RESUMEN
Alterations in estrogen-mediated cellular signaling play an essential role in the pathogenesis of endometriosis. In addition to higher estrogen receptor (ER) ß levels, enhanced ERß activity was detected in endometriotic tissues, and the inhibition of enhanced ERß activity by an ERß-selective antagonist suppressed mouse ectopic lesion growth. Notably, gain of ERß function stimulated the progression of endometriosis. As a mechanism to evade endogenous immune surveillance for cell survival, ERß interacts with cellular apoptotic machinery in the cytoplasm to inhibit TNF-α-induced apoptosis. ERß also interacts with components of the cytoplasmic inflammasome to increase interleukin-1ß and thus enhance its cellular adhesion and proliferation properties. Furthermore, this gain of ERß function enhances epithelial-mesenchymal transition signaling, thereby increasing the invasion activity of endometriotic tissues for establishment of ectopic lesions. Collectively, we reveal how endometrial tissue generated by retrograde menstruation can escape immune surveillance and develop into sustained ectopic lesions via gain of ERß function.
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Endometriosis/patología , Receptor beta de Estrógeno/metabolismo , Inflamasomas/metabolismo , Menstruación/metabolismo , Animales , Apoptosis , Adhesión Celular , Proliferación Celular , Endometriosis/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Vigilancia Inmunológica , Interleucina-1beta/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.
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Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas del Tejido Nervioso/genética , Factores de Empalme de ARN/genética , ARN/genética , Factores de Transcripción/genética , Adenosina/genética , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Humanos , Metilación , Elementos Reguladores de la Transcripción/genética , Activación Transcripcional/genéticaRESUMEN
Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. DNA damage onset most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.
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Diferenciación Celular/efectos de los fármacos , Reprogramación Celular/genética , Anemia de Fanconi/genética , Formaldehído/toxicidad , Daño del ADN/efectos de los fármacos , Reparación del ADN/genética , Anemia de Fanconi/sangre , Anemia de Fanconi/patología , Formaldehído/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Inestabilidad Genómica/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Células K562 , Transcripción GenéticaRESUMEN
Dysregulation of the ubiquitin-proteasomal system (UPS) enables pathogenic accumulation of disease-driving proteins in neurons across a host of neurological disorders. However, whether and how the UPS contributes to oligodendrocyte dysfunction and repair after white matter injury (WMI) remains undefined. Here we show that the E3 ligase VHL interacts with Daam2 and their mutual antagonism regulates oligodendrocyte differentiation during development. Using proteomic analysis of the Daam2-VHL complex coupled with conditional genetic knockout mouse models, we further discovered that the E3 ubiquitin ligase Nedd4 is required for developmental myelination through stabilization of VHL via K63-linked ubiquitination. Furthermore, studies in mouse demyelination models and white matter lesions from patients with multiple sclerosis corroborate the function of this pathway during remyelination after WMI. Overall, these studies provide evidence that a signaling axis involving key UPS components contributes to oligodendrocyte development and repair and reveal a new role for Nedd4 in glial biology.
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Diferenciación Celular , Proteínas de Microfilamentos/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Regeneración Nerviosa/genética , Enfermedades del Sistema Nervioso/genética , Oligodendroglía/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Esclerosis Múltiple/fisiopatología , Vaina de Mielina/genética , Enfermedades del Sistema Nervioso/fisiopatología , Oligodendroglía/citología , Estabilidad Proteica , Ubiquitinación/genéticaRESUMEN
Elucidation of endogenous cellular protein-protein interactions and their networks is most desirable for biological studies. Here we report our study of endogenous human coregulator protein complex networks obtained from integrative mass spectrometry-based analysis of 3290 affinity purifications. By preserving weak protein interactions during complex isolation and utilizing high levels of reciprocity in the large dataset, we identified many unreported protein associations, such as a transcriptional network formed by ZMYND8, ZNF687, and ZNF592. Furthermore, our work revealed a tiered interplay within networks that share common proteins, providing a conceptual organization of a cellular proteome composed of minimal endogenous modules (MEMOs), complex isoforms (uniCOREs), and regulatory complex-complex interaction networks (CCIs). This resource will effectively fill a void in linking correlative genomic studies with an understanding of transcriptional regulatory protein functions within the proteome for formulation and testing of future hypotheses.
