RESUMEN
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.
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Enfermedad Injerto contra Huésped , Quinasas Asociadas a rho , Humanos , Animales , Ratones , Quinasas Asociadas a rho/genética , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Transducción de Señal , FN-kappa B , Corticoesteroides/farmacología , Corticoesteroides/uso terapéuticoRESUMEN
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.
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Proteínas Serina-Treonina Quinasas , Huso Acromático , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitosis , Ciclo Celular , Células HeLa , Proteína Quinasa CDC2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
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.
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Microbioma Gastrointestinal , Intestinos , Metabolismo de los Lípidos , PPAR gamma , ARN Largo no Codificante , Sirtuina 1 , Animales , Ratones , Inmunidad Innata , Metabolismo de los Lípidos/genética , Linfocitos/inmunología , PPAR gamma/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Sirtuina 1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Mieloides/inmunología , Intestinos/metabolismo , Intestinos/microbiología , Tejido Adiposo/microbiología , HumanosRESUMEN
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.
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Proteínas de Arabidopsis , Arabidopsis , Transportadoras de Casetes de Unión a ATP/metabolismo , Arabidopsis/metabolismo , Tiazoles/metabolismo , Fitoalexinas , Proteínas de Arabidopsis/metabolismo , Enfermedades de las Plantas/microbiología , Regulación de la Expresión Génica de las PlantasRESUMEN
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.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/metabolismo , Mamíferos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
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Inflamasomas , Proteínas NLR , Animales , Inflamasomas/metabolismo , Proteínas NLR/química , Proteínas NLR/metabolismo , Receptores Inmunológicos/metabolismo , Plantas/metabolismoRESUMEN
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.
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Autofagia , Proteínas de Unión al ADN , Inflamación , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas de Unión al ADN/metabolismo , Células HeLa , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
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.
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Aparato de Golgi , Lisosomas , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas de Unión al ADN/metabolismo , Aparato de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , FosforilaciónRESUMEN
Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.
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Proteínas Adaptadoras Transductoras de Señales , Esclerosis Amiotrófica Lateral , Proteínas Relacionadas con la Autofagia , Demencia Frontotemporal , Proteínas Asociadas a Microtúbulos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteómica , ARN/genética , ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
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Hepatocitos , Secretoma , Adulto , Anciano , Animales , Colágeno/metabolismo , Hepatocitos/metabolismo , Humanos , Inflamación/metabolismo , Hígado/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Ratones , Ratones Noqueados , Proteínas Tirosina Quinasas/metabolismoRESUMEN
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.
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Proteínas Ricas en Prolina del Estrato Córneo , Staphylococcus aureus Resistente a Meticilina , Animales , Antibacterianos , Lipopolisacáridos , Proteínas de la Membrana/metabolismo , Ratones , ProlinaRESUMEN
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.
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Autofagia , Péptidos y Proteínas de Señalización Intracelular , Animales , Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Fosforilación , Proteína Fosfatasa 2RESUMEN
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.
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Homólogo de la Proteína 1 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación Fúngica de la Expresión Génica , Nutrientes , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The eukaryotic TORC1 kinase assimilates diverse environmental cues, including growth factors and nutrients, to control growth by tuning anabolic and catabolic processes. In yeast, TORC1 stimulates protein synthesis in response to abundant nutrients primarily through its proximal effector kinase Sch9. Conversely, TORC1 inhibition following nutrient limitation unlocks various distally controlled kinases (e.g., Atg1, Gcn2, Npr1, Rim15, Slt2/Mpk1, and Yak1), which cooperate through poorly defined circuits to orchestrate the quiescence program. To better define the signaling landscape of the latter kinases, we use in vivo quantitative phosphoproteomics. Through pinpointing known and uncharted Npr1, Rim15, Slt2/Mpk1, and Yak1 effectors, our study examines the architecture of the distally controlled TORC1 kinase network. Accordingly, this is built on a combination of discrete, convergent, and multilayered feedback regulatory mechanisms, which likely ensure homeostatic control of and/or robust responses by TORC1 and its effector kinases under fluctuating nutritional conditions.
