Felicitas de Tezanos Pinto

Deletions in the acidic lipid-binding region of the plasma membrane Ca2+ pump. A mutant with high affinity for Ca2+ resembling the acidic lipid-activated enzyme.

The C-terminal segment of the loop between transmembrane helices 2 and 3 (AL region) of the plasma membrane Ca2+ pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca2+pump isoform h4xb with deletions in the AL region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296–349 had full Ca2+ transport activity, but deletions involving the segment of amino acids 350–356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V max of mutant d296–349 was similar to that of the recombinant wild type pump, but its K 0.5 for Ca2+ was about 5-fold lower. The addition of calmodulin increased theV max and the apparent Ca2+ affinity of both the wild type and d296–349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca2+ and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296–349. Taken together these results suggest that (a) deletion of residues 296–349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the AL region; (b) the AL region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca2+ affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the AL region is independent of the autoinhibition by the C-terminal calmodulin-binding region.

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.

Neurochemistry of Mammalian Entrainment: Signal Transduction Pathways in the Suprachiasmatic Nuclei

Neurochemical events leading to photic entrainment of circadian rhythms are reviewed. This entrainment pathway includes the retinohypothalamic tract and a glutamate-NMDA receptor (among others) interaction in the suprachiasmatic nuclei (SCN). The model we propose involves an increase in intracellular calcium levels and the activation of specific proteins in SCN neurons, including the Ca 2+ /calmodulin dependent protein kinase (CaM kinase) and phosphatase (calcineurin), other kinases (such as the cGMP-dependent protein kinase, PKG) and enzymes (nitric oxide synthase, NOS), which in turn activate specific transcription factors, in a cascade of events that is controlled both by light and by the circadian clock itself. Although the step at which the circadian gating of this process occurs is unknown, we propose it occurs downstream of glutamate binding, calcium entrance, and NOS activation. We conclude that a promising way of studying the function of the circadian pacemaker is to investigate the signal transduction pathway(s) leading to changes in the SCN, including the biochemical activity of its components.

Neuroactive steroids alter the Crcadian system of the Syrian hamster in a phase-dependent manner

Several steroid compounds affect neuronal function, primarily by modulating the GABAA receptor complex. A circadian variation in the brain concentration of neurosteroids has been reported in rats and humans. We have previously reported that natural occurring or synthetic neuroactive steroids such as androsterone and alphaxalone also have a rhythmic effect on behavior (anesthetic and anticonvulsant activity) and GABAergic activity. In the present work, we have tested the ability of neuroactive steroids to phase shift circadian rhythms in hamsters. The GABA(A) negative modulator dehydroepiandrosterone sulphate (DHEAS) elicited phase advances when administered at CT 6, while the positive modulator androsterone lacked any effect at this time. A complete phase response curve for DHEAS revealed a nonphotic-like effect. DHEAS also blocked the circadian effects of light, while androsterone induced photic-like responses. There is also evidence that neurosteroids may be present and even synthesized in the SCN. Collectively, the results so far indicate that some neuroactive steroids might modulate the activity of the circadian clock.

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.

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.

Deletions in the AL region of the h4xb plasma membrane Ca2+ pump High apparent affinity for Ca2+ of a deletion mutant resembling the alternative spliced form h4zb

Mutants of the plasma membrane Ca2+ pump (human isoform 4xb) with deletions in the linker between domain A and transmembrane segment M3 (AL region) were constructed and expressed in Chinese hamster ovary cells. The total or partial removal of the amino acid segment 300–349 did not change the maximal Ca2+ transport activity, but mutants with deletions involving residues 300–338 exhibited a higher apparent affinity for Ca2+ than the wild type h4xb enzyme. Deletion of the putative acidic lipid interacting sequence (residues 339–349) had no observable functional consequences. The removal of either residues 300–314 or 313–338 resulted in a similar increase in the apparent Ca2+ affinity of the pump although the increase was somewhat lower than that obtained by the deletion 300–349 suggesting that both deletions affected the same structural determinant. The results show that alterations in the region of the alternative splicing site A change the sensitivity to Ca2+ of the human isoform 4 of the PMCA.

The strategic function of the P5-ATPase ATP13A2 in toxic waste disposal

The P-type ATPase ATP13A2 protein was originally associated with a form of Parkinsons Disease (PD) known as Kufor Rakeb Syndrome (KRS). However, in the last years it has been found to underlay variants of neuronal ceroid-lipofuscinoses and hereditary spastic paraplegia. These findings expand the clinical and genetic spectrum of ATP13A2-associated disorders, which are commonly characterized by lysosomal dysfunction. Nowadays it is well known that lysosomes are not merely related to the degradation and recycling of cellular waste, but are also involved in fundamental processes such as secretion, plasma membrane repair, signaling, energy metabolism and autophagy. The essential role of lysosomes in these cellular processes has significant implications for health and disease. ATP13A2 is localized in lysosomes and late endosomes and its mutation leads to lysosome dysfunction, diminishes the exosome secretion and impairs autophagic flux. In this review, we first describe ATP13A2-associated disorders and their relation with the endolysosomal pathway. We then describe the ATP13A2-involvement in iron homeostasis and its potential linkage with new pathologies like cancer, and finally, we consider the putative role of ATP13A2 in lipid processing and degradation, opening the interesting possibility of a broader role of this protein providing protection against a variety of disease-associated changes affecting cellular homeostasis.