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Proteínas/metabolismo , Proteoma/análisis , Secuencia de Aminoácidos , Proteína BRCA1/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Inmunoprecipitación , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción GenéticaRESUMEN
Wnt signaling plays an essential role in developmental and regenerative myelination in the central nervous system. The Wnt signaling pathway is composed of multiple regulatory layers; thus, how these processes are coordinated to orchestrate oligodendrocyte (OL) development remains unclear. Here, we show CK2α, a Wnt/ß-catenin signaling Ser/Thr kinase, phosphorylates Daam2, inhibiting its function and Wnt activity during OL development. Intriguingly, we found Daam2 phosphorylation differentially impacts distinct stages of OL development, accelerating early differentiation followed by decelerating maturation and myelination. Application toward white matter injury revealed CK2α-mediated Daam2 phosphorylation plays a protective role for developmental and behavioral recovery after neonatal hypoxia, while promoting myelin repair following adult demyelination. Together, our findings identify a unique regulatory node in the Wnt pathway that regulates OL development via protein phosphorylation-induced signaling complex instability and highlights a new biological mechanism for myelin restoration.
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Sustancia Blanca , Fosforilación , Vaina de Mielina , Vía de Señalización WntRESUMEN
Two sphingosine kinase isoforms, sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2), synthesize the lipid sphingosine-1-phosphate (S1P) by phosphorylating sphingosine. SPHK1 is a cytoplasmic kinase, and SPHK2 is localized to the nucleus and other organelles. In the cytoplasm, the SPHK1/S1P pathway modulates autophagy and protein ubiquitination, among other processes. In the nucleus, the SPHK2/S1P pathway regulates transcription. Here, we hypothesized that the SPHK2/S1P pathway governs protein ubiquitination in neurons. We found that ectopic expression of SPHK2 increases ubiquitinated substrate levels in cultured neurons and pharmacologically inhibiting SPHK2 decreases protein ubiquitination. With mass spectrometry, we discovered that inhibiting SPHK2 affects lipid and synaptic protein networks as well as a ubiquitin-dependent protein network. Several ubiquitin-conjugating and hydrolyzing proteins, such as the E3 ubiquitin-protein ligases HUWE1 and TRIP12, the E2 ubiquitin-conjugating enzyme UBE2Z, and the ubiquitin-specific proteases USP15 and USP30, were downregulated by SPHK2 inhibition. Using RNA sequencing, we found that inhibiting SPHK2 altered lipid and neuron-specific gene networks, among others. Genes that encode the corresponding proteins from the ubiquitin-dependent protein network that we discovered with mass spectrometry were not affected by inhibiting SPHK2, indicating that the SPHK2/S1P pathway regulates ubiquitination at the protein level. We also show that both SPHK2 and HUWE1 were upregulated in the striatum of a mouse model of Huntington's disease, the BACHD mice, indicating that our findings are relevant to neurodegenerative diseases. Our results identify SPHK2/S1P as a novel regulator of protein ubiquitination networks in neurons and provide a new target for developing therapies for neurodegenerative diseases.
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Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. Previous work showed that this organism stimulates CRC cells proliferation and tumor growth. However, the molecular mechanisms underlying these activities are not well understood. Here, we found that Sgg upregulates the expression of several type of collagens in HT29 and HCT116 cells, with type VI collagen (ColVI) being the highest upregulated type. Knockdown of ColVI abolished the ability of Sgg to induce cell proliferation and reduced the adherence of Sgg to CRC cells. The extracellular matrix (ECM) is an important regulator of cell proliferation. Therefore, we further examined the role of decellularized matrix (dc-matrix), which is free of live bacteria or cells, in Sgg-induced cell proliferation. Dc-matrix prepared from Sgg-treated cells showed a significantly higher pro-proliferative activity than that from untreated cells or cells treated with control bacteria. On the other hand, dc-matrix from Sgg-treated ColVI knockdown cells showed no difference in the capacity to support cell proliferation compared to that from untreated ColVI knockdown cells, suggesting that the ECM by itself is a mediator of Sgg-induced cell proliferation. Furthermore, Sgg treatment of CRC cells but not ColVI knockdown CRC cells resulted in significantly larger tumors in vivo, suggesting that ColVI is important for Sgg to promote tumor growth in vivo. These results highlight a dynamic bidirectional interplay between Sgg and the ECM, where Sgg upregulates collagen expression. The Sgg-modified ECM in turn affects the ability of Sgg to adhere to host cells and more importantly, acts as a mediator for Sgg-induced CRC cell proliferation. Taken together, our results reveal a novel mechanism in which Sgg stimulates CRC proliferation through modulation of the ECM.
