scholarly journals Characterisation of FADD interactome reveals novel insights into FADD recruitment and signalling at the Death Inducing Signalling Complex (DISC)

2021 ◽  
Author(s):  
Joanna L Fox ◽  
Laura S Dickens ◽  
Rebekah Jukes-Jones ◽  
Gareth J Miles ◽  
Claudia Langlais ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Specific Binding ◽  
Affinity Purification ◽  
Death Receptor ◽  
Vital Role ◽  
Death Domain ◽  
Apoptotic Pathway ◽  
Signaling Complex ◽  
Binding Partners ◽  
Molecular Events

AbstractFas-associated death domain protein (FADD) plays a vital role in the extrinsic apoptotic pathway, where it forms an essential component of the death-inducing signaling complex (DISC). However, the precise early molecular events that facilitate recruitment of FADD to the DISC remain poorly defined. Using affinity purification and mass spectrometry we investigated the FADD interactome in untreated cells and following death receptor stimulation to identify novel FADD-interacting proteins. As expected, in death receptor-stimulated samples our analysis identified key components of the DISC such as Caspase-8. In addition, we identified novel binding partners including Transferrin Receptor 1 (TfR1) and Myosin Light Chain Kinase 2 (MYLK2) that are able to modulate FADD recruitment to the DISC and consequently downstream apoptotic signaling. TfR1 is pre-associated with FADD and recruited into the DISC; moreover, our data reveal that TfR1 is also pre-associated with the death receptors, TRAIL-R1 and TRAIL-R2, thereby functioning as a key regulator of DISC formation. In the case of MYLK2, specific binding of FADD to MYLK2 in non-apoptotic cells sequesters FADD from other DISC components ensuring aberrant apoptosis is not initiated. Furthermore, MYLK2 enzymatic activity is required to for it to translocate, in complex with FADD, to sites of DISC-mediated death receptor oligimerization. Taken together, our study highlights the important role that additional novel FADD binding partners play in the regulation of death receptor-mediated apoptotic cell death, in part by modulating FADD recruitment to the DISC.

scholarly journals Regulation of the Extrinsic Apoptotic Pathway by the Extracellular Matrix Glycoprotein EMILIN2

2007 ◽  
Vol 27 (20) ◽  
pp. 7176-7187 ◽  
Author(s):  
Maurizio Mongiat ◽  
Giovanni Ligresti ◽  
Stefano Marastoni ◽  
Erica Lorenzon ◽  
Roberto Doliana ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Survival ◽  
Three Dimensional ◽  
Death Receptor ◽  
Death Receptors ◽  
Soft Agar ◽  
Apoptotic Pathway ◽  
Signaling Complex ◽  
Extrinsic Apoptotic Pathway ◽  
Trail Receptors

ABSTRACT Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.

scholarly journals RALB GTPase: a critical regulator of DR5 expression and TRAIL sensitivity in KRAS mutant colorectal cancer

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Hajrah Khawaja ◽  
Andrew Campbell ◽  
Jamie Z. Roberts ◽  
Arman Javadi ◽  
Paul O’Reilly ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Death ◽  
Apoptotic Cell ◽  
Treatment Options ◽  
Unmet Need ◽  
Death Receptor ◽  
Mrna Levels ◽  
Death Receptor 5 ◽  
Apoptotic Pathway ◽  
Cell Functions

Abstract RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.

scholarly journals Epilepsy and Apoptosis Pathways

2005 ◽  
Vol 25 (12) ◽  
pp. 1557-1572 ◽  
Author(s):  
David C Henshall ◽  
Roger P Simon
Keyword(s):  
Cell Death ◽  
Neuronal Death ◽  
Apoptotic Cell ◽  
Death Receptor ◽  
Apoptotic Pathway ◽  
Apoptosis Pathway ◽  
Cell Death Pathway ◽  
Pathway Function ◽  
Apoptosis Pathways ◽  
Molecular Machinery

Epilepsy is a common, chronic neurologic disorder characterized by recurrent unprovoked seizures. Experimental modeling and clinical neuroimaging of patients has shown that certain seizures are capable of causing neuronal death. Such brain injury may contribute to epileptogenesis, impairments in cognitive function or the epilepsy phenotype. Research into cell death after seizures has identified the induction of the molecular machinery of apoptosis. Here, the authors review the clinical and experimental evidence for apoptotic cell death pathway function in the wake of seizure activity. We summarize work showing intrinsic (mitochondrial) and extrinsic (death receptor) apoptotic pathway function after seizures, activation of the caspase and Bcl-2 families of cell death modulators and the acute and chronic neuropathologic impact of intervening in these molecular cascades. Finally, we describe evolving data on nonlethal roles for these proteins in neuronal restructuring and cell excitability that have implications for shaping the epilepsy phenotype. This review highlights the work to date on apoptosis pathway signaling during seizure-induced neuronal death and epileptogenesis, and speculates on how emerging roles in brain remodeling and excitability have enriched the number of therapeutic strategies for protection against seizure-damage and epileptogenesis.

