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During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.
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DNA Polimerase III , DNA , Histonas , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Nucleossomos/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , DNA Polimerase III/metabolismo , DNA Polimerase III/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , DNA/metabolismo , Replicação do DNA , Ligação Proteica , DNA Polimerase Dirigida por DNARESUMO
The selective oxidation of toluene to yield value-added oxygenates, such as benzyl alcohol, benzaldehyde, and benzoic acid, via dioxygen presents a chlorine-free approach under benign conditions. Metal-free catalytic processes are preferred to avoid metal ion contamination. In this study, we employed N-hydroxyphthalimide (NHPI) as a catalyst for the aerobic oxidation of toluene to its oxygenated derivatives. The choice of solvent exerted a significant impact on the catalytic activity and selectivity of the catalyst NHPI at reaction temperatures exceeding 70 °C. Notably, hexafluoroisopropanol substantially enhanced the selective production of benzaldehyde. Furthermore, we identified didecyl dimethyl ammonium bromide, featuring two symmetrical long hydrophobic chains, as a potent enhancer of NHPI for the solvent-free aerobic oxidation of toluene. This effect is ascribed to its unique symmetrical structure, extraction capabilities, and resistance to thermal and acid/base conditions. Based on the product distribution and control experiments, we proposed a plausible reaction mechanism. These findings may inform the industrial synthesis of oxygenated derivatives from toluene.
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The prevalent RNA alternative splicing (AS) contributes to molecular diversity, which has been demonstrated in cellular function regulation and disease pathogenesis. However, the contribution of AS in pancreatic islets during diabetes progression remains unclear. Here, we reanalyze the full-length single-cell RNA sequencing data from the deposited database to investigate AS regulation across human pancreatic endocrine cell types in non-diabetic (ND) and type 2 diabetic (T2D) individuals. Our analysis demonstrates the significant association between transcriptomic AS profiles and cell-type-specificity, which could be applied to distinguish the clustering of major endocrine cell types. Moreover, AS profiles are enabled to clearly define the mature subset of ß-cells in healthy controls, which is completely lost in T2D. Further analysis reveals that RNA-binding proteins (RBPs), heterogeneous nuclear ribonucleoproteins (hnRNPs) and FXR1 family proteins are predicted to induce the functional impairment of ß-cells through regulating AS profiles. Finally, trajectory analysis of endocrine cells suggests the ß-cell identity shift through dedifferentiation and transdifferentiation of ß-cells during the progression of T2D. Together, our study provides a mechanism for regulating ß-cell functions and suggests the significant contribution of AS program during diabetes pathogenesis.
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Processamento Alternativo , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Análise de Sequência de RNA , Análise de Célula Única , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Transcriptoma , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologiaRESUMO
Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) protect against diabetic cardiovascular diseases and nephropathy. However, their activity in diabetic retinopathy (DR) remains unclear. Our retrospective cohort study involving 1626 T2DM patients revealed superior efficacy of GLP-1 RAs in controlling DR compared to other glucose-lowering medications, suggesting their advantage in DR treatment. By single-cell RNA-sequencing analysis and immunostaining, we observed a high expression of GLP-1R in retinal endothelial cells, which was down-regulated under diabetic conditions. Treatment of GLP-1 RAs significantly restored the receptor expression, resulting in an improvement in retinal degeneration, vascular tortuosity, avascular vessels, and vascular integrity in diabetic mice. GO and GSEA analyses further implicated enhanced mitochondrial gene translation and mitochondrial functions by GLP-1 RAs. Additionally, the treatment attenuated STING signaling activation in retinal endothelial cells, which is typically activated by leaked mitochondrial DNA. Expression of STING mRNA was positively correlated to the levels of angiogenic and inflammatory factors in the endothelial cells of human fibrovascular membranes. Further investigation revealed that the cAMP-responsive element binding protein played a role in the GLP-1R signaling pathway on suppression of STING signaling. This study demonstrates a novel role of GLP-1 RAs in the protection of diabetic retinal vasculature by inhibiting STING-elicited inflammatory signals.
