RESUMO
Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Caspase 8/metabolismo , Morte Celular , Humanos , Modelos Biológicos , Transdução de SinaisRESUMO
Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIPL in the heterodimer caspase-8/c-FLIPL. This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2' loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIPL. In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIPL and get new insights into molecular mechanisms of its activation.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Multimerização Proteica , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/química , Linhagem Celular Tumoral , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Proteína Ligante Fas , Humanos , Modelos Moleculares , Reprodutibilidade dos Testes , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Domínio Efetor de Morte , Sequência de Aminoácidos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Células HeLa , Humanos , Células Jurkat , Modelos Moleculares , Isoformas de Proteínas/metabolismo , Transporte Proteico , Receptor fas/metabolismoRESUMO
BACKGROUND: Structural homology modeling supported by bioinformatics analysis plays a key role in uncovering new molecular interactions within gene regulatory networks. Here, we have applied this powerful approach to analyze the molecular interactions orchestrating death receptor signaling networks. In particular, we focused on the molecular mechanisms of CD95-mediated NF-κB activation and the role of c-FLIP/NEMO interaction in the induction of this pathway. RESULTS: To this end, we have created the homology model of the c-FLIP/NEMO complex using the reported structure of the v-FLIP/NEMO complex, and rationally designed peptides targeting this complex. The designed peptides were based on the NEMO structure. Strikingly, the experimental in vitro validation demonstrated that the best inhibitory effects on CD95-mediated NF-κB activation are exhibited by the NEMO-derived peptides with the substitution D242Y of NEMO. Furthermore, we have assumed that the c-FLIP/NEMO complex is recruited to the DED filaments formed upon CD95 activation and validated this assumption in silico. Further insight into the function of c-FLIP/NEMO complex was provided by the analysis of evolutionary conservation of interacting regions which demonstrated that this interaction is common in distinct mammalian species. CONCLUSIONS: Taken together, using a combination of bioinformatics and experimental approaches we obtained new insights into CD95-mediated NF-κB activation, providing manifold possibilities for targeting the death receptor network.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Quinase I-kappa B/metabolismo , Sondas Moleculares , NF-kappa B/metabolismo , Receptor fas/metabolismo , Sequência de Aminoácidos , Biologia Computacional , Humanos , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Transdução de SinaisRESUMO
Mutations of fms-like tyrosine kinase 3 (FLT3) are the most frequent mutations in acute myeloid leukemia (AML). Furthermore, the internal tandem duplication (ITD) represents the most common mutation of FLT3 in AML. To explore therapeutic strategies for AML patients carrying FLT3-ITD, we analyzed death receptor (DR) signaling networks in AML cells comprising FLT3-ITD. We have started with murine myeloid progenitor 32D cells that ectopically express human FLT3-ITD (32D- FLT3-ITD) and found that RIPK1 is strongly upregulated in these cells. Subsequently, we have shown that combinatorial treatment of 32D-FLT3-ITD cells with the SMAC mimetic BV6 and CD95L sensitizes these cells toward apoptosis and necroptosis. Moreover, combinatorial treatment with death ligands (DLs), for example, CD95L or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and BV6 enhanced cell death in primary AML blasts from patients carrying FLT3-ITD mutation. Finally, pharmacological and genetic targeting of RIPK1 inhibited DL/BV6-mediated cell death in cells with FLT3-ITD mutations. Taken together, our study suggests a promising therapeutic opportunity for AML cancer cells harboring FLT3-ITD mutation via targeting RIPK1 pathways.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Duplicação Gênica , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Mutação , Regulação para Cima , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.