Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor (R) associated with cardiac, renal and cerebral protective responses. Physiological evidence suggests that Mas R undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating R activity is unknown. We investigated the hypothesis that Mas R desensitizes and internalizes upon stimulation with Ang-(1-7). For this purpose, we generated a chimera between the Mas R and the fluorescent protein YFP (MasR-YFP). MasR-YFP transfected HEK 293T cells were incubated with Ang-(1-7) and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 min. Following the time course of [125I]Ang-(1-7) endocytosis we observed that half of MasR-YFP underwent endocytosis after 10 min and this was blocked by a Mas R antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1 and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin-1 suggesting that at some point MasR-YFP traverses caveolin-1 positive compartments. In conclusion, Mas R undergoes endocytosis upon stimulation with Ang-(1-7) and this event may explain the desensitization of Mas R responsiveness. In this way, Mas R activity and density may be tightly controlled by the cell.
The blue and green fluorescent proteins (BFP and GFP) have been fused at the N-and C-terminal ends, respectively, of the plasma membrane Ca 2؉ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca 2؉ -ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45 Å . The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca 2؉ -calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.The homeostasis of the intracellular Ca 2ϩ is crucial for cell function. The Ca 2ϩ ATPases from plasma membrane (PMCAs) 2 participate in the modulation of Ca 2ϩ signals and are responsible for the long term maintenance of the low concentration of intracellular Ca 2ϩ (1). The PMCAs belong to the P2-type ATPase superfamily of ion pumps and form an aspartyl phosphate intermediate during the transport cycle (2). Another essential feature of these ATPases is their ability to switch between two different conformational states from E 2 to E 1 in the presence of the transported ion.Human PMCAs are encoded by four separate genes, and additional variants are generated via alternative splicing of primary gene transcripts. PMCA4 is found virtually in all human tissues, and the splice variant xb is the most studied isoform. Computer modeling and sequence comparisons indicate that the overall structure of the PMCAs closely resembles that of other P-ATPases. Following the domain organization proposed for the SERCA (3, 4), the PMCA would contain a transmembrane region of 10 segments (M1-M10) and three major catalytic domains exposed to the cytosol. The nucleotide-binding (N) and the phosphorylation (P) domains contain the ATP binding site and the aspartate residue that forms the acyl phosphate intermediate, respectively, whereas the actuator domain (A) plays an essential role in the long range transmission of the conformational changes occurring during the transport cycle.Despite the clear overall homology, certain amino acid segments of the PMCA protein are not found in SERCA. The existence of these segments in the PMCA molecule has been generally associated with the extensive regulatory mechanisms that are known to alter the function of the PMCA. Indeed, the major difference between the two calcium pumps is the long C-terminal segment (C region) of the PMCA following M10, and this region is in...
Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.
Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design.
P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes, ATP13A (ATPase type 13A) 1-ATP13A5, that belong to the P5-ATPase group have been identified in humans. Mutations of the human gene ATP13A2 underlie a form of PD (Parkinson's disease). Previous studies have suggested a relation between polyamines and P5B-ATPases. We have recently shown that the cytotoxicity induced by the polyamine analogue paraquat (1,1'-dimethyl-4,4'-bipyridinium), which is an environmental agent related to PD development, was increased in ATP13A2-expressing CHO (Chinese-hamster ovary) cells. In the present study we showed that ATP13A2-expressing CHO cells exhibit a 2-fold higher accumulation of spermidine. Increasing concentrations of spermidine reduced the viability of CHO cells stably expressing ATP13A2. The higher levels of spermidine attained by the ATP13A2-expressing CHO cells were correlated with an increase in the ATP-dependent spermidine uptake in an isolated subcellular fraction containing lysosomes and late endosomes. The results of the present study support the idea that the human P5B-ATPase ATP13A2 is involved in polyamine uptake.
P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor-Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA). Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.
Background: Spf1 belongs to the least characterized group of P5-ATPases. Results: GFP-Spf1 hydrolyzes ATP and forms a phosphoenzyme that rapidly decays in the presence of ADP. Conclusion: The Spf1 performs well the E 1 steps of the reaction cycle, but progression to the E 2 forms is slow. Significance: The study extends the understanding of the catalytic mechanism of P5-ATPases.
We studied the kinetics of extracellular ATP (ATPe) in and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [P]Pi released from [γ-P]ATP. was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In, the addition of [γ-P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to increased luminal ATP 2 fold. ATPe regulation of depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. can activate ATP release from Caco-2 cells and intestinal segments, a response which might lead to intestinal release of ATP from the gut lumen.
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