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D Guerini

Publications and source records attributed to D Guerini.

At least 37 records · Page 2Linked to original sources
PubMed1996

Expression and functional characterization of isoforms 4 of the plasma membrane calcium pump.

PMCA isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII had a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but had a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone was almost 3 times higher than in PMCA4CI and was increased by La3+ less than in the PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) had no Ca2+-dependent ATPase activity, but was still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform was retained in the endoplasmic reticulum; changes in membrane architecture apparently occurred during its expression; the C-terminal portion of the isoform was located in the cytosol, indicating that the deletion of the 10th transmembrane domain resulted in the loss of at least another transmembrane domain.

Alternative Splicing↗
PubMed1996

Mutation of conserved residues in transmembrane domains 4,6 and 8 causes loss of Ca2+ transport by the plasma membrane Ca2+ pump.

Mutants of the plasma membrane Ca2+ pump (PMCA), in which amino acids in transmembrane domains (TM) 4, 6, and 8 had been replaced, have been expressed in COS-7 cells. They were analyzed functionally by measuring the uptake of Ca2+ in microsomal preparations and by following the formation of the phosphorylated intermediate from ATP and from phosphate. The mutated residues corresponded to amino acids whose mutation in the sarcoplasmic reticulum pump (SERCA) caused loss of Ca2+ transport by the pump protein: however, only four of the six SERCA residues were conserved in the PMCA pump. Mutation of Glu423 (TM4), Asn879 or Asp883 (TM6), or Gln97l (TM8) suppressed Ca2+ transport by the pump and its ability to form the phosphorylated intermediate starting from ATP. By contrast, the ability of these mutants to form the intermediate starting from phosphate was not impaired. In two mutants (Glu423 and Asp883) it was even enhanced. Two conserved Pro residues of TM4 were also mutated, leading to the loss of the ability of the pump to form the Ca2+- and ATP-dependent phosphorylated intermediate. Unexpectedly, two of the mutations (Asn879 and Gln971) led to the mistargeting of the mutated proteins, i.e., to their retention in the endoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1996

Detection of a new polymorphism in the plasma-membrane Ca2+ ATPase isoform-3 gene and its exclusion as a candidate for X-linked myotubular myopathy (MTM1).

The severe neonatal centronuclear/myotubular myopathy (XLMTM) is an X-linked disorder characterized by generalized muscle weakness, hypotonia and serious respiratory insufficiency. The gene for this disease has been assigned to the long arm of chromosome X in the Xq28 band. Ca2+ ATPase isoform-3 (ATP2B3) has also been mapped to the human Xq28 region. Moreover, it is expressed in fetal but not in adult muscle, suggesting the developmental regulation of gene transcription. These findings render the ATP2B3 gene as an interesting candidate gene for XLMTM. Four families and 7 unrelated XLMTM patients have been analysed by using cDNA and genomic probes of ATP2B3. No large deletions or duplications have been found but a new EcoRI polymorphism has been identified. In addition, the DNA of an XLMTM male deletion patient has been hybridized with the ATP2B3 gene sequences. Our results therefore support the exclusion of ATP2B3 as the causal disease gene of XLMTM. The isolation of the MTM1 gene has recently been reported by another group. However, our approach has led to the detection of a new polymorphism that is an informative marker for linkage and mutation studies in other Xq28-mapped neurological or neuromuscular disorders.

Adult↗
PubMed1996

Calcineurin A alpha (PPP3CA), calcineurin A beta (PPP3CB) and calcineurin B (PPP3R1) are located on human chromosomes 4, 10q21-->q22 and 2p16-->p15 respectively.

Calcineurin (also called protein phosphatase-2B) is a calmodulin-regulated protein phosphatase which plays an important role in signal transduction. The enzyme is a heterodimer of a 58-59 kDa calmodulin-binding catalytic subunit (calcineurin A) and a small (i.e. 19 kDa) Ca(2+)-binding regulatory subunit (calcineurin B). The highly conserved calcineurin B is encoded by a single gene in all tissues except testes, whereas there are three isoforms of calcineurin A (alpha, beta and gamma) encoded by genes on three different chromosomes. This enzyme can play a critical role in transcriptional regulation and growth control in T lymphocytes by a mechanism believed to involve dephosphorylation of the nuclear factor NF-AT which is essential for transcription of the interleukin-2 gene. To better evaluate the potential role of the calcineurin genes in human genetic disorders, we have studied their chromosome locations. Calcineurin B (PPP3R1) is located on human chromosome 2p16-->p15 and calcineurin A beta (PPP3CB, previous gene symbol CALNB) is present on 10q21-->q22. We confirm the localization of calcineurin A alpha (PPP3CA, previous gene symbol CALNA) to chromosome 4 without regional localization.

