RESUMO
BACKGROUND: The inflammatory response is indispensable for protective immunity, yet microbial pathogens often trigger an excessive response, 'cytokine storm', harmful to the host. Full T-cell activation requires interaction of costimulatory receptors B7-1(CD80) and B7-2(CD86) expressed on antigen-presenting cells with CD28 expressed on the T cells. We created short peptide mimetics of the homodimer interfaces of the B7 and CD28 receptors and examined their ability to attenuate B7/CD28 coligand engagement and signaling through CD28 for inflammatory cytokine induction in human immune cells, and to protect from lethal toxic shock in vivo. METHODS: Short B7 and CD28 receptor dimer interface mimetic peptides were synthesized and tested for their ability to attenuate the inflammatory cytokine response of human peripheral blood mononuclear cells, as well as for their ability to attenuate B7/CD28 intercellular receptor engagement. Mice were used to test the ability of such peptides to protect from lethal superantigen toxin challenge when administered in molar doses far below the toxin dose. RESULTS: B7 and CD28 homodimer interfaces are remote from the coligand binding sites, yet our finding is that by binding back into the receptor dimer interfaces, short dimer interface mimetic peptides inhibit intercellular B7-2/CD28 as well as the tighter B7-1/CD28 engagement, attenuating thereby pro-inflammatory signaling. B7 mimetic peptides exhibit tight selectivity for the cognate receptor in inhibiting intercellular receptor engagement with CD28, yet each diminishes signaling through CD28. In a prominent example of inflammatory cytokine storm, by attenuating formation of the B7/CD28 costimulatory axis, B7-1 and CD28 dimer interface mimetic peptides protect mice from lethal toxic shock induced by a bacterial superantigen even when administered in doses far submolar to the superantigen. CONCLUSIONS: Our results reveal that the B7 and CD28 homodimer interfaces each control B7/CD28 costimulatory receptor engagement and highlight the protective potential against cytokine storm of attenuating, yet not ablating, pro-inflammatory signaling via these receptor domains.
Assuntos
Antígenos CD28 , Choque Séptico , Humanos , Animais , Camundongos , Leucócitos Mononucleares , Moléculas de Adesão Celular , Síndrome da Liberação de Citocina , Citocinas , Polímeros , SuperantígenosRESUMO
Nutrient availability regulates the C. elegans life cycle as well as mitochondrial physiology. Food deprivation significantly reduces mitochondrial genome (mtDNA) numbers and leads to aging-related phenotypes. Here we show that the bZIP (basic leucine zipper) protein ATFS-1, a mediator of the mitochondrial unfolded protein response (UPRmt), is required to promote growth and establish a functional germline after prolonged starvation. We find that recovery of mtDNA copy numbers and development after starvation requires mitochondrion-localized ATFS-1 but not its nuclear transcription activity. We also find that the insulin-like receptor DAF-2 functions upstream of ATFS-1 to modulate mtDNA content. We show that reducing DAF-2 activity represses ATFS-1 nuclear function while causing an increase in mtDNA content, partly mediated by mitochondrion-localized ATFS-1. Our data indicate the importance of the UPRmt in recovering mitochondrial mass and suggest that atfs-1-dependent mtDNA replication precedes mitochondrial network expansion after starvation.
Assuntos
Proteínas de Caenorhabditis elegans , Genoma Mitocondrial , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Resposta a Proteínas não DobradasRESUMO
The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1.
Assuntos
Proteases Dependentes de ATP , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Replicação do DNA , DNA Mitocondrial , Heteroplasmia , Mitocôndrias , Proteínas Mitocondriais , Fosforilação Oxidativa , Fatores de Transcrição , Animais , Humanos , Animais Geneticamente Modificados , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , DNA Polimerase gama/genética , DNA Polimerase gama/metabolismo , DNA Mitocondrial/biossíntese , DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteólise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Caenorhabditis elegans/genética , Metabolismo Energético , Regulação da Expressão Gênica , Chaperonas Moleculares , Transporte Proteico , Transdução de Sinais , Fatores de Transcrição/metabolismo , Resposta a Proteínas não DobradasRESUMO
Cryptic transcription occurs widely across the eukaryotic genome; however, its regulation during vertebrate development is not understood. Here, we show that two class I histone deacetylases, Hdac1 and Hdac2, silence cryptic transcription to promote mitochondrial function in developing murine hearts. Mice lacking Hdac1 and Hdac2 in heart exhibit defective developmental switch from anaerobic to mitochondrial oxidative phosphorylation (OXPHOS), severe defects in mitochondrial mass, mitochondrial function, and complete embryonic lethality. Hdac1/Hdac2 promotes the transition to OXPHOS by enforcing transcriptional fidelity of metabolic gene programs. Mechanistically, Hdac1/Hdac2 deacetylates histone residues including H3K23, H3K14, and H4K16 to suppress cryptic transcriptional initiation within the coding regions of actively transcribed metabolic genes. Thus, Hdac1/2-mediated epigenetic silencing of cryptic transcription is essential for mitochondrial function during early vertebrate development.
