RESUMO
Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.
Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética , Processamento Alternativo/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Bases de Dados Genéticas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/genética , Masculino , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Especificidade por SubstratoRESUMO
The intestinal mucosa is a dynamic surface that facilitates interactions between the host and an outside world that includes trillions of microbes, collectively termed the microbiota. This fine balance is regulated by an energetically demanding physical and biochemical barrier that is formed by the intestinal epithelial cells. Additionally, this homeostasis exists at an interface between the anaerobic colonic lumen and a highly oxygenated, vascularized lamina propria. The resultant oxygen gradient within the intestine establishes "physiologic hypoxia" as a central metabolic feature of the mucosa. Although oxygen is vital for energy production to meet cellular metabolism needs, the availability of oxygen has far-reaching influences beyond just energy provision. Recent studies have shown that the intestinal mucosa has purposefully adapted to utilize differential oxygen levels largely through the presence of short-chain fatty acids (SCFA), particularly butyrate. Intestinal epithelial cells utilize butyrate for a multitude of functions that promote mucosal homeostasis. In this review, we explore how the physiologic hypoxia profile interfaces with SCFA to benefit host mucosal tissues.
RESUMO
Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.
Assuntos
Infecções por HIV , Inibidores da Protease de HIV , HIV-1 , Humanos , Inibidores da Protease de HIV/farmacologia , Inibidores da Protease de HIV/uso terapêutico , Darunavir/farmacologia , Darunavir/uso terapêutico , Sulfato de Atazanavir/farmacologia , Sulfato de Atazanavir/uso terapêutico , Farmacorresistência Viral , HIV-1/genética , Antirretrovirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Protease de HIV/metabolismoRESUMO
The intestinal mucosa exists in dynamic balance with trillions of luminal microbes. Disruption of the intestinal epithelial barrier, commonly observed in mucosal inflammation and diseases such as inflammatory bowel diseases (IBDs), is often associated with dysbiosis, particularly decreases in species producing short-chain fatty acids (SCFAs), such as butyrate. It remains unclear to what extent microbiota-derived factors contribute to the overall maintenance of intestinal homeostasis. Initial studies revealed that butyrate selectively promotes epithelial barrier function and wound healing. We aimed to define the specific mechanism(s) through which butyrate contributes to these epithelial responses. Guided by an unbiased profiling approach, we identified the dominant regulation of the actin-binding protein synaptopodin (SYNPO). Extensions of this work revealed a role for SYNPO in intestinal epithelial barrier function and wound healing. SYNPO was localized to the intestinal epithelial tight junction and within F-actin stress fibers where it is critical for barrier integrity and cell motility. Butyrate, but not other SCFAs, induced SYNPO in epithelial cell lines and murine colonic enteroids through mechanisms possibly involving histone deacetylase inhibition. Moreover, depletion of the microbiota abrogated expression of SYNPO in the mouse colon, which was rescued with butyrate repletion. Studies in Synpo-deficient mice demonstrated exacerbated disease susceptibility and increased intestinal permeability in a dextran sulfate sodium colitis model. These findings establish a critical role for the microbiota and their products, specifically butyrate, in the regulated expression of SYNPO for intestinal homeostasis and reveal a direct mechanistic link between microbiota-derived butyrate and barrier restoration.
