Biomedical subjects

E Lacanà

Publications and source records attributed to E Lacanà.

10 recordsLinked to original sources

PubMed1997

Dissociation of apoptosis and activation of IL-1beta-converting enzyme/Ced-3 proteases by ALG-2 and the truncated Alzheimer's gene ALG-3.

Recent attention has been focused on the members of the IL-1beta-converting enzyme (ICE)/Ced-3 family of cysteine protease as the key components of programmed cell death. However, the molecular events that lead to protease activation and link it to the final apoptotic processes remain poorly characterized. We have shown recently that ALG-2 is a Ca2+-binding protein required for apoptosis. ALG-2 depletion protects the mouse T cell hybridoma 3DO from programmed cell death induced by several stimuli, such as synthetic glucocorticoids, TCR, and Fas triggering. In this work, we show that in the ALG-2-depleted clones the ICE/Ced-3 proteases are normally activated upon TCR, Fas, and dexamethasone stimulation, as determined by cleavage of the endogenous substrate poly(ADP-ribose) polymerase and of a fluorogenic substrate. ALG-3, a truncated form of the familial Alzheimer's disease gene PS2, confers resistance to TCR- and Fas-induced apoptosis. Of interest, it also reduces protease activity and inhibits poly(ADP-ribose) polymerase cleavage upon Fas triggering. Our results suggest that, during apoptosis, ALG-2 functions downstream of, and that ALG-3 interferes with the sequential activation of members of the ICE family proteases.

Animals↗

PubMed1997

Functional cloning of genes involved in T-cell receptor-induced programmed cell death.

Programmed cell death (PCD) is a normal event under genetic control that regulates the life span of different cell types in multicellular organisms. Among other physiological processes, PCD plays a pivotal role in the regulation of the immune system. Using a functional selection strategy we have isolated and characterized genes involved in T-cell receptor-induced apoptosis. One, ALG-2, is a Ca(2+)-binding protein that participates in regulatory events that occur late in the apoptotic program, where several death signals converge. Another, ALG-3, is a mouse homologue of the chromosome 1 familial Alzheimer's disease gene PS2. ALG-3 codes for a truncated PS2 polypeptide that antagonizes the apoptotic role of PS2. A PS2 mutation associated with Alzheimer's disease generates a molecule with enhanced apoptotic activity indicating that it might accelerate the process of neurodegeneration that occurs in this disease.

Alzheimer Disease↗

PubMed1996

Requirement of the familial Alzheimer's disease gene PS2 for apoptosis. Opposing effect of ALG-3.

ALG-3, a truncated mouse homologue of the chromosome 1 familial Alzheimer's disease gene PS2, rescues T hybridoma 3DO cells from T-cell receptor-induced apoptosis by inhibiting Fas ligand induction and Fas signaling. Here we show that ALG-3 transfected 3DO cells express a COOH-terminal PS2 polypeptide. Overexpression of PS2 in ALG-3 transfected 3DO cells reconstitutes sensitivity to receptor-induced cell death, suggesting that the artificial PS2 polypeptide functions as a dominant negative mutant of PS2. ALG-3 and antisense PS2 protect PC12 cells from glutamate-induced apoptosis but not from death induced by hydrogen peroxide or the free radical MPP+. Thus, the PS2 gene is required for some forms of cell death in diverse cell types, and its function is opposed by ALG-3.

Alzheimer Disease↗

PubMed1996

Participation of presenilin 2 in apoptosis: enhanced basal activity conferred by an Alzheimer mutation.

Overexpression of the familial Alzheimer's disease gene Presenilin 2 (PS2) in nerve growth factor-differentiated PC12 cells increased apoptosis induced by trophic factor withdrawal or beta-amyloid. Transfection of antisense PS2 conferred protection against apoptosis induced by trophic withdrawal in nerve growth factor-differentiated or amyloid precursor protein-expressing PC12 cells. The apoptotic cell death induced by PS2 protein was sensitive to pertussis toxin, suggesting that heterotrimeric GTP-binding proteins are involved. A PS2 mutation associated with familial Alzheimer's disease was found to generate a molecule with enhanced basal apoptotic activity. This gain of function might accelerate the process of neurodegeneration that occurs in Alzheimer's disease, leading to the earlier age of onset characteristic of familial Alzheimer's disease.

