RESUMO
The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.
Assuntos
Aminoácidos/deficiência , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The eukaryotic TORC1 kinase is a homeostatic controller of growth that integrates nutritional cues and mediates signals primarily from the surface of lysosomes or vacuoles. Amino acids activate TORC1 via the Rag GTPases that combine into structurally conserved multi-protein complexes such as the EGO complex (EGOC) in yeast. Here we show that Ego1, which mediates membrane-anchoring of EGOC via lipid modifications that it acquires while traveling through the trans-Golgi network, is separately sorted to vacuoles and perivacuolar endosomes. At both surfaces, it assembles EGOCs, which regulate spatially distinct pools of TORC1 that impinge on functionally divergent effectors: vacuolar TORC1 predominantly targets Sch9 to promote protein synthesis, whereas endosomal TORC1 phosphorylates Atg13 and Vps27 to inhibit macroautophagy and ESCRT-driven microautophagy, respectively. Thus, the coordination of three key regulatory nodes in protein synthesis and degradation critically relies on a division of labor between spatially sequestered populations of TORC1.
Assuntos
Proteostase , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/enzimologia , Endossomos/genética , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Transcrição/genética , Vacúolos/enzimologia , Vacúolos/genéticaRESUMO
Vertebrate lonesome kinase (VLK) is the only known extracellular tyrosine kinase, but its physiological functions are largely unknown. We show that VLK is highly expressed in hepatocytes of neonatal mice, but downregulated during adulthood. To determine the role of VLK in liver homeostasis and regeneration, we generated mice with a hepatocyte-specific knockout of the VLK gene (Pkdcc). Cultured progenitor cells established from primary hepatocytes of Pkdcc knockout mice produced a secretome, which promoted their own proliferation in 3D spheroids and proliferation of cultured fibroblasts. In vivo, Pkdcc knockout mice developed liver steatosis with signs of inflammation and perivascular fibrosis upon aging, combined with expansion of liver progenitor cells. In response to chronic CCl4-induced liver injury, the pattern of deposited collagen was significantly altered in these mice. The liver injury marker alpha-fetoprotein (AFP) was increased in the secretome of VLK-deficient cultured progenitor cells and in liver tissues of aged or CCl4-treated knockout mice. These results support a key role for VLK and extracellular protein phosphorylation in liver homeostasis and repair through paracrine control of liver cell function and regulation of appropriate collagen deposition. This article has an associated First Person interview with the first author of the paper.
Assuntos
Hepatócitos , Secretoma , Adulto , Idoso , Animais , Colágeno/metabolismo , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/metabolismoRESUMO
AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.
Assuntos
Proteínas Serina-Treonina Quinases , Fuso Acromático , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Ciclo Celular , Células HeLa , Proteína Quinase CDC2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Macroautophagy/autophagy is a highly conserved eukaryotic molecular process that facilitates the recycling of superfluous cytoplasmic materials, damaged organelles, and invading pathogens, resulting in proper cellular homeostasis and survival during stress conditions. Autophagy is stringently regulated at multiple stages, including control at transcriptional, translational, and posttranslational levels. In this work, we identified a mechanism by which regulation of autophagy is achieved through the posttranslational modification of Atg9. Here, we show that, in order to limit autophagy to a low, basal level during normal conditions, Atg9 is ubiquitinated and subsequently targeted for degradation in a proteasome-dependent manner through the action of the E3 ligase Met30. When cells require increased autophagy flux to respond to nutrient deprivation, the proteolysis of Atg9 is significantly reduced. Overall, this work reveals an additional layer of mechanistic regulation that allows cells to further maintain appropriate levels of autophagy and to rapidly induce this process in response to stress.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Proteínas F-Box/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Autofagia/genética , Proteínas Relacionadas à Autofagia/fisiologia , Regulação para Baixo , Proteínas F-Box/fisiologia , Lisossomos/metabolismo , Proteínas de Membrana/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization. In infected mice, SAA proteins circulate in association with the vitamin A derivative retinol, suggesting that SAAs transport retinol during infection. Here we illuminate a structural basis for the retinol-SAA interaction. In the bloodstream of infected mice, most SAA is complexed with high-density lipoprotein (HDL). However, we found that the majority of the circulating retinol was associated with the small fraction of SAA proteins that circulate without binding to HDL, thus identifying free SAA as the predominant retinol-binding form in vivo. We then determined the crystal structure of retinol-bound mouse SAA3 at a resolution of 2.2 Å. Retinol-bound SAA3 formed a novel asymmetric trimeric assembly that was generated by the hydrophobic packing of the conserved amphipathic helices α1 and α3. This hydrophobic packing created a retinol-binding pocket in the center of the trimer, which was confirmed by mutagenesis studies. Together, these findings illuminate the molecular basis for retinol transport by SAA proteins during infection.
