Biomedical subjects

Angelita Rebollo

Publications and source records attributed to Angelita Rebollo.

16 recordsLinked to original sources

PubMed2007

The toxofilin-actin-PP2C complex of Toxoplasma: identification of interacting domains.

Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure-function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein-protein interactions that are important to parasite motility.

Actin Capping Proteins↗

PubMed2006

Identification of PP1alpha as a caspase-9 regulator in IL-2 deprivation-induced apoptosis.

One of the mechanisms that regulate cell death is the reversible phosphorylation of proteins. ERK/MAPK phosphorylates caspase-9 at Thr(125), and this phosphorylation is crucial for caspase-9 inhibition. Until now, the phosphatase responsible for Thr(125) dephosphorylation has not been described. Here, we demonstrate that in IL-2-proliferating cells, phosphorylated serine/threonine phosphatase type 1alpha (PP1alpha) associates with phosphorylated caspase-9. IL-2 deprivation induces PP1alpha dephosphorylation, which leads to its activation and, as a consequence, dephosphorylation and activation of caspase-9 and subsequent dissociation of both molecules. In cell-free systems supplemented with ATP caspase-9 activation is induced by addition of cytochrome c and we show that in this process PP1alpha is indispensable for triggering caspase-9 as well as caspase-3 cleavage and activation. Moreover, PP1alpha associates with caspase-9 in vitro and in vivo, suggesting that it is the phosphatase responsible for caspase-9 dephosphorylation and activation. Finally, we describe two novel phosphatase-binding sites different from the previously described PP1alpha consensus motifs, and we demonstrate that these novel sites mediate the interaction of PP1alpha with caspase-9.

Amino Acid Sequence↗

PubMed2006

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

Amino Acid Sequence↗

PubMed2005

Use of penetrating peptides interacting with PP1/PP2A proteins as a general approach for a drug phosphatase technology.

Protein phosphatase types 1 (PP1) and 2A (PP2A) represent two major families of serine/threonine protein phosphatases that have been implicated in the regulation of many cellular processes, including cell growth and apoptosis in mammalian cells. PP1 and PP2A proteins are composed of oligomeric complexes comprising a catalytic structure (PP1c or PP2AC) containing the enzymatic activity and at least one more interacting subunit. The binding of different subunits to a catalytic structure generates a broad variety of holoenzymes. We showed here that casein kinase 2alpha (Ck2alpha) and simian virus 40 small t antigen share a putative common beta-strand structure required for PP2A1 trimeric holoenzyme binding. We have also characterized DPT-sh1, a short basic peptide from Ck2alpha that interacted only in vitro with the PP2A-A subunit and behaves as a nontoxic penetrating shuttle in several cultivated human cell lines and chick embryos. In addition, DPT-sh1 specifically accumulated in human red cells infected with Plasmodium falciparum malaria parasites. We therefore designed bipartite peptides containing DPT-sh1 and PP1- or PP2A-interacting sequences. We found that DPT-5, a DPT-sh1-derived peptide containing a short sequence identified in CD28 antigen, interacts with PP2A-Balpha, and DPT-7, another DPT-sh1-derived peptide containing a short sequence identified in Bad as a PP1 catalytic consensus docking motif, induce apoptosis in cultivated cell lines. These results clearly indicate that the rational design of PP1/PP2A interacting peptides is a pertinent strategy to deregulate intracellular survival pathways.

Amino Acid Sequence↗

PubMed2005

Modulating apoptosis as a target for effective therapy.

Alterations in cell proliferation and cell death are essential determinants in the pathogenesis and progression of several diseases such as cancer, neurodegenerative disorders or autoimmune diseases among others. Complex networks of regulatory factors determine whether cells proliferate or die. Recent progress in understanding the molecular changes offer the possibility of specifically targeting molecules and pathways to achieve more effective and rational therapies. Drugs that target molecules involved in apoptosis are used as treatment against several diseases. Candidates such as TNF death receptor family, caspase inhibitors, antagonists of the p53-MDM2 interaction, NF-kappaB and PI3K pathways and Bcl-2 family members have been targeted as cancer cell killing agents. Moreover, apoptosis of tumor cells can also be achieved by targeting the inhibitor of apoptosis proteins, IAPs, in addition to the classical antiproliferative approach. Disruption of STAT activation and interferon beta therapy have been used as a treatment to prevent the progression of some autoimmune diseases. In models of Parkinson's, Alzheimer's and amyotrophic lateral sclerosis, blocking of Par-4 expression or function, as well as caspase activation, prevents neuronal cell death. Finally, it has been shown that gene therapy may be an encouraging approach for treatment of neurodegenerative disorders.