Alzheimer Disease↗

PubMed1996

Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer's disease gene ALG-3.

Two apoptosis-linked genes, named ALG-2 and ALG-3, were identified by means of a functional selection strategy. ALG-2 codes for a Ca(2+)-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death. ALG-3, a partial complementary DNA that is homologous to the familial Alzheimer's disease gene STM2, rescues a T cell hybridoma from T cell receptor- and Fas-induced apoptosis. These findings suggest that ALG-2 may mediate Ca(2+)-regulated signals along the death pathway and that cell death may play a role in Alzheimer's disease.

Alkaloids↗

PubMed1993

Evolutionary conservation of genomic sequences related to the GGP1 gene encoding a yeast GPI-anchored glycoprotein.

The GGP1 gene encodes the only GPI-anchored glycoprotein (gp115) that has been purified to-date in the budding yeast Saccharomyces cerevisiae. It is a single-copy gene whose deduced amino-acid sequence shares no significant homology to any other known protein. In this paper we report a Southern hybridization analysis of genomic DNA from different eukaryotic organisms to identify homologues of the GGP1 gene. We have analyzed DNA prepared from a unicellular green alga (Chlamydomonas eugametos), from two distantly related yeast species (Candida cylindracea and Schizosaccharomyces pombe), and from the common bean Phasoleus vulgaris. The moderate stringency of the experimental conditions and the high specificity of the probes used indicate that a single-copy of GGP1-related sequences exists in all these eukaryotic organisms. The chromosomal localization of the GGP1 gene in S. cerevisiae has also been determined.

Animals↗

PubMed1991

Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region.

gp115 is a N- and O-glycosylated protein of Saccharomyces cerevisiae. It is also modified by addition of glycosylphosphatidylinositol, which anchors the protein to the plasma membrane. The gene encoding gp115 (GGP1) has been cloned by a two-step procedure. By an immunoscreening of a yeast genomic DNA library in the expression vector lambda gt11, a 3'-terminal 0.9-kilobase portion of the gene has been isolated and then used as a molecular probe to screen a yeast genomic DNA library in YEp24. In this way, the whole GGP1 gene has been cloned. Its identity with the gp115 gene has been confirmed by gene disruption, which has also indicated that the function of gp115 is not essential for cell viability. The features of the sequence are also entirely consistent with it corresponding to the gp115 gene. The nucleotide sequence of GGP1 predicts a 60-kDa polypeptide, in agreement with the molecular mass of the gp115 precursor detected in sec53 mutant cells at restrictive temperature. Two hydrophobic sequences, one NH2- and the other COOH-terminal were found. The former has the features of the cleavable signal sequence, which allows the entry of proteins in the secretory pathway. The latter could be the signal sequence that has to be removed during the addition of glycosylphosphatidylinositol. The predicted amino acid sequence of gp115 shows 10 sequons for N-glycosylation and a high proportion of serine-threonine residues (22%) that could provide several sites for O-glycosylation. The unusual concentration of 27 serines in the COOH-terminal portion of the protein shares homology with a similar polyserine repeat of the serine repeat antigen (SERA protein) of Plasmodium falciparum. A two-dimensional analysis of the "in vitro" translational product of the GGP1 mRNA has been carried out, allowing the identification of the "in vivo" gp115 precursor in a two-dimensional gel.

Amino Acid Sequence↗

PubMed1990

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol.

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.

Acetylglucosaminidase↗

PubMed1990

Changes in the protein synthesis pattern during a nutritional shift-down transition in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae cells (strain A364A) during a shift-down from glucose to raffinose, a rapid reduction in the rate of RNA accumulation was observed whereas the rate of protein accumulation was unaffected for at least 2 h. Following the transition the percentage of unbudded cells slightly increased and the cell volume distribution showed a newly formed subpopulation of smaller cells. To study the effects of the shift-down on the protein synthesis pattern, total [35S]-methionine pulse-labeled extracts were fractionated by high-resolution two-dimensional gel electrophoresis. The synthesis of two classes of proteins (I and II) was modulated during the transitory state of growth: one positively, the other negatively. Two polypeptides of 57 kDa showed the most dramatic increase in synthesis during the shift-down. Also a heat-shock protein (HSP 256) appeared to be positively correlated to the shift-down transition.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1988

Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle.

A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.

Antibody Specificity↗