RESUMO
Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.
Assuntos
AMP Cíclico , Capacitação Espermática , Espermatozoides , Proteína com Valosina , Animais , Masculino , Capacitação Espermática/efeitos dos fármacos , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Espermatozoides/metabolismo , Camundongos , AMP Cíclico/metabolismo , Fosforilação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Copper homeostasis is a fundamental process in organisms, characterised by unique pathways that have evolved to meet specific needs while preserving core resistance mechanisms. While these systems are well-documented in model bacteria, information on copper resistance in species adapted to cold environments is scarce. This study investigates the potential genes related to copper homeostasis in the genome of Bizionia argentinensis (JUB59-T), a psychrotolerant bacterium isolated from Antarctic seawater. We identified several genes encoding proteins analogous to those crucial for copper homeostasis, including three sequences of copper-transport P1B-type ATPases. One of these, referred to as BaCopA1, was chosen for cloning and expression in Saccharomyces cerevisiae. BaCopA1 was successfully integrated into yeast membranes and subsequently extracted with detergent. The purified BaCopA1 demonstrated the ability to catalyse ATP hydrolysis at low temperatures. Structural models of various BaCopA1 conformations were generated and compared with mesophilic and thermophilic homologous structures. The significant conservation of critical residues and structural similarity among these proteins suggest a shared reaction mechanism for copper transport. This study is the first to report a psychrotolerant P1B-ATPase that has been expressed and purified in a functional form.
Assuntos
Temperatura Baixa , Cobre , Cobre/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Regiões Antárticas , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Genoma Bacteriano/genética , Água do Mar/microbiologia , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , ATPases Transportadoras de Cobre/química , Sulfolobaceae/genética , Sulfolobaceae/metabolismo , Sulfolobaceae/enzimologiaRESUMO
Oleic acid (OA) is a monounsaturated compound with many health-benefitting properties such as obesity prevention, increased insulin sensitivity, antihypertensive and immune-boosting properties, etc. The aim of this study was to analyze the effect of oleic acid (OA) and some anticancer drugs against oxidative damage induced by nitropropionic acid (NPA) in rat brain. Six groups of Wistar rats were treated as follows: Group 1, (control); group 2, OA; group 3, NPA + OA; group 4, cyclophosphamide (CPP) + OA; group 5, daunorubicin (DRB) + OA; and group 6, dexrazoxane (DXZ) + OA. All compounds were administered intraperitoneally route, every 24 h for 5 days. Their brains were extracted to measure lipoperoxidation (TBARS), H2O2, Ca+2, Mg+2 ATPase activity, glutathione (GSH) and dopamine. Glucose, hemoglobin and triglycerides were measured in blood. In cortex GSH increased in all groups, except in group 2, the group 4 showed the highest increase of this biomarker. TBARS decrease, and dopamine increase in all regions of groups 4, 5 and 6. H2O2 increased only in cerebellum/medulla oblongata of group 5 and 6. ATPase expression decreased in striatum of group 4. Glucose increased in group 6, and hemoglobin increased in groups 4 and 5. These results suggest that the increase of dopamine and the antioxidant effect of oleic acid administration during treatment with oncologic agents could result in less brain injury.
Assuntos
Antineoplásicos , Encéfalo , Glutationa , Ácido Oleico , Estresse Oxidativo , Ratos Wistar , Animais , Estresse Oxidativo/efeitos dos fármacos , Ácido Oleico/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ratos , Masculino , Glutationa/metabolismo , Antineoplásicos/farmacologia , Peróxido de Hidrogênio/metabolismo , Nitrocompostos/farmacologia , Dopamina/metabolismo , Propionatos/farmacologia , Ciclofosfamida , Peroxidação de Lipídeos/efeitos dos fármacos , Daunorrubicina/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Adenosina Trifosfatases/metabolismo , Antioxidantes/farmacologiaRESUMO
Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.
Assuntos
Archaeoglobus fulgidus , Proteínas de Membrana , Desnaturação Proteica , Dodecilsulfato de Sódio , Termodinâmica , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/farmacologia , Archaeoglobus fulgidus/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Espectrometria de Fluorescência , Estabilidade Proteica , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Naftalenossulfonato de Anilina/química , Naftalenossulfonato de Anilina/metabolismo , Triptofano/química , Triptofano/metabolismo , Cobre/química , Cobre/metabolismo , Dobramento de Proteína , Conformação ProteicaRESUMO
Adenosine triphosphate (ATP) regeneration is a significant step in both living cells and in vitro biotransformation (ivBT). Rotary motor ATP synthases (ATPases), which regenerate ATP in living cells, have been widely assembled in biomimetic structures for in vitro ATP synthesis. In this review, we present a comprehensive overview of ATPases, including the working principle, orientation and distribution density properties of ATPases, as well as the assembly strategies and applications of ATPase-based ATP regeneration modules. The original sources of ATPases for in vitro ATP regeneration include chromatophores, chloroplasts, mitochondria, and inverted Escherichia coli (E. coli) vesicles, which are readily accessible but unstable. Although significant advances have been made in the assembly methods for ATPase-artificial membranes in recent decades, it remains challenging to replicate the high density and orientation of ATPases observed in vivo using in vitro assembly methods. The use of bioproton pumps or chemicals for constructing proton motive forces (PMF) enables the versatility and potential of ATPase-based ATP regeneration modules. Additionally, overall robustness can be achieved via membrane component selection, such as polymers offering great mechanical stability, or by constructing a solid supporting matrix through layer-by-layer assembly techniques. Finally, the prospects of ATPase-based ATP regeneration modules can be expected with the technological development of ATPases and artificial membranes.
