RESUMO
Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.
Assuntos
Medula Óssea , Histona Acetiltransferases , Humanos , Histona Acetiltransferases/metabolismo , Medula Óssea/metabolismo , Histonas/metabolismo , Neutrófilos/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismoRESUMO
Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.
Assuntos
Interferon Tipo I , RNA de Cadeia Dupla , Antivirais , Doenças Autoimunes do Sistema Nervoso , Exonucleases/genética , Humanos , Imunidade Inata/genética , Interferon Tipo I/genética , Malformações do Sistema Nervoso , RNA de Cadeia Dupla/genética , Proteína 1 com Domínio SAM e Domínio HD/genéticaRESUMO
Tissue health is regulated by a myriad of exogenous or endogenous factors. Here we investigated the role of the conserved Kynurenine pathway (KP) in maintaining retinal homeostasis in the context of light stress in Drosophila melanogaster. cinnabar, cardinal and scarlet are fly genes that encode different steps in the KP. Along with white, these genes are known regulators of brown pigment (ommochrome) biosynthesis. Using white as a sensitized genetic background, we show that mutations in cinnabar, cardinal and scarlet differentially modulate light-induced retinal damage. Mass Spectrometric measurements of KP metabolites in flies with different genetic combinations support the notion that increased levels of 3-hydroxykynurenine (3OH-K) and Xanthurenic acid (XA) enhance retinal damage, whereas Kynurenic Acid (KYNA) and Kynurenine (K) are neuro-protective. This conclusion was corroborated by showing that feeding 3OH-K results in enhanced retinal damage, whereas feeding KYNA protects the retina in sensitized genetic backgrounds. Interestingly, the harmful effects of free 3OH-K are diminished by its sub-cellular compartmentalization. Sequestering of 3OH-K enables the quenching of its toxicity through conversion to brown pigment or conjugation to proteins. This work enabled us to decouple the role of these KP genes in ommochrome formation from their role in retinal homeostasis. Additionally, it puts forward new hypotheses on the importance of the balance of KP metabolites and their compartmentalization in disease alleviation.
Assuntos
Drosophila , Cinurenina , Animais , Cinurenina/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ácido Cinurênico/metabolismo , Ácido Cinurênico/farmacologia , Retina/metabolismoRESUMO
Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.
Assuntos
Nematoides , Pergelissolo , Humanos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Larva/genética , Larva/metabolismo , FilogeniaRESUMO
Prostate cancer is the most prevalent malignancy in men. While diagnostic and therapeutic interventions have substantially improved in recent years, disease relapse, treatment resistance, and metastasis remain significant contributors to prostate cancer-related mortality. Therefore, novel therapeutic approaches are needed. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway which plays an essential role in cholesterol homeostasis. Numerous preclinical studies have provided evidence for the pleiotropic antitumor effects of statins. However, results from clinical studies remain controversial and have shown substantial benefits to even no effects on human malignancies including prostate cancer. Potential statin resistance mechanisms of tumor cells may account for such discrepancies. In our study, we treated human prostate cancer cell lines (PC3, C4-2B, DU-145, LNCaP) with simvastatin, atorvastatin, and rosuvastatin. PC3 cells demonstrated high statin sensitivity, resulting in a significant loss of vitality and clonogenic potential (up to - 70%; p < 0.001) along with an activation of caspases (up to 4-fold; p < 0.001). In contrast, C4-2B and DU-145 cells were statin-resistant. Statin treatment induced a restorative feedback in statin-resistant C4-2B and DU-145 cells through upregulation of the HMGCR gene and protein expression (up to 3-folds; p < 0.01) and its transcription factor sterol-regulatory element binding protein 2 (SREBP-2). This feedback was absent in PC3 cells. Blocking the feedback using HMGCR-specific small-interfering (si)RNA, the SREBP-2 activation inhibitor dipyridamole or the HMGCR degrader SR12813 abolished statin resistance in C4-2B and DU-145 and induced significant activation of caspases by statin treatment (up to 10-fold; p < 0.001). Consistently, long-term treatment with sublethal concentrations of simvastatin established a stable statin resistance of a PC3SIM subclone accompanied by a significant upregulation of both baseline as well as post-statin HMGCR protein (gene expression up to 70-fold; p < 0.001). Importantly, the statin-resistant phenotype of PC3SIM cells was reversible by HMGCR-specific siRNA and dipyridamole. Our investigations reveal a key role of a restorative feedback driven by the HMGCR/SREBP-2 axis in statin resistance mechanisms of prostate cancer cells.