Assuntos
Butiratos/metabolismo , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos , Animais , Linhagem Celular , Homeostase/fisiologia , Humanos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Junções Íntimas/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Células-Tronco Pluripotentes Induzidas , Antígeno Neuro-Oncológico Ventral , Processamento Alternativo/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Antígeno Neuro-Oncológico Ventral/genética , Antígeno Neuro-Oncológico Ventral/metabolismo , Proteínas Nucleares/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genéticaRESUMO
Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP), and corroborated the findings with our X-ray structure of the PD-L1/macrocycle complex. Although all three approaches show that the macrocycle binds directly to PD-L1 over the regions of residues 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting in probing protein/ligand inhibitory interactions in cancer immunotherapy.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/química , Anticorpos Monoclonais/química , Antígeno B7-H1/metabolismo , Cristalografia por Raios X/métodos , Humanos , Imunoterapia , Ligantes , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Espectrometria de Massas , Modelos Moleculares , Oxirredução , Peptídeos/química , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Pegadas de Proteínas/métodos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Intestinal epithelial cells form a selectively permeable barrier to protect colon tissues from luminal microbiota and antigens and to mediate nutrient, fluid, and waste flux in the intestinal tract. Dysregulation of the epithelial cell barrier coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically depleting state of physiological hypoxia. However, studies that systematically examine energy flux and adenylate metabolism during intestinal epithelial barrier development and restoration after disruption are lacking. Here, to delineate barrier-related energy flux, we developed an HPLC-based profiling method to track changes in energy flux and adenylate metabolites during barrier development and restoration. Cultured epithelia exhibited pooling of phosphocreatine and maintained ATP during barrier development. EDTA-induced epithelial barrier disruption revealed that hypoxanthine levels correlated with barrier resistance. Further studies uncovered that hypoxanthine supplementation improves barrier function and wound healing and that hypoxanthine appears to do so by increasing intracellular ATP, which improved cytoskeletal G- to F-actin polymerization. Hypoxanthine supplementation increased the adenylate energy charge in the murine colon, indicating potential to regulate adenylate energy charge-mediated metabolism in intestinal epithelial cells. Moreover, experiments in a murine colitis model disclosed that hypoxanthine loss during active inflammation correlates with markers of disease severity. In summary, our results indicate that hypoxanthine modulates energy metabolism in intestinal epithelial cells and is critical for intestinal barrier function.
Assuntos
Colite/metabolismo , Colo/metabolismo , Metabolismo Energético , Hipoxantina/metabolismo , Mucosa Intestinal/metabolismo , Animais , Colite/patologia , Colo/patologia , Feminino , Mucosa Intestinal/patologia , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio , Permeabilidade , Junções Íntimas/metabolismo , Junções Íntimas/patologiaRESUMO
Commensal interactions between the enteric microbiota and distal intestine play important roles in regulating human health. Short-chain fatty acids (SCFAs), such as butyrate, produced through anaerobic microbial metabolism represent a major energy source for the host colonic epithelium and enhance epithelial barrier function through unclear mechanisms. Separate studies revealed that the epithelial anti-inflammatory IL-10 receptor α subunit (IL-10RA) is also important for barrier formation. Based on these findings, we examined if SCFAs promote epithelial barrier through IL-10RA-dependent mechanisms. Using human intestinal epithelial cells (IECs), we discovered that SCFAs, particularly butyrate, enhanced IEC barrier formation, induced IL-10RA mRNA, IL-10RA protein, and transactivation through activated Stat3 and HDAC inhibition. Loss and gain of IL-10RA expression directly correlates with IEC barrier formation and butyrate represses permeability-promoting claudin-2 tight-junction protein expression through an IL-10RA-dependent mechanism. Our findings provide a novel mechanism by which microbial-derived butyrate promotes barrier through IL-10RA-dependent repression of claudin-2.
Assuntos
Bactérias Anaeróbias/fisiologia , Butiratos/metabolismo , Colo/patologia , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/fisiologia , Receptores de Interleucina-10/metabolismo , Junções Íntimas/metabolismo , Butiratos/imunologia , Linhagem Celular , Células Cultivadas , Claudina-2/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Receptores de Interleucina-10/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Simbiose , Ativação Transcricional , Migração Transendotelial e Transepitelial , Regulação para CimaRESUMO
Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.