Apoptosis↗

PubMed2004

Induction of p53-independent apoptosis by the BH3-only protein ITM2Bs.

The p53 tumor suppressor protein is critically involved in cell cycle regulation and programmed cell death. Here we show that expression of the BH3-only protein ITM2Bs is able to induce apoptotic cell death in p53+/+, as well as in p53-/- cell lines. This cell death involves neither subcellular redistribution of p53 nor transcriptional regulation of p53 target genes such as Bax, Ras, Puma or Bcl-2. Together, our data provide evidence for a p53-independent apoptotic role of ITM2Bs.

Adaptor Proteins, Signal Transducing↗

PubMed2004

New insights in protein phosphorylation: a signature for protein phosphatase 1 interacting proteins.

Protein phosphatase 1 is regulated by the interaction between a catalytic subunit (PP1c) and multiple interacting proteins that allow the specific dephosphorylation of diverse cellular targets. This communication proposes to use the simultaneous presence of distinct consensus PP1c docking motifs R/K-x(0,1)-V-x-F and F-x-x-R/K-x-R/K as a signature to identify proteins putatively interacting with the PP1c. To develop this concept, we propose a new website, http://pp1 signature.pasteur.fr, which allows the identification of putative PP1-interacting proteins containing the two distinct PP1c docking consensus motifs represented in the Swissprot library. To validate the new concept of signature, we were able to characterise, by co-immunoprecipitation, four new PP1c interacting proteins randomly selected from the database in our website.

Amino Acid Motifs↗

PubMed2004

Bad-dependent rafts alteration is a consequence of an early intracellular signal triggered by interleukin-4 deprivation.

Many molecules are inducibly localized in lipid rafts, and their alteration inhibits early activation events, supporting a critical role for these domains in signaling. Using confocal microscopy and cellular fractionation, we have shown that the pool of Bad, attached to lipid rafts in proliferating cells, is released when cells undergo apoptosis. Kinetic studies indicate that rafts alteration is a consequence of an intracellular signal triggered by interleukin-4 deprivation. Growth factor deprivation in turn induces PP1alpha phosphatase activation, responsible for cytoplasmic Bad dephosphorylation as well as caspase-9 and caspase-3 activation. Caspases translocate to rafts and induce their modification followed by translocation of Bad from rafts to mitochondria, which correlates with apoptosis. Taken together, our results suggest that alteration of lipid rafts is an early event in the apoptotic cascade indirectly induced by interleukin-4 deprivation via PP1alpha activation, dephosphorylation of cytoplasmic Bad, and caspase activation.

Animals↗

PubMed2003

Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis.

The reversible phosphorylation of proteins controlled by protein kinases and protein phosphatases is a major mechanism that regulates a wide variety of cellular processes. In contrast to C. elegans, recent studies in mammalian cells have highlighted a major role of serine/threonine protein phosphorylation in apoptosis. To illustrate the importance of dephosphorylation processes in apoptosis, this review will focus on recent studies suggesting that the interaction of the serine/threonine protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with certain regulators of the Bcl-2 family is critically involved in the control of apoptosis.

Amino Acid Sequence↗

PubMed2003

Rafts: a simple way to control apoptosis by subcellular redistribution.

Apoptosis is an essential feature of development and homeostasis in higher organisms. Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and sphingolipids. In response to intra or extracellular stimuli, lipid rafts can include or exclude proteins to variable extents. This favors specific protein-protein interactions and modulates the activity of signaling cascades. Recently, a number of proteins involved in apoptotic signals have been located in lipid rafts. Among these proteins is included Bad, a pro-apoptotic molecule belonging to the Bcl-2 family. Bad is attached to lipid rafts in proliferating cells while associated to mitochondria in apoptotic cells, suggesting that the interaction of Bad with rafts is a dynamic process involved in the control of apoptosis. In this review, we briefly summarize the structure of rafts and illustrate their contribution to the control of apoptosis.

Animals↗

PubMed2003

Ikaros, Aiolos and Helios: transcription regulators and lymphoid malignancies.

Ikaros, Aiolos and Helios encode zinc finger transcription factors that are important regulators of lymphoid development and differentiation. These proteins are involved in the control of gene expression and when associated with nuclear complexes, participate in nucleosome remodeling. Because differential splicing produces multiple protein isoforms with potentially different functions, the Ikaros protein family provides a useful model for the study of whether post-transcriptional modifications are involved in tumoral transformation. Several reports reinforce the hypothesis that Ikaros, Aiolos and Helios expression is deregulated in human leukaemias. The direct involvement of aberrant protein expression of Ikaros family members in human haematological malignancies is discussed.

