scholarly journals Pro-apoptotic Bid induces membrane perturbation by inserting selected lysolipids into the bilayer

2005 ◽  
Vol 387 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Alexander GOONESINGHE ◽  
Elizabeth S. MUNDY ◽  
Melanie SMITH ◽  
Roya KHOSRAVI-FAR ◽  
Jean-Claude MARTINOU ◽  
...  
Keyword(s):  
Cell Death ◽  
Membrane Surface ◽  
Death Receptor ◽  
Mitochondrial Pathway ◽  
Full Length ◽  
Membrane Perturbation ◽  
C Terminus ◽  
Biochemical Level

Bid is a BH3-only member of the Bcl-2 family that regulates cell death at the level of mitochondrial membranes. Bid appears to link the mitochondrial pathway with the death receptor-mediated pathway of cell death. It is generally assumed that the f.l. (full-length) protein becomes activated after proteolytic cleavage, especially by apical caspases like caspase 8. The cleaved protein then relocates to mitochondria and promotes membrane permeabilization, presumably by interaction with mitochondrial lipids and other Bcl-2 proteins that facilitate the release of apoptogenic proteins like cytochrome c. Although the major action may reside in the C-terminus part, tBid (cleaved Bid), un-cleaved Bid also has pro-apoptotic potential when ectopically expressed in cells or in vitro. This pro-apoptotic action of f.l. Bid has remained unexplained, especially at the biochemical level. In the present study, we show that f.l. (full-length) Bid can insert specific lysolipids into the membrane surface, thereby priming mitochondria for the release of apoptogenic factors. This is most effective for lysophosphatidylcholine species that we report to accumulate in mitochondria during apoptosis induction. A Bid mutant that is not pro-apoptotic in vivo is defective in lysophosphatidylcholine-mediated membrane perturbation in vitro. Our results thus provide a biochemical explanation for the pro-apoptotic action of f.l. Bid.

scholarly journals A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

2009 ◽  
Vol 2 ◽  
pp. JCD.S3660
Author(s):  
Hang Fai Kwok ◽  
Julie A. Gormley ◽  
Christopher J. Scott ◽  
James A. Johnston ◽  
Shane A. Olwill
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
False Negative ◽  
Death Receptor ◽  
Annexin V ◽  
Fas Receptor ◽  
Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

scholarly journals The Caspase 8 Inhibitor c-FLIPL Modulates T-Cell Receptor-Induced Proliferation but Not Activation-Induced Cell Death of Lymphocytes

2002 ◽  
Vol 22 (15) ◽  
pp. 5419-5433 ◽  
Author(s):  
Susanne M. A. Lens ◽  
Takao Kataoka ◽  
Karen A. Fortner ◽  
Antoine Tinel ◽  
Isabel Ferrero ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
T Cell Receptor ◽  
Death Receptor ◽  
Cell Receptor ◽  
Caspase 8 ◽  
Activation Induced Cell Death

ABSTRACT The caspase 8 inhibitor c-FLIPL can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIPL in the T-cell compartment (c-FLIPL Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIPL Tg mice. In contrast, activation-induced cell death of T cells in c-FLIPL Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIPL Tg mice differed from Fas-deficient mice by showing no accumulation of B220+ CD4− CD8− T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIPL Tg mice. Thus, a major role of c-FLIPL in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

scholarly journals Characterization of cell-death pathways in Punta Toro virus-induced hepatocyte injury

2008 ◽  
Vol 89 (9) ◽  
pp. 2175-2181 ◽  
Author(s):  
Fangling Xu ◽  
Xiaodong Liang ◽  
Robert B. Tesh ◽  
Shu-Yuan Xiao
Keyword(s):  
Cell Death ◽  
Hepg2 Cells ◽  
Antigen Expression ◽  
Mitochondrial Pathway ◽  
Cellular Viability ◽  
Tnf Receptor ◽  
Extrinsic Pathway ◽  
Punta Toro Virus

Punta Toro virus (PTV; genus Phlebovirus, family Bunyaviridae) causes apoptosis of hepatocytes in vivo in experimentally infected hamsters and in vitro in cultured HepG2 cells. Screening for expression of apoptosis-related genes has shown alterations in the genes for tumour necrosis factor-α (TNF-α) and the TNF receptor family. This study examined the roles of the TNF receptor-related extrinsic pathway and the Bcl-2 family-associated mitochondrial pathway in PTV-induced cell death. The effects of caspase inhibitors (caspIs) and TNF on cellular viability, virus replication, and morphological and biochemical changes in apoptosis were examined in HepG2 cells at different time points after infection with PTV (Adames strain). The results showed that caspIs dampened the virus-induced reduction in cellular viability, partially suppressed and delayed viral titres and antigen expression, and partially decreased the expression of apoptotic genes, caspase activities and DNA fragmentation. TNF treatment further decreased cellular viability after PTV infection and increased the level of apoptosis, whilst caspIs partially inhibited these effects. These findings indicate that TNF, caspase-8 and caspase-9 contribute to PTV-induced hepatocytic apoptosis and that additional mediators are probably also involved in this process. These mediators from different pathways correlated with one another and may be interlinked.