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Neoplasias Colorrectales , Streptococcus gallolyticus subspecies gallolyticus , Proliferación Celular , Colágeno Tipo VI , Neoplasias Colorrectales/microbiología , Matriz Extracelular/patología , Humanos , Streptococcus gallolyticus subspecies gallolyticus/fisiologíaRESUMEN
BACKGROUND: Tumor recurrence and metastatic progression remains the leading cause for breast cancer related mortalities. However, the proteomes of patient- matched primary breast cancer (BC) and metastatic lesions have not yet been identified, due to the lack of clinically annotated longitudinal samples. In this study, we evaluated the global-proteomic landscape of BC patients with and without distant metastasis as well as compared the proteome of distant metastatic disease with its corresponding primary BC, within the same patient. METHODS: We performed mass spectrometry-based proteome profiling of 73 serum samples from 51 BC patients. Among the 51 patients with BC, 29 remained metastasis-free (henceforth called non-progressors), and 22 developed metastases (henceforth called progressors). For the 22 progressors, we obtained two samples: one collected within a year of diagnosis, and the other collected within a year before the diagnosis of metastatic disease. MS data were analyzed using intensity-based absolute quantification and normalized before differential expression analysis. Significantly differentially expressed proteins (DEPs; absolute fold-change ≥ 1.5, P-value < 0.05 and 30% abundance per clinical group) were subjected to pathway analyses. RESULTS: We identified 967 proteins among 73 serum samples from patients with BC. Among these, 39 proteins were altered in serum samples at diagnosis, between progressors and non-progressors. Among these, 4 proteins were further altered when the progressors developed distant metastasis. In addition, within progressors, 20 proteins were altered in serum collected at diagnosis versus at the onset of metastasis. Pathway analysis showed that these proteins encoded pathways that describe metastasis, including epithelial-mesenchymal transition and focal adhesion that are hallmarks of metastatic cascade. CONCLUSIONS: Our results highlight the importance of examining matched samples from distant metastasis with primary BC samples collected at diagnosis to unravel subset of proteins that could be involved in BC progression in serum. This study sets the foundation for additional future investigations that could position these proteins as non-invasive markers for clinically monitoring breast cancer progression in patients.
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B-cell-activating factor (BAFF) mediates B-cell survival and, when deregulated, contributes to autoimmune diseases and B-cell malignancies. The mechanism connecting BAFF receptor (BAFFR) signal to downstream pathways and pathophysiological functions is not well understood. Here we identified DYRK1a as a kinase that responds to BAFF stimulation and mediates BAFF-induced B-cell survival. B-cell-specific DYRK1a deficiency causes peripheral B-cell reduction and ameliorates autoimmunity in a mouse model of lupus. An unbiased screen identified DYRK1a as a protein that interacts with TRAF3, a ubiquitin ligase component mediating degradation of the noncanonical nuclear factor (NF)-κB-inducing kinase (NIK). DYRK1a phosphorylates TRAF3 at serine-29 to interfere with its function in mediating NIK degradation, thereby facilitating BAFF-induced NIK accumulation and noncanonical NF-κB activation. Interestingly, B-cell acute lymphoblastic leukemia (B-ALL) cells express high levels of BAFFR and respond to BAFF for noncanonical NF-κB activation and survival in a DYRK1a-dependent manner. Furthermore, DYRK1a promotes a mouse model of B-ALL through activation of the noncanonical NF-κB pathway. These results establish DYRK1a as a critical BAFFR signaling mediator and provide novel insight into B-ALL pathogenesis.
