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Ernesto Carafoli

Publications and source records attributed to Ernesto Carafoli.

At least 19 recordsLinked to original sources
PubMed2007

The role of phosphorylation on the structure and dynamics of phospholamban: a model from molecular simulations.

Phospholamban (PLB) is a small membrane protein that regulates the activity of the calcium ATP-ase in the cardiac, slow-twitch, and smooth muscle sarcoplasmic reticulum through the reversible phosphorylation of Ser16. We present here a comparative molecular dynamics study of unmodified and phosphorylated PLB immersed in a phospholipid membrane. The study has been performed under different ionic strength conditions, using the NMR structures of two PLB variants determined in mixed organic solvent and dodecylphosphocholine micelles. The simulations indicate that all PLB forms studied display a highly dynamic behavior of the N-terminal cytoplasmic moiety, with a decrease of its helical content in the phosphorylated forms. The cytoplasmic domain undergoes large collective motions sampling conformations parallel as well as perpendicular to the membrane surface in all the simulations. The transmembrane domain retains a tightly folded helical conformation with a small tilt with respect to the membrane plane probably induced by the presence of Asn30 and Asn34 within the hydrophobic environment. Furthermore, the phosphoric group on Ser16 establishes transient electrostatic interactions with the phospholipid heads. We propose a model in which phosphorylation diminishes the probability of interactions of PLB with residues near Lys400 in the SERCA pump, thus relieving its inhibition.

Asparagine↗
PubMed2006

Inhibitory interaction of the plasma membrane Na+/Ca2+ exchangers with the 14-3-3 proteins.

The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.

14-3-3 Proteins↗
PubMed2005

Downstream regulatory element antagonist modulator regulates Ca2+ homeostasis and viability in cerebellar neurons.

The Na+/Ca2+ exchangers NCX1, NCX2, and NCX3 are vital for the control of cellular Ca2+ homeostasis. Here, we show that a doublet of downstream regulatory element sites in the promoter of the NCX3 gene mediates transcriptional repression of NCX3 by the Ca2+-modulated transcriptional repressor downstream regulatory element antagonist modulator (DREAM). Overexpression of a DREAM EF-hand mutant insensitive to Ca2+ (EFmDREAM) in hippocampus and cerebellum of transgenic mice significantly reduced NCX3 mRNA and protein levels without modifying NCX1 and NCX2 expression. Cerebellar granules from EFmDREAM transgenic mice showed increased levels of cytosolic Ca2+ and were more vulnerable to increased Ca2+ influx after partial opening of voltage-gated plasma membrane Ca2+ channels induced by increasing K+ in the culture medium but survived better in the conditions of reduced Ca2+ influx prevailing in low extracellular K+. Overexpression of NCX3 in EFmDREAM transgenic granules using a lentiviral vector restored the normal survival response to high K+ observed in wild-type granules. Thus, the downregulation of the regulator of Ca2+ homeostasis NCX3 by Ca2+-regulated DREAM is a striking example of the autoregulatory property of the Ca2+ signal in neurons.

Amino Acid Motifs↗
PubMed2005

Inhibitory interaction of the 14-3-3{epsilon} protein with isoform 4 of the plasma membrane Ca(2+)-ATPase pump.

The isoform-specific interaction of plasma membrane Ca(2+)-ATPase (PMCA) pumps with partner proteins has been explored using a yeast two-hybrid technique. The 90 N-terminal residues of two pump isoforms (PMCA2 and PMCA4), which have a low degree of sequence homology, have been used as baits. Screening of 5 x 10(6) clones of a human brain cDNA library yielded approximately 100 LEU2- and galactoside-positive clones for both pumps. A clone obtained with the PMCA4 bait specified the epsilon-isoform of the 14-3-3 protein, whereas no 14-3-3epsilon clone was obtained with the PMCA2 bait. The 14-3-3epsilon protein immunoprecipitated with PMCA4 (not with PMCA2) when expressed in HeLa cells. Overexpression of 14-3-3epsilon in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca(2+) was impaired; stimulation with histamine, an inositol 1,4,5-trisphosphate-producing agonist, generated higher cytosolic [Ca(2+)] transients, higher post-transient plateaus of the cytosolic [Ca(2+)], and higher Ca(2+) levels in the endoplasmic reticulum lumen and in the subplasmalemmal domain. Thus, the interaction with 14-3-3epsilon inhibited PMCA4. Silencing of the 14-3-3epsilon gene by RNA interference significantly reduced the expression of 14-3-3epsilon, substantially decreasing the height of the histamine-induced cytosolic [Ca(2+)] transient and of the post-transient cytosolic [Ca(2+)] plateau.