Assuntos
Regulação da Expressão Gênica , Coração/embriologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Organogênese/genética , Animais , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Transcrição GênicaRESUMO
Mitochondrial function declines during ageing owing to the accumulation of deleterious mitochondrial genomes and damage resulting from the localized generation of reactive oxygen species, both of which are often exacerbated in diseases such as Parkinson disease. Cells have several mechanisms to assess mitochondrial function and activate a transcriptional response known as the mitochondrial unfolded protein response (UPRmt) when mitochondrial integrity and function are impaired. The UPRmt promotes cell survival and the recovery of the mitochondrial network to ensure optimal cellular function. Recent insights into the regulation, mechanisms and functions of the UPRmt have uncovered important and complex links to ageing and ageing-associated diseases. In this Review, we discuss the signal transduction mechanisms that regulate the UPRmt and the physiological consequences of its activation that affect cellular and organismal health during ageing.
Assuntos
Mitocôndrias/fisiologia , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologia , Envelhecimento/fisiologia , Animais , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/genética , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Mutação/genética , Doença de Parkinson/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The source of the autophagic membrane and the regulation of autophagosome biogenesis are still elusive open issues in the field of autophagy. In our recent study of the role of lipid droplets (LDs) and their constituents in autophagy, we provided evidence that both the biogenesis of LDs and its lipolysis by specific lipases are important for autophagosome biogenesis. Our study sheds new light on the source of the autophagic membrane and suggests that a flow of membranes from the endoplasmic reticulum (ER) to LDs, and from LDs to the ER, is essential for autophagosome biogenesis.
RESUMO
Autophagy is a major catabolic process responsible for the delivery of proteins and organelles to the lysosome/vacuole for degradation. Malfunction of this pathway has been implicated in numerous pathological conditions. Different organelles have been found to contribute to the formation of autophagosomes, but the exact mechanism mediating this process remains obscure. Here, we show that lipid droplets (LDs) are important for the regulation of starvation-induced autophagy. Deletion of Dga1 and Lro1 enzymes responsible for triacylglycerol (TAG) synthesis, or of Are1 and Are2 enzymes responsible for the synthesis of steryl esters (STE), results in the inhibition of autophagy. Moreover, we identified the STE hydrolase Yeh1 and the TAG lipase Ayr1 as well as the lipase/hydrolase Ldh1 as essential for autophagy. Finally, we provide evidence that the ER-LD contact-site proteins Ice2 and Ldb16 regulate autophagy. Our study thus highlights the importance of lipid droplet dynamics for the autophagic process under nitrogen starvation.
Assuntos
Autofagia , Ésteres/metabolismo , Gotículas Lipídicas/metabolismo , Fagossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Triglicerídeos/metabolismo , Redes e Vias Metabólicas/genéticaRESUMO
Autophagy, an evolutionarily conserved intracellular catabolic process, leads to the degradation of cytosolic proteins and organelles in the vacuole/lysosome. Different forms of selective autophagy have recently been described. Starvation-induced protein degradation, however, is considered to be nonselective. Here we describe a novel interaction between autophagy-related protein 8 (Atg8) and fatty acid synthase (FAS), a pivotal enzymatic complex responsible for the entire synthesis of C16- and C18-fatty acids in yeast. We show that although FAS possesses housekeeping functions, under starvation conditions it is delivered to the vacuole for degradation by autophagy in a Vac8- and Atg24-dependent manner. We also provide evidence that FAS degradation is essential for survival under nitrogen deprivation. Our results imply that during nitrogen starvation specific proteins are preferentially recruited into autophagosomes.
