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

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.

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Chromatin at X-linked repeats that guide dosage compensation in Drosophila melanogaster is modulated by the siRNA pathway

10.1101/330423 ◽

2018 ◽

Author(s):

Nikita Deshpande ◽

Victoria H. Meller

Keyword(s):

Drosophila Melanogaster ◽

X Chromosome ◽

Sex Chromosome ◽

Gene Dosage ◽

Ectopic Expression ◽

Dependent Manner ◽

Satellite Repeats ◽

Msl Complex ◽

Dependent Modification ◽

Sirna Pathway

AbstractMany heterogametic organisms adjust sex chromosome expression to accommodate differences in gene dosage. This requires selective recruitment of regulatory factors to the modulated chromosome. How these factors are localized to a chromosome with requisite accuracy is poorly understood. Drosophila melanogaster males increase expression from their single X chromosome. Identification of this chromosome involves cooperation between different classes of X-identity elements. The Chromatin Entry Sites (CES) recruit a chromatin-modifying complex that spreads into nearby genes and increases expression. In addition, a family of satellite repeats that is enriched on the X chromosome, the 1.688X repeats, promotes recruitment of the complex to nearby genes. The 1.688X repeats and CES are dissimilar, and appear to operate through different mechanisms. Interestingly, the siRNA pathway and siRNA from a 1.688X repeat also promote X recognition. We postulate that siRNA-dependent modification of 1.688X chromatin contributes to recognition of nearby genes. In accord with this, we found enrichment of the siRNA effector Argonaute2 (Ago2) at some 1.688X repeats.Mutations in several proteins that physically interact with Ago2, including the histone methyltransferase Su(var)3-9, enhance the lethality of males with defective X recognition. Su(var)3-9 deposits H3K9me2 on some 1.688X repeats, and this mark is disrupted upon ectopic expression of 1.688X siRNA. Furthermore, integration of 1.688X DNA on an autosome induces local H3K9me2 deposition, but enhances expression of nearby genes in a siRNA-dependent manner. Our findings are consistent with a model in which siRNA-directed modification of 1.688X chromatin contributes to recognition of the fly X chromosome by the MSL complex.

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.6673 ◽

1999 ◽

Vol 19 (10) ◽

pp. 6673-6681 ◽

Cited By ~ 32

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.

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p180 Is Involved in the Interaction between the Endoplasmic Reticulum and Microtubules through a Novel Microtubule-binding and Bundling Domain

Molecular Biology of the Cell ◽

10.1091/mbc.e06-12-1125 ◽

2007 ◽

Vol 18 (10) ◽

pp. 3741-3751 ◽

Cited By ~ 37

Author(s):

Kiyoko Ogawa-Goto ◽

Keiko Tanaka ◽

Tomonori Ueno ◽

Keisuke Tanaka ◽

Takeshi Kurata ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Cultured Cells ◽

Specific Interaction ◽

Endoplasmic Reticulum Membrane ◽

Dependent Manner ◽

Small Interfering Rnas ◽

Animal Cells ◽

Microtubule Binding

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

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Transcriptional Adaptor ADA3 of Drosophila melanogaster Is Required for Histone Modification, Position Effect Variegation, and Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.01307-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 376-385 ◽

Cited By ~ 18

Author(s):

Benjamin Grau ◽

Cristina Popescu ◽

Laura Torroja ◽

Daniel Ortuño-Sahagún ◽

Imre Boros ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Histone Acetyltransferase ◽