14-3-3 Proteins↗
PubMed2005

Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations.

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.

Aequorin↗
PubMed2005

Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity.

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.

Amino Acid Sequence↗
PubMed2005

Calcium--a universal carrier of biological signals. Delivered on 3 July 2003 at the Special FEBS Meeting in Brussels.

Calcium is the most universal carrier of signals to cells. Chosen by evolution because of its peculiar flexibility as a ligand, it now regulates all important aspects of cell activity, beginning with the creation of new life at fertilization and ending with the dramatic event of apoptotic suicide at the end of the life cycle. The process of signal transduction by Ca2+ displays a number of properties that make it unique among all other carriers of signals: for instance, the ability to perform both a first messenger and a second messenger function, or the frequent activation of autoregulatory mechanisms. The aspect that distinguishes the Ca2+ signaling function most dramatically is ambivalence. Cells have an absolute dependence on the messenger function of Ca2+ in order to function properly and must control its homeostasis with precision to maintain its free concentration in their interior at the appropriate low level. Catastrophy, however, invariably follows whenever protracted failures of the control mechanisms lead to sustained Ca2+ overload.

Animals↗
PubMed2003

Expression, purification, and characterization of isoform 1 of the plasma membrane Ca2+ pump: focus on calpain sensitivity.

The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms.

Adenosine Triphosphatases↗
PubMed2003

A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells.

The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of [Ca2+] in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.

Animals↗
PubMed2003

Historical review: mitochondria and calcium: ups and downs of an unusual relationship.

The discovery of Ca(2+) transport by mitochondria is conventionally credited to De Luca and Engstrom, and Vasington and Murphy, who showed in 1961-1962 that Ca(2+) was taken up by isolated mitochondria using respiratory or ATP energy. However, contributions had already appeared in the 1950s showing - albeit indirectly - that isolated mitochondria bound Ca(2+) actively. Somehow, however, these contributions failed to attract the attention that they undoubtedly deserved. The 1961-1962 findings started the ball rolling, initiating a topic that was to have a peculiar oscillatory history. It went from peaks of great enthusiasm to valleys of essential neglect, and from there to a final (hopefully permanent) robust revival.

Adenosine Triphosphate↗
PubMed2003

The calcium-signalling saga: tap water and protein crystals.

Of the approximately 1,400 grams of calcium that are in the human body, less than 10 grams manage to escape being trapped in the skeleton and teeth. These few grams might be an insignificant quantity, but they are extraordinarily significant qualitatively. They circulate in the blood and extracellular spaces, and penetrate cells to regulate their most important activities.

Biology↗
PubMed2003

Control of the Na+/Ca2+ exchanger 3 promoter by cyclic adenosine monophosphate and Ca2+ in differentiating neurons.

The human gene for member 3 of solute carrier family 8 (SLC8A3), encoding the Na+/Ca2+ exchanger isoform 3 (NCX3), was identified on chromosome 14q24.2. The minimal promoter region was predicted 250 bp upstream of exon 1. This was confirmed by luciferase reporter assays of pGL3-promoter constructs in transfected SH-SY5Y cells. The promoter activity was monitored during the differentiation of this cell line elicited by the sequential treatment with retinoic acid and brain-derived neurotrophic factor (BDNF). The activity was induced by cyclic AMP (cAMP) via the CRE (cAMP response element) and was stimulated by retinoic acid. The increase of intracellular Ca2+ induced by the partial depolarization of the plasma membrane with KCl down-regulated both the basal and the cAMP-stimulated transcription. The down-regulation of the latter may be mediated by the phosphorylation of the CRE-binding protein by a calmodulin-dependent kinase (CaMKII). The exposure of cells to BDNF after treatment with retinoic acid rapidly induced promoter activity during the initial five hours and phosphorylation of CRE-binding protein during the first two hours. The promoter activity was further enhanced by cAMP, but became insensitive to Ca2+. In BDNF-stimulated cells cAMP elevation caused the preferential phosphorylation of ATF1 instead of that of CRE-binding protein.

Activating Transcription Factors↗
PubMed2002

A structural model of the complex formed by phospholamban and the calcium pump of sarcoplasmic reticulum obtained by molecular mechanics.