Assuntos
Anticorpos Monoclonais Humanizados/química , Colite Ulcerativa/tratamento farmacológico , Metaloproteinase 9 da Matriz/química , Inibidores de Metaloproteinases de Matriz/química , Neoplasias Gástricas/tratamento farmacológico , Sítio Alostérico , Anticorpos/química , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Gelatina/química , Deleção de Genes , Células HEK293 , Humanos , Concentração Inibidora 50 , Ligação Proteica , Proteínas Recombinantes/química , Ressonância de Plasmônio de SuperfícieRESUMO
Sporadic amyotrophic lateral sclerosis (sALS) is the most common form of ALS, however, the molecular mechanisms underlying cellular damage and motor neuron degeneration remain elusive. To identify molecular signatures of sALS we performed genome-wide expression profiling in laser capture microdissection-enriched surviving motor neurons (MNs) from lumbar spinal cords of sALS patients with rostral onset and caudal progression. After correcting for immunological background, we discover a highly specific gene expression signature for sALS that is associated with phosphorylated TDP-43 (pTDP-43) pathology. Transcriptome-pathology correlation identified casein kinase 1ε (CSNK1E) mRNA as tightly correlated to levels of pTDP-43 in sALS patients. Enhanced crosslinking and immunoprecipitation in human sALS patient- and healthy control-derived frontal cortex, revealed that TDP-43 binds directly to and regulates the expression of CSNK1E mRNA. Additionally, we were able to show that pTDP-43 itself binds RNA. CK1E, the protein product of CSNK1E, in turn interacts with TDP-43 and promotes cytoplasmic accumulation of pTDP-43 in human stem-cell-derived MNs. Pathological TDP-43 phosphorylation is therefore, reciprocally regulated by CK1E activity and TDP-43 RNA binding. Our framework of transcriptome-pathology correlations identifies candidate genes with relevance to novel mechanisms of neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Caseína Quinase I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Medula Espinal/metabolismo , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios Motores/patologia , Fosforilação , Medula Espinal/patologiaRESUMO
Identification of in vivo direct RNA targets for RNA binding proteins (RBPs) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus. Further, we show that TAG-eCLIP performed using tag-specific antibodies can yield the same robust binding profiles after proper control normalization as eCLIP with antibodies against endogenous proteins. Finally, we note that antibodies against commonly used tags can immunoprecipitate significant amounts of antibody-specific RNA, emphasizing the need for paired controls alongside each experiment for normalization. TAG-eCLIP enables eCLIP profiling of new native proteins where no suitable antibody exists, expanding the RBP-RNA interaction landscape.
Assuntos
Sistemas CRISPR-Cas , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Ligação a RNA/genética , RNA/química , Coloração e Rotulagem/métodos , Anticorpos/química , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Endonucleases/química , Células HEK293 , Recombinação Homóloga , Humanos , Células K562 , Peptídeos/química , Reação em Cadeia da Polimerase , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA/métodos , TranscriptomaRESUMO
Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.
Assuntos
Fosfatidilinositol 3-Quinases/química , Purinas/química , Quinazolinonas/química , Trifosfato de Adenosina/química , Androstadienos/química , Animais , Ligação Competitiva , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/química , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Cinética , Camundongos , Modelos Moleculares , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , WortmaninaRESUMO
HIF-1α is a hypoxia-inducible protein that regulates many cell and molecular processes, including those involved in angiogenesis and stem cell maintenance. Prior studies demonstrated constitutive HIF-1α stabilization in neural stem cells (NSCs) of the adult mouse SVZ, but its role there has not been elucidated. Here, we tested the hypothesis that HIF-1α plays an essential role in the maintenance of adult NSCs and stabilization of the SVZ vascular niche using conditional, tamoxifen-inducible Hif1a knock-out mice. We generated nestin-CreER(T2)/R26R-YFP/Hif1a(fl/fl) triple transgenic mice, to enable tamoxifen-inducible Hif1a gene inactivation in nestin-expressing NSCs within the adult SVZ. Hif1a gene deletion resulted in a significant loss of YFP(+) NSCs within the SVZ by 45 d post recombination, which was preceded by significant regression of the SVZ vasculature at 14 d, and concomitant decrease of VEGF expression by NSCs. Loss of YFP(+) NSCs following Hif1a gene inactivation in vivo was likely an indirect consequence of vascular regression, since YFP(+) neurosphere formation over serial passage was unaffected. These results identify NSC-encoded HIF-1α as an essential factor in the maintenance of the adult SVZ, and demonstrate that NSCs within the SVZ maintain the integrity of their vascular niche through HIF-1α-mediated signaling mechanisms.