Assuntos
Salmonella typhimurium/metabolismo , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismo , Febre Tifoide/metabolismo , Vitamina A/metabolismo , Vitaminas/metabolismo , Animais , Cristalografia por Raios X , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação , Conformação Proteica , Proteína Amiloide A Sérica/genética , Febre Tifoide/virologiaRESUMO
Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.
Assuntos
Autofagossomos/metabolismo , Vírus da Influenza A/metabolismo , Proteínas Ribossômicas/metabolismo , Autofagia , Linhagem Celular Tumoral , Humanos , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribossomos/metabolismoRESUMO
Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the α amino group of mono-l-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A. The MS analysis suggested that all eEF1A molecules are modified by this glutaminylation and that this posttranslational modification occurs at all stages of yeast growth. The mutational studies revealed that this glutaminylation is not essential for the normal functions of eEF1A in S. cerevisiae However, eEF1A glutaminylation slightly reduced growth under antibiotic-induced translational stress conditions. Moreover, we identified the same posttranslational modification in eEF1A from Schizosaccharomyces pombe but not in various other eukaryotic organisms tested despite strict conservation of the Glu45 residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification that appears to influence protein translation.
Assuntos
Glutamina/metabolismo , Modelos Moleculares , Fator 1 de Elongação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoacilação/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Sequência Conservada , Cristalografia por Raios X , Bases de Dados de Proteínas , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Sequências Hélice-Alça-Hélice , Mutagênese Sítio-Dirigida , Mutação , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Inflammasomes are multimeric protein complexes that mediate the activation of inflammatory caspases. One central component of inflammasomes is nucleotide-binding domain (NBD)- and leucine-rich repeat (LRR)-containing proteins (NLRs) that can function as pattern recognition receptors (PRRs). In resting cells, NLR proteins exist in an auto-inhibited, monomeric, and ADP-bound state. Perception of microbial or damage-associated signals results in NLR oligomerization, thus recruiting inflammatory caspases directly or through the adaptor molecule apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). The assembled NLR inflammasomes serve as dedicated machinery to facilitate the activation of the inflammatory caspases. Here, we review current understanding of the structures of NLR inflammasomes with an emphasis on the molecular mechanisms of their assembly and activation. We also discuss implications of the self-propagation model derived from the NAIP-NLRC4 inflammasomes for the activation of other NLR inflammasomes and a potential role of the C-terminal LRR domain in the activation of an NLR protein.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/química , Proteínas Adaptadoras de Sinalização CARD/imunologia , Inflamassomos/química , Inflamassomos/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Humanos , Inflamassomos/genética , Família Multigênica , Receptores de Reconhecimento de Padrão/química , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Transdução de SinaisRESUMO
BACKGROUND: Chronic myeloid leukemia (CML) is driven by the fusion kinase Bcr-Abl. Bcr-Abl tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), revolutionized CML therapy. Nevertheless, about 20 % of CMLs display primary or acquired TKI resistance. TKI resistance can be either caused by mutations within the Bcr-Abl kinase domain or by aberrant signaling by its effectors, e.g. Lyn or Gab2. Bcr-Abl mutations are frequently observed in TKI resistance and can only in some cases be overcome by second line TKIs. In addition, we have previously shown that the formation of Gab2 complexes can be regulated by Bcr-Abl and that Gab2 signaling counteracts the efficacy of four distinct Bcr-Abl inhibitors. Therefore, TKI resistance still represents a challenge for disease management and alternative therapies are urgently needed. FINDINGS: Using different CML cell lines and models, we identified the clinically approved TKIs sorafenib (SF) and axitinib (AX) as drugs overcoming the resistance mediated by the Bcr Abl(T315I) mutant as well as the one mediated by Gab2 and Lyn(Y508F). In addition, we demonstrated that AX mainly affects the Bcr-Abl/Grb2/Gab2 axis, whereas SF seems to act independently of the fusion kinase and most likely by blocking signaling pathways up- and downstream of Gab2. CONCLUSION: We demonstrate that SF and AX show potency in various and mechanistically distinct scenarios of TKI resistance, including Bcr-Abl(T315I) as well as Lyn- and Gab2-mediated resistances. Our data invites for further evaluation und consideration of these inhibitors in the treatment of TKI resistant CML.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Imidazóis/farmacologia , Indazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Axitinibe , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Mutação , Niacinamida/farmacologia , Mutação Puntual , Mapas de Interação de Proteínas/efeitos dos fármacos , Sorafenibe , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1ß, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings.
