Gerardo R. Corradi

Angiotensin (1-7) Induces Mas Receptor Internalization

Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein–coupled receptor Mas, a receptor associated with cardiac, renal, and cerebral protective responses. Physiological evidence suggests that Mas receptor (MasR) undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating receptor activity is unknown. We investigated the hypothesis that MasR desensitizes and internalizes on stimulation with Ang-(1-7). For this purpose, we generated a chimera between the MasR and the yellow 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 minutes. Following the time course of [125I]Ang-(1-7) endocytosis, we observed that half of MasR-YFP underwent endocytosis after 10 minutes, and this was blocked by a MasR 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, MasR undergoes endocytosis on stimulation with Ang-(1-7), and this event may explain the desensitization of MasR responsiveness. In this way, MasR activity and density may be tightly controlled by the cell.

Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ 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 Ca2+-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 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-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 Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity.

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 Parkinsons 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.

Shadows of an Absent Partner: ATP Hydrolysis and Phosphoenzyme Turnover of the Spf1 (Sensitivity to Pichia farinosa killer toxin) P5-ATPase.

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 E1 steps of the reaction cycle, but progression to the E2 forms is slow. Significance: The study extends the understanding of the catalytic mechanism of P5-ATPases. The P5-ATPases are important components of eukaryotic cells. They have been shown to influence protein biogenesis, folding, and transport. The knowledge of their biochemical properties is, however, limited, and the transported ions are still unknown. We expressed in Saccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end. The GFP-Spf1 protein was localized in the yeast endoplasmic reticulum. Purified preparations of GFP-Spf1 hydrolyzed ATP at a rate of ∼0.3–1 μmol of Pi/mg/min and formed a phosphoenzyme in a simple reaction medium containing no added metal ions except Mg2+. No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. Omission of protease inhibitors from the purification buffers resulted in a high level of endogenous proteolysis at the C-terminal portion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. The Mg2+ dependence of the GFP-Spf1 ATPase was similar to that of other P-ATPases where Mg2+ acts as a cofactor. The addition of Mn2+ to the reaction medium decreased the ATPase activity. The enzyme manifested optimal activity at a near neutral pH. When chased by the addition of cold ATP, 90% of the phosphoenzyme remained stable after 5 s. In contrast, the phosphoenzyme rapidly decayed to less than 20% when chased for 3 s by the addition of ADP. The greater effect of ADP accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1∼P phosphoenzyme. This behavior may reflect a limiting countertransported substrate needed to promote turnover or a missing regulatory factor.

Tyrosine Hydroxylase Is Short-Term Regulated by the Ubiquitin-Proteasome System in PC12 Cells and Hypothalamic and Brainstem Neurons from Spontaneously Hypertensive Rats: Possible Implications in Hypertension

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.

CHO cells expressing the human P5-ATPase ATP13A2 are more sensitive to the toxic effects of herbicide Paraquat

P-ATPases are membrane transporters energized by ATP. The subfamily of P₅-ATPases is the least studied P-ATPases and the ion substrate specificity of the P₅ subfamily is not known. Mutations of the human P₅ATPase gene ATP13A2 has been shown to underlie a form of Parkinson disease (PD). We investigated the link between ATP13A2 and environmental factors related to PD development. Increasing concentrations of the synthetic polyamine analog paraquat induced a greater cytotoxic effect over CHO cells expressing ATP13A2. Paraquat-toxicity was associated with increased production of cellular reactive oxygen species and this increment was reversed by the natural polyamine spermidine. Acridine orange fluorescence intensity suggested that ATP13A2 induced the expansion of acidic vesicles that become more alkaline upon external addition of spermidine. Polyamine uptake is proposed to be initiated by a plasma membrane carrier followed by sequestration into acidic vesicles of the late endocytic compartment through an unidentified active mechanism; because ATP13A2 is located in lysosomes and late endosomes, our results open the possibility that ATP13A2 could be one of those active transporters capable of transporting polyamines like spermidine as well as its toxic analog paraquat.

High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae

The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.

Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid-protein interaction.

The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.

The plasma membrane Ca2+ pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca2+

The plasma membrane Ca2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca2+ at a rate near 1.5% of that attained at saturating concentrations of Ca2+. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca2+-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca2+ and is catalyzed by E(2)-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.

Dynamic regulation of extracellular ATP in Escherichia coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli 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 [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]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 E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli 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. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.