Gene Dosage ◽

Position Effect ◽

Position Effect Variegation ◽

Dependent Manner ◽

Histone H4 ◽

Chromosomal Pairing ◽

Effect Variegation ◽

A Subunit

ABSTRACT The Drosophila melanogaster gene diskette (also known as dik or dAda3) encodes a protein 29% identical to human ADA3, a subunit of GCN5-containing histone acetyltransferase (HAT) complexes. The fly dADA3 is a major contributor to oogenesis, and it is also required for somatic cell viability. dADA3 localizes to chromosomes, and it is significantly reduced in dGcn5 and dAda2a, but not in dAda2b, mutant backgrounds. In dAda3 mutants, acetylation at histone H3 K9 and K14, but not K18, and at histone H4 K12, but not K5, K8, and K16, is significantly reduced. Also, phosphorylation at H3 S10 is reduced in dAda3 and dGcn5 mutants. Variegation for white (w m4 ) and scute (Hw v ) genes, caused by rearrangements of X chromosome heterochromatin, is modified in a dAda3 + gene-dosage-dependent manner. The effect is not observed with rearrangements involving Y heterochromatin (bw D ), euchromatin (Scutoid), or transvection effects on chromosomal pairing (white and zeste interaction). Activity of scute gene enhancers, targets for Iroquoi transcription factors, is abolished in dAda3 mutants. Also, Iroquoi-associated phenotypes are sensitive to dAda3 + gene dosage. We conclude that dADA3 plays a role in HAT complexes which acetylate H3 and H4 at specific residues. In turn, this acetylation results in chromatin structure effects of certain rearrangements and transcription of specific genes.

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Polyomavirus Small T Antigen Induces Apoptosis in Mammalian Cells through the UNC5B Pathway in a PP2A-Dependent Manner

Journal of Virology ◽

10.1128/jvi.02187-19 ◽

2020 ◽

Vol 94 (14) ◽

Author(s):

Sameer Ahmed Bhat ◽

Zarka Sarwar ◽

Syed Qaaifah Gillani ◽

Misbah Un Nisa ◽

Irfana Reshi ◽

...

Keyword(s):

Cell Death ◽

Mammalian Cells ◽

Mitotic Arrest ◽

T Antigen ◽

Mitotic Catastrophe ◽

Mrna Levels ◽

Dependent Manner ◽

Antibody Treatment ◽

Small T Antigen ◽

Apoptotic Activity

ABSTRACT UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A. IMPORTANCE UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.

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Cell Penetrating Mechanism of Bax Inhibiting Peptides.

Blood ◽

10.1182/blood.v106.11.4298.4298 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4298-4298

Author(s):

Jose A. Gomez ◽

Tomoyuki Yoshida ◽

Minh Lam ◽

Clark W. Distelhorst ◽

Shigemi Matsuyama

Keyword(s):

Cell Death ◽

Plasma Membrane ◽

Fluorescent Dyes ◽

Cultured Cells ◽

Cell Penetrating Peptides ◽

Dependent Manner ◽

Cell Penetration ◽

New Members ◽

Cell Penetrating ◽

High Degree

Abstract Plasma membrane is known to have a high degree of selectivity for molecular trafficking, and it does not allow the penetration of peptides larger than 3 amino acids. Previously known exceptions of large peptides that penetrate the plasma membrane are the Arginine rich peptides such as human immunodeficiency virus (HIV)-tat peptides. However, the mechanism of cell penetration of these peptides is largely unknown. Bax Inhibiting Peptides (BIP) are penta-peptides derived from the Bax binding domain of Ku70. At present, three types of BIP have been developed. Those are: VPMLK, VPTLK, and VPALR. All of these three BIPs directly bind Bax and inhibit Bax-mediated cell death in cultured cells as well as in animal study. Surprisingly, BIPs are cell permeable and autonomously enter the cytoplasm of the cells within 1 hr. Therefore BIPs are recognized as new members of cell penetration peptides. In this study, we investigated the mechanism of cell penetration of BIPs. DAMI cells (a human megakaryocyte cell line) and HeLa cells were used to investigate the detailed mechanism of cell penetration of BIPs. To detect the cell entry of BIPs, fluorescent dyes (fluorescein or tetramethylrodhamine) were conjugated to the N-terminus of BIPs and the cytoplasmic localization of BIPs was confirmed by confocal microscopy. Cell Penetration activities of BIPs were detected at 1 uM concentration in the culture medium. The significant accumulation of BIPs in the cytoplasm were detected within 1 hour of incubation both at 4 °C and 37 °C, suggesting that ATP-independent mechanism of cell penetration of BIP exists. However, cellular uptake of BIPs reaches plateau at 100 uM at 4 °C, whereas it increases in a dose dependent manner up to 1 mM at 37 °C without any sign of cytotoxicity. These results suggest that there are at least two mechanisms contributing to the cell penetration of BIPs that are, “ATP-independent (4 °C)” and “ATP-dependent (37 °C)” mechanisms. In addition to BIPs, we generated a series of mutated BIPs that do not bind Bax but retain cell-penetrating activities. We performed competition assay using fluorescence dye-labeled and non-labeled BIP (and the mutant BIPs), and the preliminary results suggest that there is a specific receptor for each peptide for its delivery into the cells. Our data also indicates that BIPs can deliver a cargo molecule (e.g. fluorescent dye) with at least the same molecular weight. Unlike other cell penetrating peptides, BIP has minimum toxicity due to its nature to inhibit Bax-mediated cell death. Along with the new data showing that BIP protects cells from pathological damages in cell culture and animal model, we will discuss the potential application of BIPs as a new type of drug delivery tool.