Assuntos
Doença Enxerto-Hospedeiro , Quinases Associadas a rho , Humanos , Animais , Camundongos , Quinases Associadas a rho/genética , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transdução de Sinais , NF-kappa B , Corticosteroides/farmacologia , Corticosteroides/uso terapêuticoRESUMO
Nucleotide-binding and leucine-rich repeat (NLR) proteins are critical intracellular immune receptors in both animals and plants. Perception of pathogen-derived or stress-associated signals induces NLR oligomerization to form multiprotein complexes called inflammasomes in animals or resistosomes in plants to mediate host immune response. Significant progress has been made during the past few years in our understanding of NLR biology, particularly the structural perspective of these two types of NLR-containing complexes. In this article, we review the latest advances in our structural knowledge of how NLR inflammasomes and resistosomes are activated and assembled and how the structural information provides insight into their distinct mechanisms of action. Commonalities and differences between NLR inflammasomes and resistosomes are also discussed.
Assuntos
Inflamassomos , Proteínas NLR , Animais , Inflamassomos/metabolismo , Proteínas NLR/química , Proteínas NLR/metabolismo , Receptores Imunológicos/metabolismo , Plantas/metabolismoRESUMO
The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels activate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallett et al., 2015) reported that AMPK in yeast, that is Snf1, prevents the transient TORC1 reactivation during the early phase following acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 temporally maintains TORC1 inactive in glucose-starved cells primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phosphorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physiologically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The intestinal microbiota regulates mammalian lipid absorption, metabolism, and storage. We report that the microbiota reprograms intestinal lipid metabolism in mice by repressing the expression of long noncoding RNA (lncRNA) Snhg9 (small nucleolar RNA host gene 9) in small intestinal epithelial cells. Snhg9 suppressed the activity of peroxisome proliferator-activated receptor γ (PPARγ)-a central regulator of lipid metabolism-by dissociating the PPARγ inhibitor sirtuin 1 from cell cycle and apoptosis protein 2 (CCAR2). Forced expression of Snhg9 in the intestinal epithelium of conventional mice impaired lipid absorption, reduced body fat, and protected against diet-induced obesity. The microbiota repressed Snhg9 expression through an immune relay encompassing myeloid cells and group 3 innate lymphoid cells. Our findings thus identify an unanticipated role for a lncRNA in microbial control of host metabolism.
Assuntos
Microbioma Gastrointestinal , Intestinos , Metabolismo dos Lipídeos , PPAR gama , RNA Longo não Codificante , Sirtuína 1 , Animais , Camundongos , Imunidade Inata , Metabolismo dos Lipídeos/genética , Linfócitos/imunologia , PPAR gama/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sirtuína 1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Células Mieloides/imunologia , Intestinos/metabolismo , Intestinos/microbiologia , Tecido Adiposo/microbiologia , HumanosRESUMO
Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0-4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.
