(1-402)) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N(1-402) recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINE®12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N(1-402) in NDSB-256 was successfully used in pull-down and BIAcore™ protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N(1-402) and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called "cytosolic" tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N(1-402) should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.