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Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/farmacología , Fosfoproteínas/metabolismo , Proteínas Quinasas/química , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteoma/análisis , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
A diverse group of antimicrobial proteins (AMPs) helps protect the mammalian intestine from varied microbial challenges. We show that small proline-rich protein 2A (SPRR2A) is an intestinal antibacterial protein that is phylogenetically unrelated to previously discovered mammalian AMPs. In this study, SPRR2A was expressed in Paneth cells and goblet cells and selectively killed Gram-positive bacteria by disrupting their membranes. SPRR2A shaped intestinal microbiota composition, restricted bacterial association with the intestinal surface, and protected against Listeria monocytogenes infection. SPRR2A differed from other intestinal AMPs in that it was induced by type 2 cytokines produced during helminth infection. Moreover, SPRR2A protected against helminth-induced bacterial invasion of intestinal tissue. Thus, SPRR2A is a distinctive AMP triggered by type 2 immunity that protects the intestinal barrier during helminth infection.
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Proteínas Ricas en Prolina del Estrato Córneo/metabolismo , Microbioma Gastrointestinal , Bacterias Grampositivas/fisiología , Mucosa Intestinal/metabolismo , Intestinos/microbiología , Nematospiroides dubius , Infecciones por Strongylida/inmunología , Animales , Carga Bacteriana , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Proteínas Ricas en Prolina del Estrato Córneo/genética , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Células Caliciformes/metabolismo , Humanos , Inmunidad Innata , Mucosa Intestinal/microbiología , Listeria monocytogenes/fisiología , Listeriosis/microbiología , Ratones , Viabilidad Microbiana , Células de Paneth/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Infecciones por Strongylida/metabolismo , Infecciones por Strongylida/microbiologíaRESUMEN
Vitamin A and its derivative retinol are essential for the development of intestinal adaptive immunity. Retinoic acid (RA)producing myeloid cells are central to this process, but how myeloid cells acquire retinol for conversion to RA is unknown. Here, we show that serum amyloid A (SAA) proteinsretinol-binding proteins induced in intestinal epithelial cells by the microbiotadeliver retinol to myeloid cells. We identify low-density lipoprotein (LDL) receptorrelated protein 1 (LRP1) as an SAA receptor that endocytoses SAA-retinol complexes and promotes retinol acquisition by RA-producing intestinal myeloid cells. Consequently, SAA and LRP1 are essential for vitamin Adependent immunity, including B and T cell homing to the intestine and immunoglobulin A production. Our findings identify a key mechanism by which vitamin A promotes intestinal immunity.
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Inmunidad Adaptativa , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Células Mieloides/metabolismo , Proteína Amiloide A Sérica/metabolismo , Vitamina A/metabolismo , Animales , Linfocitos B/inmunología , Antígeno CD11c/análisis , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Endocitosis , Eliminación de Gen , Humanos , Inmunoglobulina A/biosíntesis , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Intestino Delgado/citología , Intestino Delgado/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Unión Proteica , Proteínas de Unión al Retinol/metabolismo , Salmonelosis Animal/inmunología , Salmonella typhimurium , Proteína Amiloide A Sérica/genética , Células Th17/inmunologíaRESUMEN
The evolutionarily conserved ULK1 kinase complex acts as gatekeeper of canonical autophagy and regulates induction of autophagosome biogenesis. To better understand control of ULK1 and analyze whether ULK1 has broader functions that are also linked to the later steps of autophagy, we perform comprehensive phosphoproteomic analyses. Combining in vivo with in vitro data, we identify numerous direct ULK1 target sites within autophagy-relevant proteins that are critical for autophagosome maturation and turnover. In addition, we highlight an intimate crosstalk between ULK1 and several phosphatase complexes. ULK1 is not only a PP2A target but also directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A and serving as positive feedback to promote autophagy-dependent protein turnover. Thus, ULK1 and phosphatase activities are tightly coordinated to robustly regulate protein degradation by autophagy.
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Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/fisiología , Proteínas de Unión a Calmodulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Fosfatasa 2/metabolismo , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.
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Trastorno del Espectro Autista/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Trastorno del Espectro Autista/genética , Citosol/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Microcefalia/genética , Microcefalia/metabolismo , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Quinasas DyrKRESUMEN
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.
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Aminoácidos/deficiencia , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Retroalimentación Fisiológica , Regulación Fúngica de la Expresión Génica , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.