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Trends Cell Biol ; 30(5): 354-369, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32302548


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.

BMC Genomics ; 20(Suppl 3): 293, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31815628


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.

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 Sinais
Biomed Res Int ; 2019: 4087160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31317028


Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors-chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)-or inducer-rapamycin (Rap)-can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.

Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Caseínas/farmacologia , Neoplasias/tratamento farmacológico , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/genética , Caseínas/genética , Catepsina D/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Células MCF-7 , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Morfolinas/farmacologia , Neoplasias/genética , Pironas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/genética
Cell Death Differ ; 26(6): 981-993, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30903104


Glycosylation and glycan-binding proteins such as galectins play an important role in the control of cell death signaling. Strikingly, very little attention has been given so far to the understanding of the molecular details behind this key regulatory network. Glycans attached to the death receptors such as CD95 and TRAIL-Rs, either alone or in a complex with galectins, might promote or inhibit apoptotic signals. However, we have just started to decode the functions of galectins in the modulation of extrinsic and intrinsic apoptosis. In this work, we have discussed the current understanding of the glycosylation-galectin regulatory network in CD95- as well as TRAIL-R-induced apoptosis and therapeutic strategies based on targeting galectins in cancer.

EMBO J ; 37(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29661886


Increasing evidence suggests that synaptic functions of the amyloid precursor protein (APP), which is key to Alzheimer pathogenesis, may be carried out by its secreted ectodomain (APPs). The specific roles of APPsα and APPsß fragments, generated by non-amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsß in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2. APPsα efficiently rescued deficits in spine density, synaptic plasticity (LTP and PPF), and spatial reference memory of cDKO mice. In contrast, APPsß failed to show any detectable effects on synaptic plasticity and spine density. The C-terminal 16 amino acids of APPsα (lacking in APPsß) proved sufficient to facilitate LTP in a mechanism that depends on functional nicotinic α7-nAChRs. Further, APPsα showed high-affinity, allosteric potentiation of heterologously expressed α7-nAChRs in oocytes. Collectively, we identified α7-nAChRs as a crucial physiological receptor specific for APPsα and show distinct in vivo roles for APPsα versus APPsß. This implies that reduced levels of APPsα that might occur during Alzheimer pathogenesis cannot be compensated by APPsß.

Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Cognição/fisiologia , Plasticidade Neuronal/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Coluna Vertebral/metabolismo , Coluna Vertebral/patologia , Transmissão Sináptica/genética , Receptor Nicotínico de Acetilcolina alfa7/genética
Front Mol Neurosci ; 10: 30, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28223920


Cleavage of the amyloid precursor protein (APP) by α-secretase generates an extracellularly released fragment termed secreted APP-alpha (APPsα). Not only is this process of interest due to the cleavage of APP within the amyloid-beta sequence, but APPsα itself has many physiological properties that suggest its great potential as a therapeutic target. For example, APPsα is neurotrophic, neuroprotective, neurogenic, a stimulator of protein synthesis and gene expression, and enhances long-term potentiation (LTP) and memory. While most early studies have been conducted in vitro, effectiveness in animal models is now being confirmed. These studies have revealed that either upregulating α-secretase activity, acutely administering APPsα or chronic delivery of APPsα via a gene therapy approach can effectively treat mouse models of Alzheimer's disease (AD) and other disorders such as traumatic head injury. Together these findings suggest the need for intensifying research efforts to harness the therapeutic potential of this multifunctional protein.