Assuntos
Células-Tronco Adultas/fisiologia , Circulação Cerebrovascular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Ventrículos Laterais/irrigação sanguínea , Ventrículos Laterais/fisiologia , Células-Tronco Neurais/fisiologia , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
BACKGROUND: GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays. METHODS: Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods. RESULTS: GS-9256 and vedroprevir had measured Ki values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; Ki values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was >10,000-fold against all tested off-target proteases. Association rate constants of 4×10(5)M(-1)s(-1) and 1×10(6)M(-1)s(-1), respectively, were measured, and dissociation rate constants of 4.8×10(-5)s(-1) and 2.6×10(-4)s(-1) were determined. CONCLUSIONS: GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics. GENERAL SIGNIFICANCE: The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.
RESUMO
The subcellular compartmentalization of kinase activity allows for regulation of distinct cellular processes involved in cell differentiation or survival. The PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, is a neuroprotective kinase localized to cytosolic and mitochondrial compartments. While mitochondrial targeting of PINK1 is important for its activities regulating mitochondrial homeostasis, the physiological role of the cytosolic pool of PINK1 remains unknown. Here, we demonstrate a novel role for cytosolic PINK1 in neuronal differentiation/neurite maintenance. Over-expression of wild-type PINK1, but not a catalytically inactive form of PINK1(K219M), promoted neurite outgrowth in SH-SY5Y cells and increased dendritic lengths in primary cortical and midbrain dopaminergic neurons. To identify the subcellular pools of PINK1 involved in promoting neurite outgrowth, we transiently transfected cells with PINK1 constructs designed to target PINK1 to the outer mitochondrial membrane (OMM-PINK1) or restrict PINK1 to the cytosol (ΔN111-PINK1). Both constructs blocked cell death associated with loss of endogenous PINK1. However, transient expression of ΔN111-PINK1, but not of OMM-PINK1 or ΔN111-PINK1(K219M), promoted dendrite outgrowth in primary neurons, and rescued the decreased dendritic arborization of PINK1-deficient neurons. Mechanistically, the cytosolic pool of PINK1 regulated neurite morphology through enhanced anterograde transport of dendritic mitochondria and amplification of protein kinase A-related signaling pathways. Our data support a novel role for PINK1 in regulating dendritic morphogenesis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dendritos/fisiologia , Mitocôndrias/metabolismo , Proteínas Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Córtex Cerebral/citologia , Citosol/metabolismo , Feminino , Humanos , Masculino , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroblastoma , Doença de Parkinson/metabolismo , Gravidez , Cultura Primária de Células , Proteínas Quinases/genética , Transdução de Sinais/fisiologiaRESUMO
Urticarial vasculitis is a rare autoimmune disorder characterized by persistent edematous papules and plaques on the skin that last longer than 24 hours, often accompanied by systemic symptoms such as joint pain and fever. Unlike common urticaria, this condition involves inflammation of small blood vessels, leading to more severe and long-lasting skin lesions with a tendency to leave a bruiselike appearance. Diagnosis is challenging and may require a skin biopsy. Associated with underlying autoimmune diseases, treatment involves managing symptoms with medications such as antihistamines and corticosteroids, addressing the immune system's dysfunction, and treating any concurrent autoimmune conditions.