Assuntos
Acil Coenzima A , Inibidores de Hidroximetilglutaril-CoA Redutases , Neoplasias da Próstata , Masculino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Sinvastatina/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Caspases , DipiridamolRESUMO
Shotgun analysis provides a quantitative snapshot of the lipidome composition of cells, tissues, or model organisms; however, it does not elucidate the spatial distribution of lipids. Here we demonstrate that shotgun analysis could quantify low-picomole amounts of lipids isolated by laser capture microdissection (LCM) of hundred micrometer-sized histological zones visualized at the cryosections of tissues. We identified metabolically distinct periportal (pp) and pericentral (pc) zones by immunostaining of 20 µm thick cryosections of a healthy mouse liver. LCM was used to ablate, catapult, and collect the tissue material from 10 to 20 individual zones covering a total area of 0.3-0.5 mm2 and containing ca. 500 cells. Top-down shotgun profiling relying upon computational stitching of 61 targeted selective ion monitoring ( t-SIM) spectra quantified more than 200 lipid species from 17 lipid classes including glycero- and glycerophospholipids, sphingolipids, cholesterol esters, and cholesterol. Shotgun LCM revealed the overall commonality of the full lipidome composition of pp and pc zones along with significant ( p < 0.001) difference in the relative abundance of 13 lipid species. Follow-up proteomics analyses of pellets recovered from an aqueous phase saved after the lipid extraction identified 13 known and 7 new protein markers exclusively present in pp or in pc zones and independently validated the specificity of their visualization, isolation, and histological assignment.
Assuntos
Bioquímica/métodos , Microdissecção e Captura a Laser/métodos , Lipídeos/sangue , Fígado/citologia , Animais , Humanos , Masculino , Camundongos , ProteômicaRESUMO
Nine mammalian adenylyl cyclases (AC) are pseudoheterodimers with two hexahelical membrane domains, which are isoform-specifically conserved. Previously we proposed that these membrane domains are orphan receptors (https://doi.org/10.7554/eLife.13098; https://doi.org/10.1016/j.cellsig.2020.109538). Lipids extracted from fetal bovine serum at pH 1 inhibited several mAC activities. Guided by a lipidomic analysis we tested glycerophospholipids as potential ligands. Contrary to expectations we surprisingly discovered that 1-stearoyl-2-docosahexaenoyl-phosphatidic acid (SDPA) potentiated Gsα-activated activity of human AC isoform 3 seven-fold. The specificity of fatty acyl esters at glycerol positions 1 and 2 was rather stringent. 1-Stearoyl-2-docosahexaenoyl-phosphatidylserine and 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine significantly potentiated several Gsα-activated mAC isoforms to different extents. SDPA appears not interact with forskolin activation of AC isoform 3. SDPA enhanced Gsα-activated AC activities in membranes from mouse brain cortex. The action of SDPA was reversible. Unexpectedly, SDPA did not affect cAMP generation in HEK293 cells stimulated by isoproterenol, PGE2 and adenosine, virtually excluding a role as an extracellular ligand and, instead, suggesting an intracellular role. In summary, we discovered a new dimension of intracellular AC regulation by chemically defined glycerophospholipids.
Assuntos
Adenilil Ciclases , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Adenilil Ciclases/metabolismo , Animais , Colforsina/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Glicerofosfolipídeos , Células HEK293 , Humanos , Mamíferos/metabolismo , CamundongosRESUMO
Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic ß-cells. ß-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between ß-cells via gap junctions. Here, we identify the importance of signaling between ß-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4-positive cells, being more mature while Wnt4-negative cells are more proliferative. Knock-out in adult ß-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between ß-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating ß-cells.
Assuntos
Sinalização do Cálcio , Insulinas , Glucose/metabolismo , Trifosfato de Adenosina/metabolismo , Insulinas/metabolismo , Difosfato de Adenosina/metabolismoRESUMO
BACKGROUND: SARS-CoV-2 infection causes acute respiratory distress, which may progress to multiorgan failure and death. Severe COVID-19 disease is accompanied by reduced erythrocyte turnover, low hemoglobin levels along with increased total bilirubin and ferritin serum concentrations. Moreover, expansion of erythroid progenitors in peripheral blood together with hypoxia, anemia, and coagulopathies highly correlates with severity and mortality. We demonstrate that SARS-CoV-2 directly infects erythroid precursor cells, impairs hemoglobin homeostasis and aggravates COVID-19 disease. METHODS: Erythroid precursor cells derived from peripheral CD34+ blood stem cells of healthy donors were infected in vitro with SARS-CoV-2 alpha variant and differentiated into red blood cells (RBCs). Hemoglobin and iron metabolism in hospitalized COVID-19 patients and controls were analyzed in plasma-depleted whole blood samples. Raman trapping spectroscopy rapidly identified diseased cells. RESULTS: RBC precursors express ACE2 receptor and CD147 at day 5 of differentiation, which makes them susceptible to SARS-CoV-2 infection. qPCR analysis of differentiated RBCs revealed increased HAMP mRNA expression levels, encoding for hepcidin, which inhibits iron uptake. COVID-19 patients showed impaired hemoglobin biosynthesis, enhanced formation of zinc-protoporphyrine IX, heme-CO2, and CO-hemoglobin as well as degradation of Fe-heme. Moreover, significant iron dysmetablolism with high serum ferritin and low serum iron and transferrin levels occurred, explaining disturbances of oxygen-binding capacity in severely ill COVID-19 patients. CONCLUSIONS: Our data identify RBC precursors as a direct target of SARS-CoV-2 and suggest that SARS-CoV-2 induced dysregulation in hemoglobin- and iron-metabolism contributes to the severe systemic course of COVID-19. This opens the door for new diagnostic and therapeutic strategies.