Animals↗

PubMed2003

BH3-only proteins: the lords of death.

Although the mechanisms by which Bcl-2 family proteins control the apoptotic machinery of the cell are not fully understood, it becomes clear that the role of BH3-only proteins consists in serving as sensors or sentinels of cellular damage, transducing the apoptotic stimuli to the mitochondria. For this reason, mammalian cells have developed several strategies for their strict regulation throughout evolution. This review aims to highlight the different ways by which BH3-only proteins are controlled, including transcriptional regulation, post-translational modifications and subcellular localization.

Animals↗

PubMed2002

Proapoptotic activity of ITM2B(s), a BH3-only protein induced upon IL-2-deprivation which interacts with Bcl-2.

Growth factor deprivation is a physiological mechanism to induce apoptosis. We used an IL-2-dependent murine T cell line to identify proteins that trigger apoptosis. Here we report the identification, the cloning and characterization of ITM2B(s), a protein induced upon IL-2-deprivation. ITM2B(s), which shares the BH3 domain of Bcl-2 family members, is a cytoplasmic and mitochondrial protein. Expression of ITM2B(s) induces apoptosis in IL-2-stimulated cells, but not in IL-4-stimulated cells, while overexpression of the long form of the protein is not able to induce apoptosis. In IL-2-stimulated cells, ITM2B(s) interacts with the antiapoptotic protein Bcl-2, and does not interact with the proapoptotic Bad. Mutation of the critical L and D residues within the BH3 domain abolished the ability of ITM2B(s) to promote apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Segregation of Bad from lipid rafts is implicated in the induction of apoptosis.

Many molecules relocate subcellularly in cells undergoing apoptosis. Using coimmunoprecipitation experiments we demonstrate that Bad is not associated to 14-3-3 protein, suggesting a new mechanism for the control of the proapoptotic role of Bad. Here we show, by confocal microscopy and cellular fractionation, that Bad is attached to lipid rafts in IL-4-stimulated cells and thymocytes while associated with mitochondria in IL-4-deprived cells. Disruption of lipid rafts by methyl-beta-cyclodextrin treatment induces segregation of Bad from rafts, which correlates with apoptosis. Our results suggest that the interaction of Bad with rafts is a dynamic process regulated by IL-4 and involved in the control of apoptosis.

Animals↗

PubMed2002

The anti-apoptotic molecules Bcl-xL and Bcl-w target protein phosphatase 1alpha to Bad.

Bcl-xL and Bcl-w specifically interact with PP1alpha and Bad. A phosphatase activity sensitive to okadaic acid was detected in Bcl-xL, Bcl-w and Bad immunoprecipitates. Serine phosphorylation of Bcl-xL and Bcl-w correlates with the number of trimolecular complexes formed. Depletion of Bcl-xL and Bcl-w decreases the remaining Bad-associated phosphatase activity and association of protein phosphatase 1 (PP1)alpha to Bad. Bcl-xL and Bcl-w contain the R/K X V/I X F consensus motif shared by PP1 targeting subunits. This motif, in addition to F X X R X R motif, is involved in binding of Bcl-xL and Bcl-w to PP1alpha. Disruption of Bcl-xL/PP1alpha or Bcl-w/PP1alpha association strongly decreases Bad-associated phosphataseactivity and stability of trimolecular complexes. These results suggest that Bcl-xL and Bcl-w are PP1alpha targeting subunits and this trimolecular complex may be involved in the control of apoptosis.

Animals↗

PubMed2002

ITM2BS regulates apoptosis by inducing loss of mitochondrial membrane potential.

Apoptosis is a conserved and essential feature of homeostasis. We have found that expression of the short form of integral membrane protein 2B (ITM2B(S)) in IL-2-stimulated T cells, as well as in COS-7 cells, induces apoptosis. Biochemical and confocal studies demonstrate that association of ITM2B(S) with mitochondria correlates with loss of mitochondrial membrane potential, release of cytochrome c to the cytosol and, as a final consequence, induction of apoptosis in IL-2-stimulated cells. Moreover, the apoptosis-inducing activity of ITM2B(S) correlates with caspase 9 and caspase 3 activation. Taken together, our results demonstrate that ITM2B(S) induces apoptosis via a caspase-dependent mitochondrial pathway.

Adaptor Proteins, Signal Transducing↗