scholarly journals c-FLIP Mediates Resistance of Hodgkin/Reed-Sternberg Cells to Death Receptor–induced Apoptosis

2004 ◽  
Vol 199 (8) ◽  
pp. 1041-1052 ◽  
Author(s):  
Stephan Mathas ◽  
Andreas Lietz ◽  
Ioannis Anagnostopoulos ◽  
Franziska Hummel ◽  
Burkhard Wiesner ◽  
...  
Keyword(s):  
Death Receptor ◽  
B Cell Lymphoma ◽  
Immunohistochemical Analysis ◽  
Death Domain ◽  
Inhibitory Protein ◽  
Antiapoptotic Proteins ◽  
Signaling Complex ◽  
Induced Apoptosis ◽  
High Level ◽  
Interfering Rna

Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

scholarly journals A Death Receptor-associated Anti-apoptotic Protein, BRE, Inhibits Mitochondrial Apoptotic Pathway

2004 ◽  
Vol 279 (50) ◽  
pp. 52106-52116 ◽  
Author(s):  
Qing Li ◽  
Arthur Kar-Keung Ching ◽  
Ben Chung-Lap Chan ◽  
Stephanie Ka-Yee Chow ◽  
Pak-Leong Lim ◽  
...  
Keyword(s):  
Tumor Necrosis Factor Receptor ◽  
Death Receptor ◽  
Cellular Level ◽  
Down Regulation ◽  
Apoptotic Pathway ◽  
Signaling Complex ◽  
Apoptotic Protein ◽  
Tnf Α ◽  
Receptor Ligation ◽  
Interfering Rna

BRE,brain andreproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-α-induced activation of NF-κB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-α, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-α, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.

scholarly journals Reovirus-Induced Apoptosis Is Mediated by TRAIL

2000 ◽  
Vol 74 (17) ◽  
pp. 8135-8139 ◽  
Author(s):  
Penny Clarke ◽  
Suzanne M. Meintzer ◽  
Spencer Gibson ◽  
Christian Widmann ◽  
Timothy P. Garrington ◽  
...  
Keyword(s):  
Death Receptor ◽  
Tnf Receptor ◽  
Death Domain ◽  
Apoptotic Pathway ◽  
Cellular Receptors ◽  
Trail Receptors ◽  
Infected Cells ◽  
Soluble Trail ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

scholarly journals Differential Inhibition of TRAIL-Mediated DR5-DISC Formation by Decoy Receptors 1 and 2

2006 ◽  
Vol 26 (19) ◽  
pp. 7046-7055 ◽  
Author(s):  
Delphine Mérino ◽  
Najoua Lalaoui ◽  
Alexandre Morizot ◽  
Pascal Schneider ◽  
Eric Solary ◽  
...  
Keyword(s):  
Death Receptor ◽  
Death Domain ◽  
Transmembrane Receptors ◽  
Signaling Complex ◽  
Cancer Cell Death ◽  
Decoy Receptors ◽  
Additional Level ◽  
Induced Apoptosis ◽  
Death Receptor 4 ◽  
Trail Signaling

ABSTRACT Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.

scholarly journals Edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 711-719 ◽  
Author(s):  
Consuelo Gajate ◽  
Faustino Mollinedo
Keyword(s):  
Multiple Myeloma ◽  
Cytochrome C ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Death Receptor ◽  
Death Receptors ◽  
Death Domain ◽  
Cytochrome C Release ◽  
Signaling Complex ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) is an incurable B-cell malignancy, requiring new therapeutic strategies. We have found that synthetic alkyl-lysophospholipids (ALPs) edelfosine and perifosine induced apoptosis in MM cell lines and patient MM cells, whereas normal B and T lymphocytes were spared. ALPs induced recruitment of Fas/CD95 death receptor, Fas-associated death domain–containing protein, and procaspase-8 into lipid rafts, leading to the formation of the death-inducing signaling complex (DISC) and apoptosis. TNF-related apoptosis-inducing ligand receptor-1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5, as well as Bid, were also recruited into lipid rafts, linking death receptor and mitochondrial signaling pathways. ALPs induced mitochondrial cytochrome c release. Bcl-XL overexpression prevented cytochrome c release and apoptosis. A Fas/CD95-deficient MM subline expressing DR4 and DR5 was resistant to edelfosine. Fas/CD95 retrovirus transduction bestowed edelfosine sensitivity in these cells. A Fas/CD95 mutant lacking part of the intracellular domain was ineffective. Lipid raft disruption prevented ALP-induced Fas/CD95 clustering, DISC formation, and apoptosis. ALP-induced apoptosis was Fas/CD95 ligand (FasL/CD95L) independent. ALP-induced recruitment of death receptors in lipid rafts potentiated MM cell killing by FasL/CD95L and TRAIL. These data uncover a novel lipid raft–mediated therapy in MM involving concentration of death receptors in membrane rafts, with Fas/CD95 playing a major role in ALP-mediated apoptosis.