Assuntos
Autofagia , Ácido Graxo Sintases/metabolismo , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteólise , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In this issue, Randow and coworkers (von Muhlinen et al., 2012) report that LC3C plays an essential role in Salmonella clearance by interacting with the autophagic receptor NDP52, with consequent recruitment of all other ATG8 proteins to assist in the buildup of the autophagic membrane.
Assuntos
Autofagia , Células/citologia , Ubiquitinas/metabolismo , Animais , Células/metabolismo , Humanos , Ligação Proteica , Ubiquitinas/genéticaRESUMO
Autophagy is a unique membrane trafficking process whereby newly formed membranes, termed phagophores, engulf parts of the cytoplasm leading to the production of double-membraned autophagosomes that get delivered to lysosomes for degradation. This catabolic pathway has been linked to numerous physiological and pathological conditions, such as development, programmed cell death, cancer, pathogen infection, neurodegenerative disorders, and myopathies. In this review, we will focus on recent studies in yeast and mammalian systems that have provided insights into two critical areas of autophagosome biogenesis - the source of the autophagosomal membranes, and the mechanisms regulating the fusion of the edges of the double-membraned phagophores to form autophagosomes.
Assuntos
Autofagia/fisiologia , Fagossomos/metabolismo , Proteínas Relacionadas à Autofagia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved ß-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the ß-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.
Assuntos
Antígenos CD28/imunologia , Citocinas/genética , Choque Séptico/imunologia , Superantígenos/imunologia , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/imunologia , Antígenos CD28/genética , Linhagem Celular Tumoral , Citocinas/imunologia , Enterotoxinas/imunologia , Mapeamento de Epitopos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Imunidade Celular , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ligação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Choque Séptico/genética , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Superantígenos/administração & dosagem , Ressonância de Plasmônio de SuperfícieRESUMO
Autophagy-related (Atg) proteins are eukaryotic factors participating in various stages of the autophagic process. Thus far 34 Atgs have been identified in yeast, including the key autophagic protein Atg8. The Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal α helices and a ubiquitin-like core. Atg8 family members are expressed in various tissues, where they participate in multiple cellular processes, such as intracellular membrane trafficking and autophagy. Their role in autophagy has been intensively studied. Atg8 proteins undergo a unique ubiquitin-like conjugation to phosphatidylethanolamine on the autophagic membrane, a process essential for autophagosome formation. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs. Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), γ-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals. Although genes from all three subfamilies are found in vertebrates, some invertebrate lineages have lost the genes from one or two subfamilies. The amino terminus of Atg8 proteins varies between the subfamilies and has a regulatory role in their various functions. Here we discuss the evolution of Atg8 proteins and summarize the current view of their function in intracellular trafficking and autophagy from a structural perspective.
Assuntos
Autofagia , Ubiquitinas/genética , Ubiquitinas/fisiologia , Animais , Evolução Molecular , Humanos , Transporte Proteico , Ubiquitinas/químicaRESUMO
Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for the proteins role in autophagosome biogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Fusão de Membrana , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Família da Proteína 8 Relacionada à Autofagia , Membrana Celular/metabolismo , Células Cultivadas , Células HeLa , Humanos , Fusão de Membrana/genética , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
ESCRT complexes are implicated in mediating membrane protein degradation, whereas hsc70 mediates cytosolic protein degradation via chaperone-mediated autophagy. In this issue of Developmental Cell, Sahu et al. (2011) describe in mammalian cells the involvement of ESCRT complexes and hsc70 in the degradation of cytosolic proteins in a process resembling microautophagy.
RESUMO
Autophagy, a critical process for bulk degradation of proteins and organelles, requires conjugation of Atg8 proteins to phosphatidylethanolamine on the autophagic membrane. At least eight different Atg8 orthologs belonging to two subfamilies (LC3 and GATE-16/GABARAP) occur in mammalian cells, but their individual roles and modes of action are largely unknown. In this study, we dissect the activity of each subfamily and show that both are indispensable for the autophagic process in mammalian cells. We further show that both subfamilies act differently at early stages of autophagosome biogenesis. Accordingly, our results indicate that LC3s are involved in elongation of the phagophore membrane whereas the GABARAP/GATE-16 subfamily is essential for a later stage in autophagosome maturation.