RESUMO
Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Substituição de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Cricetinae , Ativação Enzimática , Retroalimentação Fisiológica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Lipossomos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
Members of the Arf family of small G proteins are involved in membrane traffic and organelle structure. They control the recruitment of coat proteins, and modulate the structure of actin filaments and the lipid composition of membranes. The ADP-ribosylation factor 6 (Arf6) isoform and the exchange factor for Arf6 (EFA6) are known to regulate the endocytic pathway of many different receptors. To determine the molecular mechanism of the EFA6/Arf6 function in vesicular transport, we searched for new EFA6 partners. In a two-hybrid screening using the catalytic Sec7 domain as a bait, we identified endophilin as a new partner of EFA6. Endophilin contains a Bin/Amphiphysin/Rvs (BAR) domain responsible for membrane bending, and an SH3 domain responsible for the recruitment of dynamin and synaptojanin, two proteins involved, respectively, in the fission and uncoating of clathrin-coated vesicles. By using purified proteins, we confirmed the direct interaction, and identified the N-BAR domain as the binding motif to EFA6A. We showed that endophilin stimulates the catalytic activity of EFA6A on Arf6. In addition, we observed that the Sec7 domain competes with flat but not with highly curved lipid membranes to bind the N-BAR. In cells, expression of EFA6A recruits endophilin to EFA6A-positive plasma membrane ruffles, whereas expression of endophilin rescues the EFA6A-mediated inhibition of transferrin internalization. Overall, our results support a model whereby EFA6 recruits endophilin on flat areas of the plasma membrane to control Arf6 activation and clathrin-mediated endocytosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Endocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Recombinantes/metabolismo , Fator 6 de Ribosilação do ADP , Clatrina/metabolismo , Clonagem Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , XantenosRESUMO
Acid-sensing ion channels (ASICs) are neuronal proton-gated cation channels associated with nociception, fear, depression, seizure, and neuronal degeneration, suggesting roles in pain and neurological and psychiatric disorders. We have recently discovered black mamba venom peptides called mambalgin-1 and mambalgin-2, which are new three-finger toxins that specifically inhibit with the same pharmacological profile ASIC channels to exert strong analgesic effects in vivo. We now combined bioinformatics and functional approaches to uncover the molecular mechanism of channel inhibition by the mambalgin-2 pain-relieving peptide. Mambalgin-2 binds mainly in a region of ASIC1a involving the upper part of the thumb domain (residues Asp-349 and Phe-350), the palm domain of an adjacent subunit, and the ß-ball domain (residues Arg-190, Asp-258, and Gln-259). This region overlaps with the acidic pocket (pH sensor) of the channel. The peptide exerts both stimulatory and inhibitory effects on ASIC1a, and we propose a model where mambalgin-2 traps the channel in a closed conformation by precluding the conformational change of the palm and ß-ball domains that follows proton activation. These data help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Analgésicos/química , Venenos Elapídicos/química , Simulação de Acoplamento Molecular , Peptídeos/química , Animais , Sítios de Ligação , Estrutura Terciária de Proteína , Ratos , Relação Estrutura-AtividadeRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Chaperone-mediated autophagy (CMA) is a highly selective form of autophagy. During CMA, the HSC70 chaperone carries target proteins endowed with a KFERQ-like motif to the lysosomal receptor LAMP2A, which then translocate them into lysosomes for degradation. In the present study, we scrutinized the mechanisms underlying the response and resistance to Azacytidine (Aza) in MDS/AML cell lines and bone marrow CD34+ blasts from MDS/AML patients. In engineered Aza-resistant MDS cell lines and some AML cell lines, we identified a profound defect in CMA linked to the absence of LAMP2A. LAMP2 deficiency was responsible for Aza resistance and hypersensitivity to lysosome and autophagy inhibitors. Accordingly, gain of function of LAMP2 in deficient cells or loss of function in LAMP2-expressing cells rendered them sensitive or resistant to Aza, respectively. A strict correlation was observed between the absence of LAMP2, resistance to Aza and sensitivity to lysosome inhibitors. Low levels of LAMP2 expression in CD34+ blasts from MDS/AML patients correlated with lack of sensitivity to Aza and were predictive of poor overall survival. We propose that CD34+/LAMP2Low patients at diagnosis or who become CD34+/LAMP2Low during the course of treatment with Aza might benefit from a lysosome inhibitor already used in the clinic.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Polo-like kinases (Plks) define a highly conserved family of Ser/Thr kinases with crucial roles in the regulation of cell division. Here we show that Plk1 is cleaved by caspase 3, but not by other caspases in different hematopoietic cell lines treated with competitive inhibitors of the ATP-binding pocket of Plk1. Intriguingly, Plk1 was not cleaved in cells treated with Rigosertib, a non-competitive inhibitor of Plk1, suggesting that binding of the inhibitor to the ATP binding pocket of Plk1 triggers a conformational change and unmasks a cryptic caspase 3 cleavage site on the protein. Cleavage occurs after Asp-404 in a DYSD/K sequence and separates the kinase domain from the two PBDs of Plk1. All Plk1 inhibitors triggered G2/M arrest, activation of caspases 2 and 3, polyploidy, multiple nuclei and mitotic catastrophe, albeit at higher concentrations in the case of Rigosertib. Upon BI-2536 treatment, Plk1 cleavage occurred only in the cytosolic fraction and cleaved Plk1 accumulated in this subcellular compartment. Importantly, the cleaved N-Terminal fragment of Plk1 exhibited a higher enzymatic activity than its non-cleaved counterpart and accumulated into the cytoplasm conversely to the full length and the C-Terminal Plk1 fragments that were found essentially into the nucleus. Finally, the DYSD/K cleavage site was highly conserved during evolution from c. elegans to human. In conclusion, we described herein for the first time a specific cleavage of Plk1 by caspase 3 following treatment of cancer cells with ATP-competitive inhibitors of Plk1.
RESUMO
CSF-1 and IL-34 share the CSF-1 receptor and no differences have been reported in the signaling pathways triggered by both ligands in human monocytes. IL-34 promotes the differentiation and survival of monocytes, macrophages and osteoclasts, as CSF-1 does. However, IL-34 binds other receptors, suggesting that differences exist in the effect of both cytokines. In the present study, we compared the differentiation and polarization abilities of human primary monocytes in response to CSF-1 or IL-34. CSF-1R engagement by one or the other ligands leads to AKT and caspase activation and autophagy induction through expression and activation of AMPK and ULK1. As no differences were detected on monocyte differentiation, we investigated the effect of CSF-1 and IL-34 on macrophage polarization into the M1 or M2 phenotype. We highlighted a striking increase in IL-10 and CCL17 secretion in M1 and M2 macrophages derived from IL-34 stimulated monocytes, respectively, compared to CSF-1 stimulated monocytes. Variations in the secretome induced by CSF-1 or IL-34 may account for their different ability to polarize naïve T cells into Th1 cells. In conclusion, our findings indicate that CSF-1 and IL-34 exhibit the same ability to induce human monocyte differentiation but may have a different ability to polarize macrophages.