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BACKGROUND: Diabetic retinopathy (DR) is a major cause of blindness and is characterized by dysfunction of the retinal microvasculature. Neutrophil stasis, resulting in retinal inflammation and the occlusion of retinal microvessels, is a key mechanism driving DR. These plugging neutrophils subsequently release neutrophil extracellular traps (NETs), which further disrupts the retinal vasculature. Nevertheless, the primary catalyst for NETs extrusion in the retinal microenvironment under diabetic conditions remains unidentified. In recent studies, cellular communication network factor 1 (CCN1) has emerged as a central molecule modulating inflammation in pathological settings. Additionally, our previous research has shed light on the pathogenic role of CCN1 in maintaining endothelial integrity. However, the precise role of CCN1 in microvascular occlusion and its potential interaction with neutrophils in diabetic retinopathy have not yet been investigated. METHODS: We first examined the circulating level of CCN1 and NETs in our study cohort and analyzed related clinical parameters. To further evaluate the effects of CCN1 in vivo, we used recombinant CCN1 protein and CCN1 overexpression for gain-of-function, and CCN1 knockdown for loss-of-function by intravitreal injection in diabetic mice. The underlying mechanisms were further validated on human and mouse primary neutrophils and dHL60 cells. RESULTS: We detected increases in CCN1 and neutrophil elastase in the plasma of DR patients and the retinas of diabetic mice. CCN1 gain-of-function in the retina resulted in neutrophil stasis, NETs extrusion, capillary degeneration, and retinal leakage. Pre-treatment with DNase I to reduce NETs effectively eliminated CCN1-induced retinal leakage. Notably, both CCN1 knockdown and DNase I treatment rescued the retinal leakage in the context of diabetes. In vitro, CCN1 promoted adherence, migration, and NETs extrusion of neutrophils. CONCLUSION: In this study, we uncover that CCN1 contributed to retinal inflammation, vessel occlusion and leakage by recruiting neutrophils and triggering NETs extrusion under diabetic conditions. Notably, manipulating CCN1 was able to hold therapeutic promise for the treatment of diabetic retinopathy.
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Proteína Rica em Cisteína 61 , Retinopatia Diabética , Armadilhas Extracelulares , Neutrófilos , Animais , Feminino , Humanos , Masculino , Camundongos , Proteína Rica em Cisteína 61/metabolismo , Proteína Rica em Cisteína 61/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/genética , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/metabolismo , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Retina/patologia , Retina/metabolismoRESUMO
Brown adipose tissue (BAT) is the main site of nonshivering thermogenesis which plays an important role in thermogenesis and energy metabolism. However, the regulatory factors that inhibit BAT activity remain largely unknown. Here, cardiotrophin-like cytokine factor 1 (CLCF1) is identified as a negative regulator of thermogenesis in BAT. Adenovirus-mediated overexpression of CLCF1 in BAT greatly impairs the thermogenic capacity of BAT and reduces the metabolic rate. Consistently, BAT-specific ablation of CLCF1 enhances the BAT function and energy expenditure under both thermoneutral and cold conditions. Mechanistically, adenylate cyclase 3 (ADCY3) is identified as a downstream target of CLCF1 to mediate its role in regulating thermogenesis. Furthermore, CLCF1 is identified to negatively regulate the PERK-ATF4 signaling axis to modulate the transcriptional activity of ADCY3, which activates the PKA substrate phosphorylation. Moreover, CLCF1 deletion in BAT protects the mice against diet-induced obesity by promoting BAT activation and further attenuating impaired glucose and lipid metabolism. Therefore, our results reveal the essential role of CLCF1 in regulating BAT thermogenesis and suggest that inhibiting CLCF1 signaling might be a potential therapeutic strategy for improving obesity-related metabolic disorders.
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Tecido Adiposo Marrom , Metabolismo Energético , Animais , Camundongos , Adenoviridae , Interleucinas , Obesidade/genética , Termogênese/genéticaRESUMO
Senescence of vascular smooth muscle cells (VSMCs) is a key contributor to plaque vulnerability in atherosclerosis (AS), which is affected by endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. However, the crosstalk between ER stress and ROS production in the pathogenesis of VSMC senescence remains to be elucidated. ER-associated degradation (ERAD) is a complex process that clears unfolded or misfolded proteins to maintain ER homeostasis. HRD1 is the major E3 ligase in mammalian ERAD machineries that catalyzes ubiquitin conjugation to the unfolded or misfolded proteins for degradation. Our results showed that HRD1 protein levels were reduced in human AS plaques and aortic roots from ApoE-/- mice fed with high-fat diet (HFD), along with the increased ER stress response. Exposure to cholesterol in VSMCs activated inflammatory signaling and induced senescence, while reduced HRD1 protein expression. CRISPR Cas9-mediated HRD1 knockout (KO) exacerbated cholesterol- and thapsigargin-induced cell senescence. Inhibiting ER stress with 4-PBA (4-Phenylbutyric acid) partially reversed the ROS production and cell senescence induced by HRD1 deficiency in VSMCs, suggesting that ER stress alone could be sufficient to induce ROS production and senescence in VSMCs. Besides, HRD1 deficiency led to mitochondrial dysfunction, and reducing ROS production from impaired mitochondria partly reversed HRD1 deficiency-induced cell senescence. Finally, we showed that the overexpression of HDR1 reversed cholesterol-induced ER stress, ROS production, and cellular senescence in VSMCs. Our findings indicate that HRD1 protects against senescence by maintaining ER homeostasis and mitochondrial functionality. Thus, targeting HRD1 function may help to mitigate VSMC senescence and prevent vascular aging related diseases. TRIAL REGISTRATION: A real-world study based on the discussion of primary and secondary prevention strategies for coronary heart disease, URL:https://www.clinicaltrials.gov, the trial registration number is [2022]-02-121-01.
