scholarly journals The BH3 Domain of Bcl-xS Is Required for Inhibition of the Antiapoptotic Function of Bcl-xL

1999 ◽  
Vol 19 (10) ◽  
pp. 6673-6681 ◽  
Author(s):  
Brian S. Chang ◽  
Ameeta Kelekar ◽  
Marian H. Harris ◽  
John E. Harlan ◽  
Stephen W. Fesik ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Survival Function ◽  
Family Members ◽  
Protein Product ◽  
Deletion Mutants ◽  
Major Protein ◽  
Bh3 Domain ◽  
Acid Protein

ABSTRACT bcl-x is a member of the bcl-2 family of genes. The major protein product, Bcl-xL, is a 233-amino-acid protein which has antiapoptotic properties. In contrast, one of the alternatively spliced transcripts of the bcl-xgene codes for the protein Bcl-xS, which lacks 63 amino acids present in Bcl-xL and has proapoptotic activity. Unlike other proapoptotic Bcl-2 family members, such as Bax and Bak, Bcl-xS does not seem to induce cell death in the absence of an additional death signal. However, Bcl-xS does interfere with the ability of Bcl-xL to antagonize Bax-induced death in transiently transfected 293 cells. Mutational analysis of Bcl-xS was conducted to identify the domains necessary to mediate its proapoptotic phenotype. Deletion mutants of Bcl-xS which still contained an intact BH3 domain retained the ability to inhibit survival through antagonism of Bcl-xL. Bcl-xS was able to form heterodimers with Bcl-xL in mammalian cells, and its ability to inhibit survival correlated with the ability to heterodimerize with Bcl-xL. Deletion mutants of Bax and Bcl-2, which lacked BH1 and BH2 domains but contained a BH3 domain, were able to antagonize the survival effect conferred by Bcl-xL. The results suggest that BH3 domains from both pro- and antiapoptotic Bcl-2 family members, while lacking an intrinsic ability to promote programmed cell death, can be potent inhibitors of Bcl-xL survival function.

scholarly journals Debcl, a Proapoptotic Bcl-2 Homologue, Is a Component of the Drosophila melanogaster Cell Death Machinery

2000 ◽  
Vol 148 (4) ◽  
pp. 703-714 ◽  
Author(s):  
Paul A. Colussi ◽  
Leonie M. Quinn ◽  
David C.S. Huang ◽  
Michelle Coombe ◽  
Stuart H. Read ◽  
...  
Keyword(s):  
Cell Death ◽  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Gene Dosage ◽  
Family Members ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Proapoptotic Protein ◽  
Main Components

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

scholarly journals Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells.

1997 ◽  
Vol 17 (5) ◽  
pp. 2468-2474 ◽  
Author(s):  
B Ink ◽  
M Zörnig ◽  
B Baum ◽  
N Hajibagheri ◽  
C James ◽  
...  
Keyword(s):  
Cell Death ◽  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Yeast Cells ◽  
Bh3 Domain ◽  
Evolutionarily Conserved ◽  
Unicellular Organisms ◽  
Induction Of Apoptosis ◽  
Multicellular Organisms

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.

scholarly journals Identification and Characterization of a Putative Human Iodide Transporter Located at the Apical Membrane of Thyrocytes

2002 ◽  
Vol 87 (7) ◽  
pp. 3500-3503 ◽  
Author(s):  
Anne-Marie Rodriguez ◽  
Barbara Perron ◽  
Ludovic Lacroix ◽  
Bernard Caillou ◽  
Gérard Leblanc ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Human Kidney ◽  
Protein Product ◽  
Sodium Iodide Symporter ◽  
Pcr Cloning ◽  
Thyroid Cells ◽  
Iodide Transport ◽  
Acid Protein

Iodide transport by thyrocytes is a two step process involving transporters located either in the basal or in the apical membranes of the cell. The sodium iodide symporter (NIS) is localized in the basolateral membrane facing the bloodstream and mediates iodide accumulation into thyrocytes. Pendrin has been proposed as an apical transporter. In order to identify new iodide transporters, we developed a PCR cloning strategy based on NIS sequence homologies. From a human kidney cDNA library, we characterized a gene, located on chromosome 12q23, that encodes a 610 amino acid protein sharing 46% identity (70% similarity) with the human NIS. Functional analysis of the protein expressed in mammalian cells indicates that it catalyzes a passive iodide transport. The protein product was immunohistochemically localized at the apical pole of the thyroid cells facing the colloid lumen. These results suggest that this new identified protein mediates iodide transport from the thyrocyte into the colloid lumen through the apical membrane. It was designated hAIT for human Apical Iodide Transporter.