Base Sequence↗
PubMed1996

The targeting of the plasma membrane calcium pump in the cell.

The information on the structural determinants that control the cellular distribution of P-type pumps is very scarce. However, recent experiments on the membrane targeting of the plasma membrane Ca2+ pump (PMCA) have provided interesting leads on the problem: they will be discussed in this succinct review. A general introduction on the biochemical properties of the PMCA pump will preface the discussion of the specific findings on the role of three distinct regions of the molecule in the targeting process.

Biological Transport↗
PubMed1995

Isolation and characterization of a stable Chinese hamster ovary cell line overexpressing the plasma membrane Ca(2+)-ATPase.

Stable Chinese hamster ovary (CHO) cell lines overexpressing the human plasma membrane Ca(2+)-ATPase (PMCA) were generated, and three independent cell clones were characterized in details. They overexpressed high amounts of active PMCA pump (15-20 times over the amount of endogenous PMCA) as indicated by experiments in which the formation of the phosphoenzyme intermediate and the uptake of Ca2+ by microsomes were measured. Immunocytochemistry experiments coupled to the biotinylation of the pump in the intact cells indicated the correct deliver of the expressed pump to the plasma membrane. The expressed pump was purified by affinity chromatography on calmodulin sepharose. The PMCA of transfected CHO cells promoted an increase of Ca2+ into the medium, after induction of Ca2+ release from the internal stores by activation of a purinergic receptor. An evident decrease of the activity of the endogenous sarcoplasmic reticulum Ca(2+)-ATPase pump was observed, probably related to the down-regulation of its expression. The cells overexpressing the PMCA pump had delayed recovery after trypsinization and plating. Their doubling time was, however, the same as CHO cells.

Animals↗
PubMed1995

Tissue distribution of the four gene products of the plasma membrane Ca2+ pump. A study using specific antibodies.

Antibodies against the four isoforms of the human plasma membrane Ca(2+)-ATPase (PMCA) were raised using an N-terminal sequence of the pump as epitope. The antibodies against PMCA isoforms 1, 2, and 3 were not species-specific, e.g. they also recognized the corresponding proteins in rat, whereas that against the human PMCA isoform 4 failed to do so. The tissue distribution of the four isoforms was estimated by Western blot analysis. Two, PMCA1 and PMCA4, were expressed in all tissues tested (with the exception of the choroid plexus, where the former was not detected). In most tissues the signal from the PMCA1 protein exceeded that of PMCA4, the exception being the erythrocyte. The PMCA2 and PMCA3 proteins were only found in neuronal tissues; the PMCA2 protein was present in high concentrations in the cerebellum and in the cerebral cortex. At variance with previous results on mRNA (e.g. the kidney) no other tissues contained the PMCA2 protein. PMCA3 was the other tissue-specific isoform; in agreement with results in the rat, the protein was found in human neuronal tissues, particularly in the choroid plexus, but was practically absent in all other tissues tested.

Animals↗
PubMed1995

A signal for endoplasmic reticulum retention located at the carboxyl terminus of the plasma membrane Ca(2+)-ATPase isoform 4CI.

The plasma membrane Ca(2+)-ATPase isoform 4b (PMCA4CI) with truncations in the cytoplasmically exposed COOH-terminal tail was expressed in COS and HeLa cells and in Sf9 cells using the baculovirus system. The truncated protein terminating with the acidic sequence Glu1067-Arg1087 was retained within the endoplasmic reticulum (ER), whereas mutants lacking this sequence or having it at a distance from the COOH terminus were delivered to the plasma membrane. Although the truncated protein retained in the endoplasmic reticulum was still able to form a Ca(2+)-dependent phosphoenzyme, it underwent partial degradation. Substitution of glutamic and aspartic residue(s) in the acidic region promoted rescue of the protein to the plasma membrane. The results suggest that the sequence Glu1067-Arg1087 encodes a masked signal for ER retention and for the degradation of the protein. However, its presence at the COOH terminus was not sufficient to induce ER-retention and degradation; when the sequence was attached to the full-length PMCA protein, normal plasma delivery was observed. Evidently, ER retention and degradation required the presence of the sequence in its specific location within the PMCA structure. The degradation of the protein retained in the endoplasmic reticulum occurred through the proteolytic attack at cytoplasmically exposed residues (amino acid sequence 720-750) by a cytoplasmic PEST sequence-related protease different from calpain.