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Aterosclerose , Músculo Liso Vascular , Animais , Humanos , Camundongos , Aterosclerose/metabolismo , Senescência Celular , Estresse do Retículo Endoplasmático/fisiologia , Degradação Associada com o Retículo Endoplasmático , Mamíferos/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Global temperatures have risen as a result of climate change, and the resulting warmer seawater will exert physiological stresses on many aquatic animals, including Apostichopus japonicus. It has been suggested that the sensitivity of aquatic poikilothermal animals to climate change is closely related to mitochondrial function. Therefore, understanding the interaction between elevated temperature and mitochondrial functioning is key to characterizing organisms' responses to heat stress. However, little is known about the mitochondrial response to heat stress in A. japonicus. In this work, we investigated the morphological and functional changes of A. japonicus mitochondria under three representative temperatures, control temperature (18 °C), aestivation temperature (25 °C) and heat stress temperature (32 °C) temperatures using transmission electron microscopy (TEM) observation of mitochondrial morphology combined with proteomics and metabolomics techniques. The results showed that the mitochondrial morphology of A. japonicus was altered, with decreases in the number of mitochondrial cristae at 25 °C and mitochondrial lysis, fracture, and vacuolization at 32 °C. Proteomic and metabolomic analyses revealed 103 differentially expressed proteins and 161 differential metabolites at 25 °C. At 32 °C, the levels of 214 proteins and 172 metabolites were significantly altered. These proteins and metabolites were involved in the tricarboxylic acid (TCA) cycle, substance transport, membrane potential homeostasis, anti-stress processes, mitochondrial autophagy, and apoptosis. Furthermore, a hypothetical network of proteins and metabolites in A. japonicus mitochondria in response to temperature changes was constructed based on proteomic and metabolomic data. These results suggest that the dynamic regulation of mitochondrial energy metabolism, resistance to oxidative stress, autophagy, apoptosis, and mitochondrial morphology in A. japonicus may play important roles in the response to elevated temperatures. In summary, this study describes the response of A. japonicus mitochondria to temperature changes from the perspectives of morphology, proteins, and metabolites, which provided a better understanding the mechanisms of mitochondrial regulation under environment stress in marine echinoderms.
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Stichopus , Animais , Stichopus/metabolismo , Temperatura , Proteômica/métodos , Estresse Fisiológico , MitocôndriasRESUMO
Phenotypic change of vascular smooth muscle cells (VSMCs) is the main contributor of vascular pathological remodeling in atherosclerosis. The endoplasmic reticulum (ER) is critical for maintaining VSMC function through elimination of misfolded proteins that impair VSMC cellular function. ER-associated degradation (ERAD) is an ER-mediated process that controls protein quality by clearing misfolded proteins. One of the critical regulators of ERAD is HRD1, which also plays a vital role in lipid metabolism. However, the function of HRD1 in VSMCs of atherosclerotic vessels remains poorly understood. The level of HRD1 expression was analyzed in aortic tissues of mice fed with a high-fat diet (HFD). The H&E and EVG (VERHOEFF'S VAN GIESON) staining were used to demonstrate pathological vascular changes. IF (immunofluorescence) and WB (western blot) were used to explore the signaling pathways in vivo and in vitro. The wound closure and transwell assays were also used to test the migration rate of VSMCs. CRISPR gene editing and transcriptomic analysis were applied in vitro to explore the cellular mechanism. Our data showed significant reduction of HRD1 in aortic tissues of mice under HFD feeding. VSMC phenotypic change and HRD1 downregulation were detected by cholesterol supplement. Transcriptomic and further analysis of HRD1-KO VSMCs showed that HRD1 deficiency induced the expression of genes related to ER stress response, proliferation and migration, but reduced the contractile-related genes in VSMCs. HRD1 deficiency also exacerbated the proliferation, migration and ROS production of VSMCs induced by cholesterol, which promoted the VSMC dedifferentiation. Our results showed that HRD1 played an essential role in the contractile homeostasis of VSMCs by negatively regulating ER stress response. Thus, HRD1 in VSMCs could serve as a potential therapeutic target in metabolic disorder-induced vascular remodeling.