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids that modulates the activity of the reticular Ca(2+) ion pump. We recently solved the three-dimensional structure of chemically synthesized, unphosphorylated, monomeric PLN (C41F) by high-resolution nuclear magnetic resonance spectroscopy in chloroform/methanol. The structure is composed of two alpha-helical regions connected by a beta turn (Type III). We used this structure and the crystallographic structure of the sarcoplasmic reticulum calcium pump (SERCA) recently determined by Toyoshima and co-workers and modeled into its E(2) form by Stokes (1KJU) or by Toyoshima (1FQU). We applied restrained and unrestrained energy optimizations and used the AMBER molecular mechanics force field to model the complex formed between PLN and the pump. The results indicate that transmembrane helix 6 (M6) of the SERCA pump is energetically favored, with respect to the other transmembrane helices, as the PLN binding partner within the membrane and is the only one of these helices that also permits contact between the N-terminal residues of PLN and the critical cytosolic binding loop region of the pump. This result is in agreement with published biochemical data and with the predictions of previous mutagenesis work on the membrane sector of the pump. The model reveals that PLN does not span the entire width of the membrane, that is, its hydrophobic C-terminal end is located near the center of the transmembrane region of the SERCA pump. The model also shows that interaction with M6 is stabilized by additional contacts made by PLN to M4. The contact between the N-terminal portion of PLN and the pump is stabilized by a number of salt and hydrogen-bond bridges, which may be abolished by phosphorylation of PLN. The contacts between the cytosolic portions of PLN and the pump are only observed in the E(2) conformation of the pump. Our model of the complex also offers a plausible structural explanation for the preference of protein kinase A for phosphorylation of Ser16 of PLN.

Animals↗
PubMed2002

The human SLC8A3 gene and the tissue-specific Na+/Ca2+ exchanger 3 isoforms.

We have identified the human gene for member 3 of Solute Carrier family 8 (SLC8A3) by bioinformatic analysis of human genomic sequences. The gene is located on chromosome 14q24.2, and spans a region of about 150 kb. The full-length DNA complementary to RNA encoding the Na(+)/Ca(2+) exchanger isoform 3 (NCX3), amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from the human neuroblastoma SH-SY5Y RNA, includes seven exons and encodes a protein of about 100 kDa. RT-PCR analysis was performed in different tissues to determine the exon composition in the region encoding the large intracellular loop of the protein. The region underwent modifications by alternative tissue-specific splicing. NCX3.2, including exon 4 but not exon 5, was found in human brain and in the neuroblastoma cell line. In human skeletal muscle two additional isoforms were identified: NCX3.3, including exons 4 and 5, and a truncated isoform (NCX3.4) produced by the skipping of both exons 3 and 4. The skipping causes a frame shift downstream of the exon 2 sequence. The new coding sequence of 25 amino acids terminates with a stop codon in exon 6. The NCX3.4 isoform (68 kDa) is truncated in the C-terminal portion of the domain first found in Drosophila Na(+)/Ca(2+) exchanger domain (Calxbeta) and lacks the C-terminal hydrophobic segments.

Alternative Splicing↗
PubMed2002

Recombinant expression of the plasma membrane Na(+)/Ca(2+) exchanger affects local and global Ca(2+) homeostasis in Chinese hamster ovary cells.

The cardiac type Na(+)/Ca(2+) exchanger (NCX1) has been transiently expressed in Chinese hamster ovary cells, which do not contain an endogenous exchanger, together with aequorin chimeras that are targeted to different intracellular compartments to investigate intracellular Ca(2+) homeostasis. The expression of NCX decreased the endoplasmic reticulum Ca(2+) concentration, [Ca(2+)](er), in resting cells, showing that the exchanger was operative under these conditions. It induced a greater reduction in the height of the mitochondrial and cytosolic Ca(2+) transients in agonist-stimulated cells than would have been expected from the [Ca(2+)](er) decrease. It also had a major effect on the sub-plasma membrane Ca(2+) concentration, [Ca(2+)](pm): after a transient [Ca(2+)](pm) rise induced by the activation of capacitative Ca(2+) influx, [Ca(2+)](pm) settled to a value about 3-fold higher than in controls. The sustained [Ca(2+)](pm) increase after the transient was due to the operation of the exchanger, either directly by operating in the Ca(2+) entry mode, or indirectly by removing the Ca(2+) inhibition on the capacitative Ca(2+) influx channels.

Adenosine Triphosphate↗