Amino Acid Sequence↗
PubMed1995

The 70 kD component of the heart sarcolemmal Na+/Ca(2+)-exchanger preparation is the C-terminal portion of the protein.

The cardiac sarcolemmal Na+/Ca(2+)-exchanger was expressed in COS-7 cells by the vaccinia virus system as a fusion protein with a poly-His tag at its C-terminus. Extracts of cells expressing the exchanger construct without the His-tag reacted with an antiserum against the C-terminal portion of the main intracellular loop of the exchanger: in agreement with the finding routinely made on heart sarcolemma and on plasma membrane of cells expressing the cardiac exchanger gene, three bands of about 160, 120, and 70 kD were detected in Western blots. All three bands shifted to higher molecular masses when the construct with the His-tag was expressed, indicating that the three proteins had the same C-terminus. Thus, the 70 kD protein, whose nature has always been obscure, appears to be a degradation product of one of the two larger proteins. N-terminal sequencing of partially purified exchanger preparations has identified the cleavage site(s) producing the 70 kD protein in the 257-269 residue region of the exchanger molecule.

Amino Acid Sequence↗
PubMed1995

Subcellular targeting of the endoplasmic reticulum and plasma membrane Ca2+ pumps: a study using recombinant chimeras.

ATP-powered Ca2+ pumps are located in the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). The two pump types share numerous structural and functional features; nevertheless, they are strictly targeted to different cell compartments. Chimeric SERCA/PMCA pumps were constructed to investigate the structural determinants responsible for their specific cellular location. The level of expression of the chimeric constructs and of the wild-type pumps in COS-7 and Sf9 cells was the same and so was their stability. One exception was chimera D, which showed special propensity to degradation. The chimeric constructs had no Ca(2+)-dependent ATPase activity, although in one trace amounts of the Ca(2+)-dependent phosphoenzyme intermediate were detected. Thus, the exchange of the regions encompassing the (NH2-terminal) transmembrane domains apparently is incompatible with the activity of the pump. The immunofluorescence experiments showed that the 85 NH2-terminal residues of the SERCA pump, encompassing the first transmembrane domain, contain a signal-promoting retention of chimeric constructs otherwise consisting of the PMCA pump structure in the endoplasmic reticulum. Additional structural determinants most likely also contribute to the retention of the SERCA pump in the endoplasmic reticulum: one chimeric construct (E) composed of the first two transmembrane domains of the PMCA pump, followed by the remainder of the SERCA pump structure, was still retained, even if not completely, in the endoplasmic reticulum.

Animals↗
PubMed1994

Cloning and expression of isoform 2 of the human plasma membrane Ca2+ ATPase. Functional properties of the enzyme and its splicing products.

Full-length cDNAs for the three human plasma membrane Ca2+ pump isoforms 2 (PMCA2) differently spliced at the A site were constructed and transferred to baculovirus. The corresponding proteins were expressed after infection in Sf9 insect cells. The proteins were expressed at high levels and retained the canonical properties of the plasma membrane Ca2+ pump. The alternative splicing process failed to produce functional differences detectable with the methods used. The Ca(2+)-dependent ATPase activity of the PMCA2 pumps had a 5-10-fold higher affinity for calmodulin than the PMCA4 pump expressed in the same system. Experiments on the formation of the phosphoenzyme intermediate from ATP revealed that the PMCA2 pumps had higher affinity for ATP than did the PMCA4 counterpart. The response of the two pump types to activating acidic phospholipids was the same.

Adenosine Triphosphate↗
PubMed1993

Calmodulin can modulate protein phosphorylation in rat liver cells nuclei.