A cytotoxic nitrido-osmium(vi) complex induces caspase-mediated apoptosis in HepG2 cancer cells

2020 ◽  
Vol 49 (47) ◽  
pp. 17173-17182
Author(s):  
Wan-Qiong Huang ◽  
Chuan-Xian Wang ◽  
Tao Liu ◽  
Zi-Xin Li ◽  
Chen Pan ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Hepg2 Cell ◽  
Cell Apoptosis ◽  
Death Receptor ◽  
Mitochondrial Pathway ◽  
Anticancer Activities ◽  
Death Receptor Pathway

A structurally fine-tuned nitridoosmium(vi) complex induces HepG2 cell apoptosis through activation of the mitochondrial pathway and death receptor pathway, showing promising in vitro and in vivo anticancer activities.

scholarly journals Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Roland Chanet ◽  
Dorothée Baïlle ◽  
Marie-Pierre Golinelli-Cohen ◽  
Sylvie Riquier ◽  
Olivier Guittet ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerase Delta ◽  
Chromosomal Dna ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus

Abstract B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

scholarly journals Crystallographic studies for the folding of an extending peptide

2014 ◽  
Vol 70 (a1) ◽  
pp. C1150-C1150
Author(s):  
Yuya Hanazono ◽  
Kazuki Takeda ◽  
Kunio Miki
Keyword(s):  
Protein Folding ◽  
Full Length ◽  
Helical Conformation ◽  
Stable Conformation ◽  
Ww Domain ◽  
X Ray Crystallography ◽  
C Terminus ◽  
Amino Acid Length

Full-length proteins can fold into thermodynamically stable structures at an exceptionally fast rate as shown by in vitro experiments. In contrast, it takes much more time to finish nascent protein folding than full-length protein folding, because nascent protein folding depends on the rate of ribosome biosynthesis in the living cell. Therefore nascent polypeptide chains in vivo fold co-translationally in different manners from the full-length proteins. However, the transient structures and the co-translational folding pathway are not well understood. In order to reveal the atomic details of nascent protein folding, we studied the hPin1 WW domain, which consists of two beta-hairpins between the three-stranded beta-sheets. Here we report a series of WW domain N-terminal fragment structures with increasing amino acid length by using circular dichroism spectroscopy and X-ray crystallography. In crystallization, maltose-binding protein was fused just behind the WW domain fragments to fix the C-terminus as nascent proteins are anchored to the ribosome. Co-translational folding of beta-sheet-rich proteins is discussed based on our finding that intermediate-length fragments unexpectedly take a helical conformation, even though the full-length protein has no helical regions. Furthermore, in a region of one of the loop structures of the full-length protein, these fragments take different formations. Our results suggest that the newly synthesized polypeptides adopt the most stable conformation during the course of peptide extension and fold into the native structures, eventually.

scholarly journals Central role of defective apoptosis in autoimmunity

2003 ◽  
Vol 31 (3) ◽  
pp. 373-399 ◽  
Author(s):  
WM Kuhtreiber ◽  
T Hayashi ◽  
EA Dale ◽  
DL Faustman
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Autoimmune Disease ◽  
Death Receptor ◽  
Specific Gene ◽  
Lymphocyte Development ◽  
Nf Kappab ◽  
Autoreactive Cells

Lymphocyte development, selection and education represent tightly controlled immune processes that normally prevent autoimmunity. Lymphocyte development likely induces cellular selection through apoptosis to remove potentially autoreactive cells. Dysregulated apoptosis, both interrupted as well as accelerated apoptosis, are now demonstrated as central defects in diverse murine autoimmune disease. In murine models of autoimmune lupus, mutations in cell death receptor Fas (CD95) and its ligand, FasL (CD95 L), have been identified. These errors create a lymphoid system resistant to apoptosis. In contrast, select lymphoid subpopulations of maturing autoimmune prone non-obese diabetic mice have identifiable and pathogenic T cells with both in vivo and in vitro heightened apoptosis after drug interventions. In part, these defects are due to faulty activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) that normally protect against apoptotic death. The genetic basis of interrupted NF-kappaB in pathogenic memory T cells in diabetes is attributable to a developmentally controlled gene defect in an essential subunit of the proteasome. No specific gene in most common forms of human autoimmune disease has yet been identified. Functional assays from diverse laboratories repeatedly demonstrate heightened apoptosis in multiple cellular signaling pathways for cell death, suggesting a common theme in disease causality.

