RESUMO
Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker, but can be significantly enhanced through phosphorylation by the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}. In the present study we describe the NMR and crystal structures of the autophagy modifier LC3B (microtubule-associated protein light chain 3 beta) in complex with the LC3 interaction region of optineurin either phosphorylated or bearing phospho-mimicking mutations. The structures show that the negative charge induced by phosphorylation is recognized by the side chains of Arg¹¹ and Lys5¹ in LC3B. Further mutational analysis suggests that the replacement of the canonical tryptophan residue side chain of autophagy receptors with the smaller phenylalanine side chain in optineurin significantly weakens its interaction with the autophagy modifier LC3B. Through phosphorylation of serine residues directly N-terminally located to the phenylalanine residue, the affinity is increased to the level normally seen for receptor-modifier interactions. Phosphorylation, therefore, acts as a switch for optineurin-based selective autophagy.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos/química , Salmonella/fisiologia , Fator de Transcrição TFIIIA/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Proteínas de Ciclo Celular , Cristalografia por Raios X , Interações Hospedeiro-Patógeno , Humanos , Ligação de Hidrogênio , Proteínas de Membrana Transportadoras , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Termodinâmica , Fator de Transcrição TFIIIA/genéticaRESUMO
Faster than death: NMR techniques that make use of nonlinear sampling and hyperdimensional processing enable the recording of complete NMR data sets for the automated assignment of the backbone and side-chain resonances of short-lived protein samples of cell lysates.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Pesquisa BiomédicaRESUMO
Modified ubiquitin sequences, each completed with a His tag and a TEV cleavage site, were designed to enhance the expression of protein/peptide targets. With this new system we have been able to characterize several peptide-protein interactions by ITC and by NMR and CD spectroscopic methods, including the interactions of LIR domains with autophagy modifiers.
Assuntos
Etiquetas de Sequências Expressas/química , Proteínas/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Sequência de Bases , Vetores Genéticos/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Selective autophagy requires the specific segregation of targeted proteins into autophagosomes. The selectivity is mediated by autophagy receptors, such as p62 and NBR1, which can bind to autophagic effector proteins (Atg8 in yeast, MAP1LC3 protein family in mammals) anchored in the membrane of autophagosomes. Recognition of autophagy receptors by autophagy effectors takes place through an LC3 interaction region (LIR). The canonical LIR motif consists of a WXXL sequence, N-terminally preceded by negatively charged residues. The LIR motif of NBR1 presents differences to this classical LIR motif with a tyrosine residue and an isoleucine residue substituting the tryptophan residue and the leucine residue, respectively. We have determined the structure of the GABARAPL-1/NBR1-LIR complex and studied the influence of the different residues belonging to the LIR motif for the interaction with several mammalian autophagy modifiers (LC3B and GABARAPL-1). Our results indicate that the presence of a tryptophan residue in the LIR motif increases the binding affinity. Substitution by other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation. This indicates that different LIRs can interact with autophagy modifiers with unique binding properties.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Proteínas Associadas aos Microtúbulos/química , Proteínas/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Autofagia , Sítios de Ligação/genética , Ligação Competitiva , Calorimetria , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Espectroscopia de Ressonância Magnética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Triptofano/química , Triptofano/genética , Triptofano/metabolismoRESUMO
Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.
Assuntos
Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Família da Proteína 8 Relacionada à Autofagia , Sítios de Ligação , Western Blotting , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Receptores de GABA-A/metabolismo , Reticulócitos/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
TANK-binding kinase 1 (TBK1/NAK/T2K) and I-kappaB Kinase (IKK-i/IKK-epsilon) play important roles in the regulation of interferon (IFN)-inducible genes during the immune response to bacterial and viral infections. Cell stimulation with ssRNA virus, dsDNA virus or gram-negative bacteria leads to activation of TBK1 or IKK-i, which in turn phosphorylates the transcription factors, IFN-regulatory factor (IRF) 3 and IRF7, promoting their translocation in the nucleus. To understand the molecular basis of activation of TBK1, we analyzed the sequence of TBK1 and IKK-i and identified a ubiquitin-like domain (ULD) adjacent to their kinase domains. Deletion or mutations of the ULD in TBK1 or IKK-i impaired activation of respective kinases, failed to induce IRF3 phosphorylation and nuclear localization and to activate IFN-beta or RANTES promoters. The importance of the ULD of TBK1 in LPS- or poly(I:C)-stimulated IFN-beta production was demonstrated by reconstitution experiments in TBK1-IKK-i-deficient cells. We propose that the ULD is a regulatory component of the TBK1/IKK-i kinases involved in the control of the kinase activation, substrate presentation and downstream signaling pathways.