This report describes the immunological identification of a 60-kDa calmodulin-binding protein, previously detected in the nuclei of rat liver cells (Bachs, O., Lanini, L., Serratosa, J., Coll, M.J., Bastos, R., Aligué, R., Rius, E., and Carafoli, E. (1990) J. Biol. Chem. 265, 18595-18600), as the calmodulin-dependent protein phosphatase calcineurin. Calcineurin could be extracted from the nuclei by incubation with DNase and RNase, indicating that it is associated with nuclear structures sensitive to the action of nucleases (chromatin or/and ribonucleoproteins). The presence of calcineurin in the nuclei of rat liver cells indicates that calmodulin may modulate the phosphorylation level of nuclear proteins by promoting their dephosphorylation. This report also shows that calmodulin inhibits the activity of casein kinase-2 in the nuclear fractions obtained by nuclease extraction. Phosphorylation experiments indicate that casein kinase-2 phosphorylates three major substrates of 100, 42-44, and 37 kDa as well as other minor proteins in the nuclease extracts. Calmodulin reduces the phosphorylation level of the two latter major proteins and of a minor band of 50 kDa. Thus, nuclear calmodulin in rat liver cells could regulate phosphorylation of nuclear proteins by at least two mechanisms: 1) activation of calcineurin and 2) inhibition of casein kinase-2.

Animals↗
PubMed1993

The effect on retirees of losing retirement and health insurance benefits.

BACKGROUND AND OBJECTIVES: The recent economic downturn has led to instability in the private health insurance industry. Although loss of medical benefits is assumed to have a negative effect on health, documentation is lacking. LTV Corporation (Ling-Temco-Vought) filed for bankruptcy and interrupted medical insurance for its retirees for six months. METHODS: Using a structured interview format, we surveyed community-living LTV retirees whose medical insurance had been interrupted. We sought to predict health status using a variety of measures. RESULTS: The vast majority of retirees were generally unaffected by the loss of medical benefits. Of the 191 LTV retirees from Youngstown, Ohio, who were interviewed shortly after health benefit loss, 13.8% reported longer-term health effects (continued decline in subjective health status), whereas 8.5% had short-term effects (decline followed by return to good or excellent health status). Although 10.5% of workers experienced serious health problems during the crisis, only one worker was unable to pay for health care as a result of the benefits loss. A discriminant analysis yielded excellent results in predicting longer-term deteriorating health status. CONCLUSIONS: Implications for community-oriented primary care service models are discussed, as well as the utility of demographic targeting for retirees losing health benefits.

Aged↗
PubMed1992

Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment.

Isoform 4b of the human plasma membrane Ca2+ pump was expressed in COS cells and in the baculovirus system (Sf9 cells). A 105-kDa pump fragment lacking the first two transmembrane domains and the so-called transduction domain was also expressed. The expression level was 2-4 times the background in COS cells and at least 7 times in the baculovirus system. Tests on membranes from both systems showed that the expressed pump was active. The expressed pump and the 105-kDa fragment were isolated from Sf9 cell membranes by calmodulin affinity chromatography. The pump had Ca(2+)-dependent ATPase activity with a calmodulin stimulation factor of 3, formed a La(3+)-stabilized phosphoenzyme, and had a KM (Ca2+) in the presence of calmodulin of about 1 microM. The 105-kDa fragment, assayed by the phosphoenzyme test on COS or Sf9 cell membranes or by ATPase measurements after isolation from Sf9 cells, proved inactive. Laser confocal microscopy on Sf9 cells showed that both the pump and the 105-kDa fragment were apparently associated with the plasma membrane. The expressed pump in COS and Sf9 cells and the endogenous pump in a number of other cell lines had a slower gel mobility (i.e. a higher apparent molecular mass) than the erythrocyte pump.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.

Amino Acid Sequence↗
PubMed1989

Cloning of human calcineurin A: evidence for two isozymes and identification of a polyproline structural domain.

Two types (I and II) of cDNAs encoding the large (A) subunit of calcineurin, a calmodulin-regulated protein phosphatase, were isolated from human basal ganglia and brainstem mRNA. The complete sequences of the two calcineurin clones are identical except for a 54-base-pair insert in the type I clone and different 3' ends including part of the coding sequence for the C termini of the two proteins. These findings suggest that calcineurin A consists of at least two isozymes that may result from alternative splicing events. The two forms of the enzyme differ in the C terminus, which contains an inhibitory domain rapidly severed by limited proteolysis. With the exception of an 18-amino acid insert, the central parts of the molecules, which harbor the catalytic domains, are identical and show extended similarities with the entire catalytic subunits of protein phosphatases 1 and 2A, defining a distinct family of protein phosphatases. The 40-residue N-terminal fragment, specific for calcineurin, contains a sequence of 11 successive prolines that is also found in bovine brain calcineurin by peptide sequencing. A role in the calmodulin activation of calcineurin is proposed for this novel structural element.

Amino Acid Sequence↗