scholarly journals Bcl-xL inhibition enhances Dinaciclib-induced cell death in soft-tissue sarcomas

2018 ◽  
Author(s):  
Santi Rello-Varona ◽  
Miriam Fuentes-Guirado ◽  
Roser López-Alemany ◽  
Aida Contreras-Pérez ◽  
Núria Mulet-Margalef ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Soft Tissue ◽  
Liver Toxicity ◽  
Soft Tissue Sarcomas ◽  
Mitochondrial Pathway ◽  
Chemotherapy Drugs ◽  
Model Drug

AbstractSoft-tissue sarcomas (STS) are an uncommon and heterogeneous group of malignancies that result in high mortality. Metastatic STS have very bad prognosis due to the lack of effective treatments. Dinaciclib is a model drug for the family of CDK inhibitors. Its main targets are cell cycle regulator CDK1 and protein synthesis controller CDK9. We present data supporting Dinaciclib ability to inactivate in vitro different STS models at nanomolar concentrations. Moreover, the different rhythms of cell death induction allow us to further study into the mechanism of action of the drug. Cell death was found to respond to the mitochondrial pathway of apoptosis. Anti-apoptotic Bcl-xL was identified as the key regulator of this process. Bcl-xL showed a slower decay curve after protein synthesis disruption that in tolerant cell lines was enough to delay apoptosis, as its action cannot be countered by the relative low levels of pro-apoptotic BH3 proteins BIM and PUMA. Combination of Dinaciclib with BH3-mimetics led to quick and massive apoptosis induction in vitro, but in vivo assessment was prevented due to liver toxicity. Additionally, Bcl-xL inhibitor A-1331852 also synergized with conventional chemotherapy drugs as Gemcitabine. Thus, Bcl-xL targeted therapy arises as a major opportunity to the treatment of STS.

scholarly journals Castration-resistant prostate cancer: Androgen receptor inactivation induces telomere DNA damage, and damage response inhibition leads to cell death

2019 ◽  
Author(s):  
Vidyavathi Reddy ◽  
Asm Iskander ◽  
Clara Hwang ◽  
George Divine ◽  
Mani Menon ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cell Death ◽  
Combined Treatment ◽  
Full Length ◽  
Castration Resistant Prostate Cancer ◽  
Castration Resistant ◽  
Damage Response

AbstractTelomere stability is important for cell viability, as cells with telomere DNA damage that is not repaired do not survive. We reported previously that androgen receptor (AR) antagonist induces telomere DNA damage in androgen-sensitive LNCaP prostate cancer cells; this triggers a DNA damage response (DDR) at telomeres that includes activation of ATM, and blocking ATM activation prevents telomere DNA repair and leads to cell death. Remarkably, AR antagonist induces telomere DNA damage and triggers ATM activation at telomeres also in 22Rv1 castration-resistant prostate cancer (CRPC) cells that are not growth inhibited by AR antagonist. Treatment with AR antagonist enzalutamide (ENZ) or ATM inhibitor (ATMi) by itself had no effect on growth in vitro or in vivo, but combined treatment with ENZ plus ATMi significantly inhibited cell survival in vitro and tumor growth in vivo. By inducing telomere DNA damage and activating a telomere DDR, an opportunity to inhibit DNA repair and promote cell death was created, even in CRPC cells. 22Rv1 cells express both full-length AR and AR splice variant AR-V7, but full-length AR was found to be the predominant form of AR associated with telomeres and required for telomere stability. Although 22Rv1 growth of untreated 22Rv1 cells appears to be driven by AR-V7, it is, ironically, expression of full-length AR that makes them sensitive to growth inhibition by combined treatment with ENZ plus ATMi. Notably, this combined treatment approach to induce telomere DNA damage and inhibit the DDR was effective in inducing cell death also in other CRPC cell lines (LNCaP/AR and C4-2B). Thus, the use of ENZ in combination with a DDR inhibitor, such as ATMi, may be effective in prolonging disease-free survival of patients with AR-positive metastatic CRPC, even those that co-express AR splice variant.

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