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RNA dependent suppression of C9orf72 ALS/FTD associated neurodegeneration by Matrin-3

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01060-y ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Nandini Ramesh ◽

Elizabeth L. Daley ◽

Amanda M. Gleixner ◽

Jacob R. Mann ◽

Sukhleen Kour ◽

...

Keyword(s):

Motor Neurons ◽

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Ectopic Expression ◽

Dependent Manner ◽

Matrin 3 ◽

Rna Foci ◽

Ran Translation

Abstract The most common genetic cause of amyotrophic lateral sclerosis (ALS) is a GGGGCC (G4C2) hexanucleotide repeat expansions in first intron of the C9orf72 gene. The accumulation of repetitive RNA sequences can mediate toxicity potentially through the formation of intranuclear RNA foci that sequester key RNA-binding proteins (RBPs), and non-ATG mediated translation into toxic dipeptide protein repeats. However, the contribution of RBP sequestration to the mechanisms underlying RNA-mediated toxicity remain unknown. Here we show that the ALS-associated RNA-binding protein, Matrin-3 (MATR3), colocalizes with G4C2 RNA foci in patient tissues as well as iPSC-derived motor neurons harboring the C9orf72 mutation. Hyperexpansion of C9 repeats perturbed subcellular distribution and levels of endogenous MATR3 in C9-ALS patient-derived motor neurons. Interestingly, we observed that ectopic expression of human MATR3 strongly mitigates G4C2-mediated neurodegeneration in vivo. MATR3-mediated suppression of C9 toxicity was dependent on the RNA-binding domain of MATR3. Importantly, we found that expression of MATR3 reduced the levels of RAN-translation products in mammalian cells in an RNA-dependent manner. Finally, we have shown that knocking down endogenous MATR3 in C9-ALS patient-derived iPSC neurons decreased the presence of G4C2 RNA foci in the nucleus. Overall, these studies suggest that MATR3 genetically modifies the neuropathological and the pathobiology of C9orf72 ALS through modulating the RNA foci and RAN translation.

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Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200712091 ◽

2008 ◽

Vol 182 (6) ◽

pp. 1127-1139 ◽

Cited By ~ 108

Author(s):

Ying-Chen Claire Hou ◽

Suganthi Chittaranjan ◽

Sharon González Barbosa ◽

Kimberly McCall ◽

Sharon M. Gorski

Keyword(s):

Cell Death ◽

Drosophila Melanogaster ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Mitogen Activated Protein Kinase ◽

Nuclear Condensation ◽

Effector Caspase ◽

Apoptosis Protein ◽

Egg Chambers

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

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Assessment of fat-specific protein 27 in the adipocyte lineage suggests a dual role for FSP27 in adipocyte metabolism and cell death

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00104.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. E654-E667 ◽

Cited By ~ 43

Author(s):

Ji Young Kim ◽

Kun Liu ◽

Shengli Zhou ◽

Kristin Tillison ◽

Yu Wu ◽

...