scholarly journals Bcl-2 does not require Raf kinase activity for its death-protective function

1997 ◽  
Vol 324 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Reynald OLIVIER ◽  
Isabelle OTTER ◽  
Laurent MONNEY ◽  
Markus WARTMANN ◽  
Christoph BORNER
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Survival Function ◽  
Active Form ◽  
Signal Transduction Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Protective Function

It has been widely accepted that the oncogene product bcl-2 protects mammalian cells from programmed cell death (apoptosis). The molecules and signalling pathways upon which bcl-2 acts are, however, still ill-defined. Recently, bcl-2 was shown to interact with c-raf-1 in vitro. Furthermore, an active form of c-raf-1 delayed apoptosis induced by trophic factor deprivation and enhanced the death-suppressive function of bcl-2 when co-expressed. This has led to the hypothesis that bcl-2 communicates cell-death protection via a raf-dependent signal transduction pathway. Here we show, by various immunological and biochemical methods, that bcl-2 does not stably associate with c-raf-1 in cellular extracts prepared from fibroblasts before or after treatment with agents that induce apoptosis. Unexpectedly, bcl-2 function is entirely maintained, if not improved, when raf-dependent signalling is experimentally abrogated. In fact, bcl-2 allows the stable overexpression of a kinase-defective dominant-negative raf mutant that usually interferes with cell viability and/or proliferation. Our results indicate that bcl-2 does not require c-raf-1 kinase activity and an associated mitogen-activated protein kinase signalling pathway for its survival function. This property may be exploited to dissect cellular events that are dependent or independent of c-raf-1 kinase activity.

scholarly journals Identification of Novel Isoforms of the BH3 Domain Protein Bim Which Directly Activate Bax To Trigger Apoptosis

2002 ◽  
Vol 22 (11) ◽  
pp. 3577-3589 ◽  
Author(s):  
Michela Marani ◽  
Tencho Tenev ◽  
David Hancock ◽  
Julian Downward ◽  
Nicholas R. Lemoine
Keyword(s):  
Cell Death ◽  
Splice Variants ◽  
Family Members ◽  
Cell Types ◽  
Cell Death Pathway ◽  
Dynein Motor ◽  
Bh3 Domain ◽  
Protein Isoform ◽  
Novel Isoforms

ABSTRACT Bim (Bcl-2-interacting mediator of cell death) is a member of the BH3 domain-only subgroup of Bcl-2 family members, for which three splice variants have been described. Bim is expressed in many healthy cell types, where it is maintained in an inactive conformation through binding to the microtubule-associated dynein motor complex. Upon certain apoptotic stimuli, Bim is released from microtubules and mediates caspase-dependent apoptosis through a mechanism that is still unclear. Here, we have identified and characterized novel splice variants of human Bim mRNA. In particular, we show that a newly discovered, small protein isoform, BimAD, is also able to induce apoptosis strongly in several human cell lines. BimAD and the previously characterized isoform BimS are shown to be capable of heterodimerizing in vivo with both death antagonists (Bcl-2 and Bcl-XL) and death agonists (Bax). Mutants of BimAD that bind to Bax but not to Bcl-2 still promote apoptosis, indicating that Bim can regulate apoptosis through direct activation of the Bax-mediated cell death pathway without interaction with antiapoptotic Bcl-2 family members. Furthermore, we have shown that the interaction of the BimS and BimAD isoforms with Bax leads to a conformational change in this protein analogous to that triggered by the BH3-only protein Bid.

scholarly journals Contribution of GADD45 Family Members to Cell Death Suppression by Cellular Bcl-xL and Cytomegalovirus vMIA

2005 ◽  
Vol 79 (23) ◽  
pp. 14923-14932 ◽  
Author(s):  
Geoffrey B. Smith ◽  
Edward S. Mocarski
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Mammalian Cells ◽  
Regulatory Protein ◽  
Family Members ◽  
Genotoxic Stress ◽  
Cell Cycle Regulatory Protein ◽  
Protein Levels ◽  
A Cell ◽  
Interfering Rna