Assuntos
Urticária , Vasculite , Humanos , Urticária/diagnóstico , Urticária/etiologia , Urticária/imunologia , Vasculite/diagnóstico , Pele/patologia , Pele/imunologia , Diagnóstico Diferencial , Antagonistas dos Receptores Histamínicos/uso terapêutico , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Biópsia , Vasculite Leucocitoclástica Cutânea/diagnóstico , Vasculite Leucocitoclástica Cutânea/imunologia , Vasculite Leucocitoclástica Cutânea/etiologiaRESUMO
Active episodes of inflammatory bowel disease (IBD), which include ulcerative colitis and Crohn's disease, coincide with profound shifts in the composition of the microbiota and host metabolic energy demand. Intestinal epithelial cells (IEC) that line the small intestine and colon serve as an initial point for contact for the microbiota and play a central role in innate immunity. In the 1980s, Roediger et al proposed the hypothesis that IBD represented a disease of diminished mucosal nutrition and energy deficiency ("starved gut") that strongly coincided with the degree of inflammation. These studies informed the scientific community about the important contribution of microbial-derived metabolites, particularly short-chain fatty acids (SCFA) such as butyrate, to overall energy homeostasis. Decades later, it is appreciated that disease-associated shifts in the microbiota, termed dysbiosis, places inordinate demands on energy acquisition within the mucosa, particularly during active inflammation. Here, we review the topic of tissue energetics in mucosal health and disease from the original perspective of that proposed by the starved gut hypothesis.
RESUMO
Microbiota-derived short-chain fatty acids, including butyrate (BA), have multiple beneficial health effects. In the colon, BA concentrations range from 10 to 20 mM and up to 95% is utilized as energy by the mucosa. BA plays a key role in epithelial-barrier regulation and anti-inflammation, and regulates cell growth and differentiation, at least in part, due to its direct influence on stabilization of the transcription factor hypoxia-inducible factor (HIF). It remains unclear whether BA is the optimal metabolite for such a response. In this study, we explored metabolite mimicry as an attractive strategy for the biological response to HIF. We discovered that 4-mercapto butyrate (MBA) stabilizes HIF more potently and has a longer biological half-life than BA in intestinal epithelial cells (IECs). We validated the MBA-mediated HIF transcriptional activity through the induction of classic HIF gene targets in IECs and enhanced epithelial barrier formation in vitro. In-vivo studies with MBA revealed systemic HIF stabilization in mice, which was more potent than its parent BA metabolite. Mechanistically, we found that MBA enhances oxygen consumption and that the sulfhydryl group is essential for HIF stabilization, but exclusively as a four-carbon SCFA. These findings reveal a combined biochemical mechanism for HIF stabilization and provide a foundation for the discovery of potent metabolite-like scaffolds.
Assuntos
Butiratos , Microbioma Gastrointestinal , Camundongos , Animais , Butiratos/farmacologia , Butiratos/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Ácidos Graxos Voláteis/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
IL-38 is a recently discovered cytokine and member of the IL-1 Family. In the IL-1 Family, IL-38 is unique because the cytokine is primarily a B lymphocyte product and functions to suppress inflammation. Studies in humans with inflammatory bowel disease (IBD) suggest that IL-38 may be protective for ulcerative colitis or Crohn's disease, and that IL-38 acts to maintain homeostasis in the intestinal tract. Here we investigated the role of endogenous IL-38 in experimental colitis in mice deficient in IL-38 by deletion of exons 1-4 in C57 BL/6 mice. Compared to WT mice, IL-38 deficient mice subjected to dextran sulfate sodium (DSS) showed greater severity of disease, more weight loss, increased intestinal permeability, and a worse histological phenotype including increased neutrophil influx in the colon. Mice lacking IL-38 exhibited elevated colonic Nlrp3 mRNA and protein levels, increased caspase-1 activation, and the concomitant increased processing of IL-1ß precursor into active IL-1ß. Expression of IL-1α, an exacerbator of IBD, was also upregulated. Colonic myleloperoxidase protein and Il17a, and Il17f mRNA levels were higher in the IL-38 deficient mice. Daily treatment of IL-38 deficient mice with an NLRP3 inhibitor attenuated diarrhea and weight loss during the recovery phase. These data implicate endogenous IL-38 as an anti-inflammatory cytokine that reduces DSS colitis severity. We propose that a relative deficiency of IL-38 contributes to IBD by disinhibition of the NLRP3 inflammasome.