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Autoinmunidad , Factor Activador de Células B/inmunología , Leucemia de Células B/inmunología , FN-kappa B/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Tirosina Quinasas/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Linfocitos B/inmunología , Linfocitos B/patología , Carcinogénesis/inmunología , Carcinogénesis/patología , Línea Celular Tumoral , Humanos , Leucemia de Células B/patología , Ratones , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Quinasas DyrKRESUMEN
Pexidartinib (PEX, TURALIO), a selective and potent inhibitor of the macrophage colony-stimulating factor-1 receptor, has been approved for the treatment of tenosynovial giant cell tumor. However, frequent and severe adverse effects have been reported in the clinic, resulting in a boxed warning on PEX for its risk of liver injury. The mechanisms underlying PEX-related hepatotoxicity, particularly metabolism-related toxicity, remain unknown. In the current study, the metabolic activation of PEX was investigated in human/mouse liver microsomes (HLM/MLM) and primary human hepatocytes (PHH) using glutathione (GSH) and methoxyamine (NH2OMe) as trapping reagents. A total of 11 PEX-GSH and 7 PEX-NH2OMe adducts were identified in HLM/MLM using an LC-MS-based metabolomics approach. Additionally, 4 PEX-GSH adducts were detected in the PHH. CYP3A4 and CYP3A5 were identified as the primary enzymes responsible for the formation of these adducts using recombinant human P450s and CYP3A chemical inhibitor ketoconazole. Overall, our studies suggested that PEX metabolism can produce reactive metabolites mediated by CYP3A, and the association of the reactive metabolites with PEX hepatotoxicity needs to be further studied.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Citocromo P-450 CYP3A , Ratones , Humanos , Animales , Citocromo P-450 CYP3A/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores del Citocromo P-450 CYP3A/farmacología , Microsomas Hepáticos/metabolismo , Metabolómica , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Glutatión/metabolismoRESUMEN
Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease-modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock-enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB-containing diets. NOB significantly alleviated ß-amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel-running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock-controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time-dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis-associated neuroinflammation.
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Enfermedad de Alzheimer/tratamiento farmacológico , Antiinflamatorios/farmacología , Flavonas/farmacología , Gliosis/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Citocinas/genética , Citocinas/metabolismo , Flavonas/uso terapéutico , Gliosis/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones , Fármacos Neuroprotectores/uso terapéuticoRESUMEN
Astrocytes display extraordinary morphological complexity that is essential to support brain circuit development and function. Formin proteins are key regulators of the cytoskeleton; however, their role in astrocyte morphogenesis across diverse brain regions and neural circuits is unknown. Here, we show that loss of the formin protein Daam2 in astrocytes increases morphological complexity in the cortex and olfactory bulb, but elicits opposing effects on astrocytic calcium dynamics. These differential physiological effects result in increased excitatory synaptic activity in the cortex and increased inhibitory synaptic activity in the olfactory bulb, leading to altered olfactory behaviors. Proteomic profiling and immunoprecipitation experiments identify Slc4a4 as a binding partner of Daam2 in the cortex, and combined deletion of Daam2 and Slc4a4 restores the morphological alterations seen in Daam2 mutants. Our results reveal new mechanisms regulating astrocyte morphology and show that congruent changes in astrocyte morphology can differentially influence circuit function.
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Astrocitos , Proteínas de Microfilamentos/genética , Proteínas de Unión al GTP rho/genética , Forminas , Morfogénesis , Bulbo Olfatorio/metabolismo , Proteómica , Simportadores de Sodio-BicarbonatoRESUMEN
Inflammatory environments provide vital biochemical stimuli (i.e., oxidative stress, pH, and enzymes) for triggered drug delivery in a controlled manner. Inflammation alters the local pH within the affected tissues. As a result, pH-sensitive nanomaterials can be used to effectively target drugs to the site of inflammation. Herein, we designed pH-sensitive nanoparticles in which resveratrol (an anti-inflammatory and antioxidant compound (RES)) and urocanic acid (UA) were complexed with a pH-sensitive moiety using an emulsion method. These RES-UA NPs were characterized by transmission electron microscopy, dynamic light scattering, zeta potential, and FT-IR spectroscopy. The anti-inflammatory and antioxidant activities of the RES-UA NPs were assessed in RAW 264.7 macrophages. The NPs were circular in shape and ranged in size from 106 to 180 nm. The RES-UA NPs suppressed the mRNA expression of the pro-inflammatory molecules inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in a concentration-dependent manner. Incubation of LPS-stimulated macrophages with RES-UA NPs reduced the generation of reactive oxygen species (ROS) in a concentration-dependent manner. These results suggest that pH-responsive RES-UA NPs can be used to decrease ROS generation and inflammation.