Assuntos
Adenosina Trifosfatases , Trifosfato de Adenosina , Biotransformação , Trifosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMO
BACKGROUND: Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown. RESULTS: Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome. CONCLUSIONS: Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA , Nucleossomos , Nucleossomos/metabolismo , Nucleossomos/genética , Montagem e Desmontagem da Cromatina/fisiologia , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histonas/metabolismoRESUMO
Paraquat (1,1'-dimethyl-4,4'-bipyridyl dichloride) is an herbicide widely used worldwide and officially banned in Brazil in 2020. Kidney lesions frequently occur, leading to acute kidney injury (AKI) due to exacerbated reactive O2 species (ROS) production. However, the consequences of ROS exposure on ionic transport and the regulator local renin-angiotensin-aldosterone system (RAAS) still need to be elucidated at a molecular level. This study evaluated how ROS acutely influences Na+-transporting ATPases and the renal RAAS. Adult male Wistar rats received paraquat (20 mg/kg; ip). After 24 h, we observed body weight loss and elevation of urinary flow and serum creatinine. In the renal cortex, paraquat increased ROS levels, NADPH oxidase and (Na++K+)ATPase activities, angiotensin II-type 1 receptors, tumor necrosis factor-α (TNF-α), and interleukin-6. In the medulla, paraquat increased ROS levels and NADPH oxidase activity but inhibited (Na++K+)ATPase. Paraquat induced opposite effects on the ouabain-resistant Na+-ATPase in the cortex (decrease) and medulla (increase). These alterations, except for increased serum creatinine and renal levels of TNF-α and interleukin-6, were prevented by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol; 1 mmol/L in drinking water), a stable antioxidant. In summary, after paraquat poisoning, ROS production culminated with impaired medullary function, urinary fluid loss, and disruption of Na+-transporting ATPases and angiotensin II signaling.
Assuntos
Paraquat , Sistema Renina-Angiotensina , Ratos , Animais , Masculino , Espécies Reativas de Oxigênio/metabolismo , Paraquat/metabolismo , Paraquat/farmacologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Creatinina/metabolismo , Creatinina/urina , Interleucina-6 , Fator de Necrose Tumoral alfa/metabolismo , Ratos Wistar , Rim , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/farmacologia , Sódio/metabolismo , Sódio/farmacologia , NADPH Oxidases/metabolismo , NADPH Oxidases/farmacologiaRESUMO
Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, we elaborated the characterization of Mt-PhoH2 through biophysical, biochemical, and computational methods. In addition to its intrinsic ATPase activity, the biochemical experiments revealed its GTPase activity and both activities are metal ion dependent. Magnesium, manganese, copper, iron, nickel, zinc, cesium, calcium, and lithium were examined for their effect on activity, and the optimum activity was found with 10 mM of Mg2+ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as Km 4.873 ± 0.44 µM, Vmax 12.3817 ± 0.084 µM/min/mg, Kcat 0.0075 ± 0.00005 s-1, and Kcat/Km 0.0015 ± 0.000001 µM-1 s-1 with GTP. In the case of GTP as a substrate, a 20% decrease in enzymatic activity and a 50% increase in binding affinity of Mt-PhoH2 were observed. The substrates ADP and GDP inhibit the ATPase and GTPase activity of Mt-PhoH2. CD spectroscopy showed the dominance of alpha helix in the secondary structure of Mt-PhoH2, and this structural pattern was altered upon addition of ATP and GTP. In silico inhibitor screening revealed ML141 and NAV_2729 as two potential inhibitors of the catalytic activity of Mt-PhoH2. Mt-PhoH2 is essential for mycobacterial growth as its knockdown strain showed a decreased growth effect. Overall, the present article emphasizes the factors essential for the proper functioning of Mt-PhoH2 which is a participant in the toxin-antitoxin machinery and may also play an important role in phosphate starvation.
Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cinética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/químicaRESUMO
J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.
Assuntos
Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico , Humanos , Adenosina Trifosfatases/metabolismo , Ácido Edético , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico HSP70/química , Chaperonas Moleculares/químicaRESUMO
The communication between the intestinal epithelium and the enteric nervous system has been considered indirect. Mechanical or chemical stimuli activate enteroendocrine cells inducing hormone secretion, which act on sub-epithelial nerve ends, activating the enteric nervous system. However, we identified an epithelial cell that expresses NKAIN4, a neuronal protein associated with the ß-subunit of Na+/K+-ATPase. This cell overexpresses Na+/K+-ATPase and ouabain-insensitive Na+-ATPase, enzymes involved in active sodium transport. NKAIN4-positive cells also express neuronal markers as NeuN, acetylcholine-esterase, acetylcholine-transferase, α3- and α7-subunits of ACh receptors, glutamic-decarboxylase, and serotonin-receptor-7, suggesting they are neurons. NKAIN4-positive cells show a polarized shape with an oval body, an apical process finished in a knob-like terminal in contact with the lumen, a basal cilia body at the base of the apical extension, and basal axon-like soma projections connecting sub-epithelial nerve terminals, lymphoid nodules, glial cells, and enterochromaffin cells, forming a network that reaches the epithelial surface. We also showed, using retrograde labeling and immunofluorescence, that these cells receive afferent signals from the enteric nervous system. Finally, we demonstrated that acetylcholine activates NKAIN4-positive cells inducing Ca2+ mobilization and probably serotonin secretion in enterochromaffin cells. NKAIN4-positive cells are neurons that would form a part of a duodenal sensory network for physiological or noxious luminal stimuli.