Keyword(s):

Cell Death ◽

Mammalian Cells ◽

Ectopic Expression ◽

Transcript Level ◽

Specific Protein ◽

Fatty Acid Binding ◽

Brown Adipose ◽

Tnf Α ◽

Human Preadipocytes

Fat-specific protein 27 (FSP27)/CIDEC was initially identified by its upregulation in TA1 adipogenesis and is one of three cell death-inducing DFF45-like effector (CIDE) family proapoptotic proteins. Ectopic expression of CIDEs promotes apoptosis of mammalian cells. On the other hand, FSP27 has very recently been illustrated to regulate lipid droplet size and promote lipid storage in adipocytes. Regulation of endogenous FSP27 expression is unknown. We assessed the FSP27 transcript level in the well-characterized 3T3-L1 in vitro adipocyte differentiation model and found its emergence parallels the adipocyte-enriched transcript adipocyte fatty acid binding protein and stearoyl Co-A desaturase 1. Furthermore, FSP27 is a differentiation-dependent transcript in adipogenesis of primary rodent and human preadipocytes and in brown adipogenesis. The FSP27 transcript is inversely regulated by TNF-α and insulin, consistent with an antilipolytic function. It is nearly abolished with a 4-h exposure of 3T3-L1 adipocytes to 10 ng/ml TNF-α, while treatment with 100 nM insulin increased the FSP27 transcript eightfold. In the latter case LY-294002 blocked this response, indicating involvement of phosphatidylinositol 3-kinase signals. Northern blot analysis of murine tissues indicated exclusive expression of FSP27 in white and brown adipose tissue; however, a dramatic upregulation occurred in the liver of ob/ob mice. Ectopic expression of murine FSP27 in 293T cells and in 3T3-L1 preadipocytes led to the appearance of key apoptotic hallmarks and cell death. However, despite the upregulation for FSP27 in adipogenesis, we failed to detect DNA laddering indicative of apoptosis in 3T3-L1 adipocytes. This suggests that adipogenesis is accompanied by decreased susceptibility to the proapoptotic effects of FSP27. Overall, our findings support roles for FSP27 in cell death and in adipocyte function.

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Calpain is required for macroautophagy in mammalian cells

The Journal of Cell Biology ◽

10.1083/jcb.200601024 ◽

2006 ◽

Vol 175 (4) ◽

pp. 595-605 ◽

Cited By ~ 103

Author(s):

Francesca Demarchi ◽

Cosetta Bertoli ◽

Tamara Copetti ◽

Isei Tanida ◽

Claudio Brancolini ◽

...

Keyword(s):

Cell Death ◽

Protein Degradation ◽

Mammalian Cells ◽

Apoptotic Cell ◽

Nuclear Factor Κb ◽

Early Endosome ◽

Regulatory Subunit ◽

Dependent Manner ◽

Nuclear Factor ◽

Salvage Pathway

Ubiquitously expressed micro- and millicalpain, which both require the calpain small 1 (CAPNS1) regulatory subunit for function, play important roles in numerous biological and pathological phenomena. We have previously shown that the product of GAS2, a gene specifically induced at growth arrest, is an inhibitor of millicalpain and that its overexpression sensitizes cells to apoptosis in a p53-dependent manner (Benetti, R., G. Del Sal, M. Monte, G. Paroni, C. Brancolini, and C. Schneider. 2001. EMBO J. 20:2702–2714). More recently, we have shown that calpain is also involved in nuclear factor κB activation and its relative prosurvival function in response to ceramide, in which calpain deficiency strengthens the proapoptotic effect of ceramide (Demarchi, F., C. Bertoli, P.A. Greer, and C. Schneider. 2005. Cell Death Differ. 12:512–522). Here, we further explore the involvement of calpain in the apoptotic switch and find that in calpain-deficient cells, autophagy is impaired with a resulting dramatic increase in apoptotic cell death. Immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments demonstrate that autophagy is impaired in CAPNS1-deficient cells. Accordingly, the enhancement of lysosomal activity and long-lived protein degradation, which normally occur upon starvation, is also reduced. In CAPNS1-depleted cells, ectopic LC3 accumulates in early endosome-like vesicles that may represent a salvage pathway for protein degradation when autophagy is defective.

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