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Antiinflamatorios , Antioxidantes , Nanopartículas , Resveratrol , Ácido Urocánico , Humanos , Antiinflamatorios/química , Antiinflamatorios/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Ciclooxigenasa 2/metabolismo , Concentración de Iones de Hidrógeno , Inflamación/metabolismo , Lipopolisacáridos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Resveratrol/química , Resveratrol/farmacología , Espectroscopía Infrarroja por Transformada de Fourier , Factor de Necrosis Tumoral alfa/metabolismo , Ácido Urocánico/química , Ácido Urocánico/farmacologíaRESUMEN
Duloxetine (DLX) is a dual serotonin and norepinephrine reuptake inhibitor, widely used for the treatment of major depressive disorder. Although DLX has shown good efficacy and safety, serious adverse effects (e.g., liver injury) have been reported. The mechanisms associated with DLX-induced toxicity remain elusive. Drug metabolism plays critical roles in drug safety and efficacy. However, the metabolic profile of DLX in mice is not available, although mice serve as commonly used animal models for mechanistic studies of drug-induced adverse effects. Our study revealed 39 DLX metabolites in human/mouse liver microsomes and mice. Of note, 13 metabolites are novel, including five N-acetyl cysteine adducts and one reduced glutathione (GSH) adduct associated with DLX. Additionally, the species differences of certain metabolites were observed between human and mouse liver microsomes. CYP1A2 and CYP2D6 are primary enzymes responsible for the formation of DLX metabolites in liver microsomes, including DLX-GSH adducts. In summary, a total of 39 DLX metabolites were identified, and species differences were noticed in vitro. The roles of CYP450s in DLX metabolite formation were also verified using human recombinant cytochrome P450 (P450) enzymes and corresponding chemical inhibitors. Further studies are warranted to address the exact role of DLX metabolism in its adverse effects in vitro (e.g., human primary hepatocytes) and in vivo (e.g., Cyp1a2-null mice). SIGNIFICANCE STATEMENT: This current study systematically investigated Duloxetine (DLX) metabolism and bioactivation in liver microsomes and mice. This study provided a global view of DLX metabolism and bioactivation in liver microsomes and mice, which are very valuable to further elucidate the mechanistic study of DLX-related adverse effects and drug-drug interaction from metabolic aspects.
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Trastorno Depresivo Mayor , Inhibidores de Captación de Serotonina y Norepinefrina , Animales , Trastorno Depresivo Mayor/metabolismo , Clorhidrato de Duloxetina/metabolismo , Ratones , Microsomas Hepáticos/metabolismo , Serotonina/metabolismo , Inhibidores de Captación de Serotonina y Norepinefrina/metabolismoRESUMEN
Gene discovery efforts in autism spectrum disorder have identified heterozygous defects in chromatin remodeller genes, the 'readers, writers and erasers' of methyl marks on chromatin, as major contributors to this disease. Despite this advance, a convergent aetiology between these defects and aberrant chromatin architecture or gene expression has remained elusive. Recently, data have begun to emerge that chromatin remodellers also function directly on the cytoskeleton. Strongly associated with autism spectrum disorder, the SETD2 histone methyltransferase for example, has now been shown to directly methylate microtubules of the mitotic spindle. However, whether microtubule methylation occurs in post-mitotic cells, for example on the neuronal cytoskeleton, is not known. We found the SETD2 α-tubulin lysine 40 trimethyl mark occurs on microtubules in the brain and in primary neurons in culture, and that the SETD2 C-terminal SRI domain is required for binding and methylation of α-tubulin. A CRISPR knock-in of a pathogenic SRI domain mutation (Setd2SRI) that disables microtubule methylation revealed at least one wild-type allele was required in mice for survival, and while viable, heterozygous Setd2SRI/wtmice exhibited an anxiety-like phenotype. Finally, whereas RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) showed no concomitant changes in chromatin methylation or gene expression in Setd2SRI/wtmice, primary neurons exhibited structural deficits in axon length and dendritic arborization. These data provide the first demonstration that microtubules of neurons are methylated, and reveals a heterozygous chromatin remodeller defect that specifically disables microtubule methylation is sufficient to drive an autism-associated phenotype.
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Ansiedad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/metabolismo , Histonas/metabolismo , Metilación , Ratones , FenotipoRESUMEN
DNA replication is executed only when cells have sufficient metabolic resources and undamaged DNA. Nutrient limitation and DNA damage cause a metabolic checkpoint and DNA damage checkpoint, respectively. Although SIRT1 activity is regulated by metabolic stress and DNA damage, its function in these stress-mediated checkpoints remains elusive. Here we report that the SIRT1-TopBP1 axis functions as a switch for both checkpoints. With glucose deprivation, SIRT1 is activated and deacetylates TopBP1, resulting in TopBP1-Treslin disassociation and DNA replication inhibition. Conversely, SIRT1 activity is inhibited under genotoxic stress, resulting in increased TopBP1 acetylation that is important for the TopBP1-Rad9 interaction and activation of the ATR-Chk1 pathway. Mechanistically, we showed that acetylation of TopBP1 changes the conformation of TopBP1, thereby facilitating its interaction with distinct partners in DNA replication and checkpoint activation. Taken together, our studies identify the SIRT1-TopBP1 axis as a key signaling mode in the regulation of the metabolic checkpoint and the DNA damage checkpoint.