scholarly journals Impaired c-Jun Amino Terminal Kinase Activity and T Cell Differentiation in Death Receptor 6–deficient Mice

2001 ◽  
Vol 194 (10) ◽  
pp. 1441-1448 ◽  
Author(s):  
Haoran Zhao ◽  
Minhong Yan ◽  
Hua Wang ◽  
Sharon Erickson ◽  
Iqbal S. Grewal ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
Death Receptor ◽  
Death Domain ◽  
Th2 Response ◽  
Apoptotic Pathway ◽  
Th Cells ◽  
Amino Terminal ◽  
Deficient Mice

During an immune response naive T helper (Th) cells differentiate into two functionally distinct subsets, Th1 and Th2, based on their cytokine secretion profile and immunomodulatory function. c-Jun amino terminal kinase (JNK) regulates Th cell differentiation by activating a transcriptional program required for cytokine production. We have recently identified a TNFR superfamily death domain–containing molecule, death receptor (DR)6, which potently activates JNK. T cells from DR6-deficient mice are substantially impaired in JNK activation. When DR6−/− mice were challenged with protein antigen, their T cells hyperproliferate and display a profound polarization toward a Th2 response whereas Th1 differentiation is not equivalently affected. In addition, DR6−/− T cells showed preference toward Th2 differentiation in vitro. The phenotype seen in the DR6−/− mice is not due to the apoptotic pathway. Therefore, DR6, working through JNK, rather than apoptosis, functions to attenuate the Th2 response. This is the first demonstration of a role in the activation and differentiation of Th cells by DR6 in particular and DRs in general.

Activation of the intrinsic-apoptotic pathway in LNCaP prostate cancer cells by genistein- topotecan combination treatments

2013 ◽  
Vol 3 (3) ◽  
pp. 66 ◽  
Author(s):  
Vanessa Hörmann ◽  
Sivanesan Dhandayuthapani ◽  
James Kumi-Diaka ◽  
Appu Rathinavelu
Keyword(s):  
Prostate Cancer ◽  
Cell Death ◽  
Cancer Cells ◽  
Apoptotic Cell ◽  
Treatment Options ◽  
Attractive Alternative ◽  
Intrinsic Apoptotic Pathway ◽  
Prostate Cancer Cells ◽  
Apoptotic Pathway ◽  
Combination Treatments

Background: Prostate cancer is the second most common cancer in American men. The development of alternative preventative and/or treatment options utilizing a combination of phytochemicals and chemotherapeutic drugs could be an attractive alternative compared to conventional carcinoma treatments. Genistein isoflavone is the primary dietary phytochemical found in soy and has demonstrated anti-tumor activities in LNCaP prostate cancer cells. Topotecan Hydrochloride (Hycamtin) is an FDA-approved chemotherapy for secondary treatment of lung, ovarian and cervical cancers. The purpose of this study was to detail the potential activation of the intrinsic apoptotic pathway in LNCaP prostate cancer cells through genistein-topotecan combination treatments. Methods: LNCaP cells were cultured in complete RPMI medium in a monolayer (70-80% confluency) at 37ºC and 5% CO2. Treatment consisted of single and combination groups of genistein and topotecan for 24 hours. The treated cells were assayed for i) growth inhibition through trypan blue exclusion assay and microphotography, ii) classification of cellular death through acridine/ ethidium bromide fluorescent staining, and iii) activation of the intrinsic apoptotic pathway through Jc-1: mitochondrial membrane potential assay, cytochrome c release and Bcl-2 protein expression.Results: The overall data indicated that genistein-topotecan combination was significantly more efficacious in reducing the prostate carcinoma’s viability compared to the single treatment options. In all treatment groups, cell death occurred primarily through the activation of the intrinsic apoptotic pathway.Conclusion: The combination of topotecan and genistein has the potential to lead to treatment options with equal therapeutic efficiency as traditional chemo- and radiation therapies, but lower cell cytotoxicity and fewer side effects in patients. Key words: topotecan; genistein; intrinsic apoptotic cell death

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