Assuntos
Autofagia , Proteínas de Ligação a DNA , Inflamação , Proteínas Serina-Treonina Quinases , Humanos , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The exporter of the auxin precursor indole-3-butyric acid (IBA), ABCG36/PDR8/PEN3, from the model plant Arabidopsis has recently been proposed to also function in the transport of the phytoalexin camalexin. Based on these bonafide substrates, it has been suggested that ABCG36 functions at the interface between growth and defense. Here, we provide evidence that ABCG36 catalyzes the direct, ATP-dependent export of camalexin across the plasma membrane. We identify the leucine-rich repeat receptor kinase, QIAN SHOU KINASE1 (QSK1), as a functional kinase that physically interacts with and phosphorylates ABCG36. Phosphorylation of ABCG36 by QSK1 unilaterally represses IBA export, allowing camalexin export by ABCG36 conferring pathogen resistance. As a consequence, phospho-dead mutants of ABCG36, as well as qsk1 and abcg36 alleles, are hypersensitive to infection with the root pathogen Fusarium oxysporum, caused by elevated fungal progression. Our findings indicate a direct regulatory circuit between a receptor kinase and an ABC transporter that functions to control transporter substrate preference during plant growth and defense balance decisions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Transportadores de Cassetes de Ligação de ATP/metabolismo , Arabidopsis/metabolismo , Tiazóis/metabolismo , Fitoalexinas , Proteínas de Arabidopsis/metabolismo , Doenças das Plantas/microbiologia , Regulação da Expressão Gênica de PlantasRESUMO
ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome biogenesis. To uncover if ULK1 has broader functions also regulating subsequent steps of autophagosome turnover, i.e., maturation, lysosomal fusion, and degradation, we performed a set of unbiased phosphoproteomic experiments employing mouse and human cells in combination with genetic and environmental perturbations. We characterized more than 1,000 potential ULK1 target sites of which many affect proteins known to be involved in all phases of the autophagosome life cycle. To better understand which of these 1,000 phosphosites were directly phosphorylated by ULK1, in contrast to downstream kinases being activated or phosphatases being inhibited by ULK1, we developed a proteome-scale in vitro kinase assay and characterized 187 phosphosites on 157 proteins as bona fide ULK1 target sites. Interestingly, our results highlight an intricate crosstalk between ULK1 and protein phosphatases. Focusing on STRN (striatin), a regulatory subunit of PPP2/PP2A (protein phosphatase 2), we identified a positive feedback loop linked to ULK1 and promoting autophagy.
Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular , Animais , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Fosforilação , Proteína Fosfatase 2RESUMO
Human skin functions as a physical barrier, preventing the entry of foreign pathogens while also accommodating a myriad of commensal microorganisms. A key contributor to the skin landscape is the sebaceous gland. Mice devoid of sebocytes are prone to skin infection, yet our understanding of how sebocytes function in host defense is incomplete. Here, we show that the small proline-rich proteins, SPRR1 and SPRR2 are bactericidal in skin. SPRR1B and SPPR2A were induced in human sebocytes by exposure to the bacterial cell wall component lipopolysaccharide (LPS). Colonization of germ-free mice was insufficient to trigger increased SPRR expression in mouse skin, but LPS injected into mouse skin stimulated increased expression of the mouse SPRR orthologous genes, Sprr1a and Sprr2a, through activation of MYD88. Both mouse and human SPRR proteins displayed potent bactericidal activity against MRSA (methicillin-resistant Staphylococcus aureus), Pseudomonas aeruginosa, and skin commensals. Thus, Sprr1a-/-;Sprr2a-/- mice are more susceptible to MRSA and P. aeruginosa skin infection. Lastly, mechanistic studies demonstrate that SPRR proteins exert their bactericidal activity through binding and disruption of the bacterial membrane. Taken together, these findings provide insight into the regulation and antimicrobial function of SPRR proteins in skin and how the skin defends the host against systemic infection.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo , Staphylococcus aureus Resistente à Meticilina , Animais , Antibacterianos , Lipopolissacarídeos , Proteínas de Membrana/metabolismo , Camundongos , ProlinaRESUMO
Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas Quinases , Proteínas de Saccharomyces cerevisiae , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Fúngica da Expressão Gênica , Nutrientes , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.