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Proteínas Portadoras/metabolismo , Daño del ADN , Sirtuina 1/metabolismo , Estrés Fisiológico , Acetilación , Animales , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Reparación del ADN , Replicación del ADN , Células HEK293 , Humanos , Ratones , Conformación Proteica , Transducción de SeñalRESUMEN
Adenosine monophosphate-activated protein kinase (AMPK) is an obligate heterotrimer that consists of a catalytic subunit (α) and two regulatory subunits (ß and γ). AMPK is a key enzyme in the regulation of cellular energy homeostasis. It has been well studied and is known to function in many cellular pathways. However, the interactome of AMPK has not yet been systematically established, although protein-protein interaction is critically important for protein function and regulation. Here, we used tandem-affinity purification, coupled with mass spectrometry (TAP-MS) analysis, to determine the interactome of AMPK and its functions. We conducted a TAP-MS analysis of all seven AMPK subunits. We identified 138 candidate high-confidence interacting proteins (HCIPs) of AMPK, which allowed us to build an interaction network of AMPK complexes. Five candidate AMPK-binding proteins were experimentally validated, underlining the reliability of our data set. Furthermore, we demonstrated that AMPK acts with a strong AMPK-binding protein, Artemis, in non-homologous end joining. Collectively, our study established the first AMPK interactome and uncovered a new function of AMPK in DNA repair.
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Proteínas Quinasas Activadas por AMP/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Células HEK293 , Humanos , Mapeo de Interacción de ProteínasRESUMEN
An early hallmark of Alzheimer's disease is the accumulation of amyloid-ß (Aß), inspiring numerous therapeutic strategies targeting this peptide. An alternative approach is to destabilize the amyloid beta precursor protein (APP) from which Aß is derived. We interrogated innate pathways governing APP stability using a siRNA screen for modifiers whose own reduction diminished APP in human cell lines and transgenic Drosophila. As proof of principle, we validated PKCß-a known modifier identified by the screen-in an APP transgenic mouse model. PKCß was genetically targeted using a novel adeno-associated virus shuttle vector to deliver microRNA-adapted shRNA via intracranial injection. In vivo reduction of PKCß initially diminished APP and delayed plaque formation. Despite persistent PKCß suppression, the effect on APP and amyloid diminished over time. Our study advances this approach for mining druggable modifiers of disease-associated proteins, while cautioning that prolonged in vivo validation may be needed to reveal emergent limitations on efficacy.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidosis/metabolismo , Proteína Quinasa C beta/antagonistas & inhibidores , Enfermedad de Alzheimer/genética , Amiloidosis/terapia , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Drosophila , Pruebas Genéticas , Terapia Genética , Humanos , Ratones , Ratones Transgénicos , Células 3T3 NIH , Fosforilación , Placa Amiloide/patología , Proteína Quinasa C beta/genética , Proteína Quinasa C beta/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Bile acids have recently emerged as key metabolic hormones with beneficial impacts in multiple metabolic diseases. We previously discovered that hepatic bile acid overload distally modulates glucose and fatty acid metabolism in adipose tissues to exert anti-obesity effects. However, the detailed mechanisms that explain the salutary effects of serum bile acid elevation remain unclear. Here, proteomic profiling identified a new hepatokine, Orosomucoid (ORM) that governs liver-adipose tissue crosstalk. Hepatic ORMs were highly induced by both genetic and dietary bile acid overload. To address the direct metabolic effects of ORM, purified ORM proteins were administered during adipogenic differentiation of 3T3-L1 cells and mouse stromal vascular fibroblasts. ORM suppressed adipocyte differentiation and strongly inhibited gene expression of adipogenic transcription factors such as C/EBPß, KLF5, C/EBPα, and PPARγ. Taken together, our data clearly suggest that bile acid-induced ORM secretion from the liver blocks adipocyte differentiation, potentially linked to anti-obesity effect of bile acids.