ABSTRACT Mammalian cells and viruses encode inhibitors of programmed cell death that localize to mitochondria and suppress apoptosis initiated by a wide variety of inducers. Mutagenesis was used to probe the role of a predicted α-helical region within the hydrophobic antiapoptotic domain (AAD) of cytomegalovirus vMIA, the UL37x1 gene product. This region was found to be essential for cell death suppression activity. A screen for proteins that interacted with the AAD of functional vMIA but that failed to interact with mutants identified growth arrest and DNA damage 45 (GADD45α), a cell cycle regulatory protein activated by genotoxic stress, as a candidate cellular binding partner. GADD45α interaction required the AAD α-helical character that also dictated GADD45α-mediated enhancement of death suppression. vMIA mutants that failed to interact with GADD45α were completely nonfunctional in cell death suppression, and any of the three GADD45 family members (GADD45α, GADD45β/MyD118, or GADD45γ/OIG37/CR6/GRP17) was able to cooperate with vMIA; however, none influenced cell death when introduced into cells alone. GADD45α was found to increase vMIA protein levels comparably to treatment with protease inhibitors MG132 and ALLN. Targeted short interfering RNA knockdown of all three GADD45 family members maximally reduced vMIA activity, and this reduction was abrogated by additional GADD45α. Interestingly, GADD45 family members were also able to bind and enhance cell death suppression by Bcl-xL, a member of the Bcl-2 family of cell death suppressors, suggesting a direct cooperative link between apoptosis and the proteins that regulate the DNA damage response.

The P-MAPA immunomodulator partially prevents apoptosis induced by Zika virus infection in THP-1 cells

2020 ◽  
Vol 21 ◽  
Author(s):  
Morganna C. Lima ◽  
Elisa A. N. Azevedo ◽  
Clarice N. L. de Morais ◽  
Larissa I. O. de Sousa ◽  
Bruno M. Carvalho ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Virus Infection ◽  
Virus Replication ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Neurological Complications ◽  
Zika Virus Infection

Background: Zika virus is an emerging arbovirus of global importance. ZIKV infection is associated with a range of neurological complications such as the Congenital Zika Syndrome and Guillain Barré Syndrome. Despite the magnitude of recent outbreaks, there is no specific therapy to prevent or to alleviate disease pathology. Objective: To investigate the role of P-MAPA immunomodulator in Zika-infected THP-1 cells. Methods: THP-1 cells were subjected at Zika virus infection (Multiplicity of Infection = 0.5) followed by treatment with P-MAPA for until 96 hours post-infection. After that, the cell death was analyzed by annexin+/ PI+ and caspase 3/ 7+ staining by flow cytometry. In addition, the virus replication and cell proliferation were accessed by RT-qPCR and Ki67 staining, respectively. Results: We demonstrate that P-MAPA in vitro treatment significantly reduces Zika virus-induced cell death and caspase-3/7 activation on THP-1 infected cells, albeit it has no role in virus replication and cell proliferation. Conclusions: Our study reveals that P-MAPA seems to be a satisfactory alternative to inhibits the effects of Zika virus infection in mammalian cells.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

scholarly journals Biochemical and Genetic Analysis of the Mitochondrial Response of Yeast to BAX and BCL-XL

2000 ◽  
Vol 20 (9) ◽  
pp. 3125-3136 ◽  
Author(s):  
Atan Gross ◽  
Kirsten Pilcher ◽  
Elizabeth Blachly-Dyson ◽  
Emy Basso ◽  
Jennifer Jockel ◽  
...  
Keyword(s):  
Cell Death ◽  
Mitochondrial Genome ◽  
Atp Synthase ◽  
Mitochondrial Permeability Transition ◽  
Family Members ◽  
Growth Arrest ◽  
Anion Channel ◽  
Cell Killing ◽  
Β Subunit ◽  
Voltage Dependent Anion Channel

ABSTRACT The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-XL) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-XL using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-XL prevented all BAX-mediated responses. We also assessed the function of BCL-XL and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic β subunit or the δ subunit of the F0F1-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho 0] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the β subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-XL protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-XL: cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.

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