Assuntos
COVID-19 , SARS-CoV-2 , Eritrócitos/metabolismo , Ferritinas , Heme/metabolismo , Hemoglobinas/metabolismo , Humanos , Ferro/metabolismoRESUMO
Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.
Assuntos
Homem de Neandertal , Neocórtex , Neurogênese , Transcetolase , Animais , Células Ependimogliais/citologia , Furões , Humanos , Camundongos , Homem de Neandertal/embriologia , Homem de Neandertal/genética , Neocórtex/embriologia , Neurogênese/genética , Neurogênese/fisiologia , Transcetolase/genética , Transcetolase/metabolismoRESUMO
Upon exposure to excessive reactive oxygen species (ROS), organismal survival depends on the strength of the endogenous antioxidant defense barriers that prevent mitochondrial and cellular deterioration. Previously, we showed that glycolic acid can restore the mitochondrial membrane potential of C. elegans treated with paraquat, an oxidant that produces superoxide and other ROS species, including hydrogen peroxide. Here, we demonstrate that glycolate fully suppresses the deleterious effects of peroxide on mitochondrial activity and growth in worms. This endogenous compound acts by entering serine/glycine metabolism. In this way, conversion of glycolate into glycine and serine ameliorates the drastically decreased NADPH/NADP+ and GSH/GSSG ratios induced by H2O2 treatment. Our results reveal the central role of serine/glycine metabolism as a major provider of reducing equivalents to maintain cellular antioxidant systems and the fundamental function of glycolate as a natural antioxidant that improves cell fitness and survival.
Assuntos
Antioxidantes/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Glicolatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Peróxido de Hidrogênio/toxicidade , Longevidade/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Paraquat/toxicidade , Fatores de TempoRESUMO
All organisms encounter abiotic stress but only certain organisms are able to cope with extreme conditions and enter into cryptobiosis (hidden life). Previously, we have shown that C. elegans dauer larvae can survive severe desiccation (anhydrobiosis), a specific form of cryptobiosis. Entry into anhydrobiosis is preceded by activation of a set of biochemical pathways by exposure to mild desiccation. This process called preconditioning induces elevation of trehalose, intrinsically disordered proteins, polyamines and some other pathways that allow the preservation of cellular functionality in the absence of water. Here, we demonstrate that another stress factor, high osmolarity, activates similar biochemical pathways. The larvae that acquired resistance to high osmotic pressure can also withstand desiccation. In addition, high osmolarity significantly increases the biosynthesis of glycerol making larva tolerant to freezing. Thus, to survive abiotic stress, C. elegans activates a combination of genetic and biochemical pathways that serve as a general survival program.
Assuntos
Caenorhabditis elegans/metabolismo , Diapausa/fisiologia , Estresse Fisiológico/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dessecação , Proteínas Intrinsicamente Desordenadas/metabolismo , Larva/metabolismo , Larva/fisiologia , Concentração Osmolar , Poliaminas/metabolismo , Torpor/fisiologia , Água/metabolismoRESUMO
Septins (SEPTs) form a family of GTP-binding proteins implicated in cytoskeleton and membrane organization, cell division and host/pathogen interactions. The precise function of many family members remains elusive. We show that SEPT6 and SEPT7 complexes bound to F-actin regulate protein sorting during multivesicular body (MVB) biogenesis. These complexes bind AP-3, an adapter complex sorting cargos destined to remain in outer membranes of maturing endosomes, modulate AP-3 membrane interactions and the motility of AP-3-positive endosomes. These SEPT-AP interactions also influence the membrane interaction of ESCRT (endosomal-sorting complex required for transport)-I, which selects ubiquitinated cargos for degradation inside MVBs. Whereas our findings demonstrate that SEPT6 and SEPT7 function in the spatial, temporal organization of AP-3- and ESCRT-coated membrane domains, they uncover an unsuspected coordination of these sorting machineries during MVB biogenesis. This requires the E3 ubiquitin ligase LRSAM1, an AP-3 interactor regulating ESCRT-I sorting activity and whose mutations are linked with Charcot-Marie-Tooth neuropathies.