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In the context of non-alcoholic fatty liver disease (NAFLD), characterized by dysregulated lipid metabolism in hepatocytes, the quest for safe and effective therapeutics targeting lipid metabolism has gained paramount importance. Sanhuang Xiexin Tang (SXT) and Baihu Tang (BHT) have emerged as prominent candidates for treating metabolic disorders. SXT combined with BHT plus Cangzhu (SBC) has been used clinically for Weihuochisheng obese patients. This retrospective analysis focused on assessing the anti-obesity effects of SBC in Weihuochisheng obese patients. We observed significant reductions in body weight and hepatic lipid content among obese patients following SBC treatment. To gain further insights, we investigated the effects and underlying mechanisms of SBC in HFD-fed mice. The results demonstrated that SBC treatment mitigated body weight gain and hepatic lipid accumulation in HFD-fed mice. Pharmacological network analysis suggested that SBC may affect lipid metabolism, mitochondria, inflammation, and apoptosis-a hypothesis supported by the hepatic transcriptomic analysis in HFD-fed mice treated with SBC. Notably, SBC treatment was associated with enhanced hepatic mitochondrial biogenesis and the inhibition of the c-Jun N-terminal kinase (JNK)/nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK)/NF-κB pathways. In conclusion, SBC treatment alleviates NAFLD in both obese patients and mouse models by improving lipid metabolism, potentially through enhancing mitochondrial biogenesis. These effects, in turn, ameliorate inflammation in hepatocytes.
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Hepatopatia Gordurosa não Alcoólica , Humanos , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , NF-kappa B/metabolismo , Biogênese de Organelas , Estudos Retrospectivos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Fígado , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Peso Corporal , Metabolismo dos Lipídeos , Lipídeos , Dieta Hiperlipídica/efeitos adversosRESUMO
Cardiac development involves large-scale rearrangements of the proteome. How the developing cardiac cells maintain the integrity of the proteome during the rapid lineage transition remains unclear. Here it is shown that proteotoxic stress visualized by the misfolded and/or aggregated proteins appears during early cardiac differentiation of human pluripotent stem cells and is resolved by activation of the PERK branch of unfolded protein response (UPR). PERK depletion increases misfolded and/or aggregated protein accumulation, leading to pluripotency exit defect and impaired mesendoderm specification of human pluripotent stem cells. Mechanistically, it is found that PERK safeguards mesendoderm specification through its conserved downstream effector ATF4, which subsequently activates a novel transcriptional target WARS1, to cope with the differentiation-induced proteotoxic stress. The results indicate that protein quality control represents a previously unrecognized core component of the cardiogenic regulatory network. Broadly, these findings provide a framework for understanding how UPR is integrated into the developmental program by activating the PERK-ATF4-WARS1 axis.
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Células-Tronco Pluripotentes , eIF-2 Quinase , Humanos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteoma/metabolismo , Proteostase , Resposta a Proteínas não DobradasRESUMO
Parental histone recycling is vital for maintaining chromatin-based epigenetic information during replication, yet its underlying mechanisms remain unclear. Here, we uncover an unexpected role of histone chaperone FACT and its N-terminus of the Spt16 subunit during parental histone recycling and transfer in budding yeast. Depletion of Spt16 and mutations at its middle domain that impair histone binding compromise parental histone recycling on both the leading and lagging strands of DNA replication forks. Intriguingly, deletion of the Spt16-N domain impairs parental histone recycling, with a more pronounced defect observed on the lagging strand. Mechanistically, the Spt16-N domain interacts with the replicative helicase MCM2-7 and facilitates the formation of a ternary complex involving FACT, histone H3/H4 and Mcm2 histone binding domain, critical for the recycling and transfer of parental histones to lagging strands. Lack of the Spt16-N domain weakens the FACT-MCM interaction and reduces parental histone recycling. We propose that the Spt16-N domain acts as a protein-protein interaction module, enabling FACT to function as a shuttle chaperone in collaboration with Mcm2 and potentially other replisome components for efficient local parental histone recycling and inheritance.
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Histonas , Proteínas de Saccharomyces cerevisiae , Fatores de Elongação da Transcrição , Cromatina/genética , DNA Helicases/genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Complexos Multiproteicos/metabolismoRESUMO
Cobalt is an alternative catalyst for furfural hydrogenation but suffers from the strong binding of H and furan ring on the surface, resulting in low catalytic activity and chemoselectivity. Herein, by constructing a Pd-Co interface in cobalt oxide-supported Pd catalysts to tailor the d-band center of Co, the concerted effort of Pd and Co boosts the catalytic performance for the hydroconversion of furfural to cyclopentanone and cyclopentanol. The increased dispersion of Pd on acid etching Co3O4 promotes the reduction of Co3+ to Co0 by enhancing hydrogen spillover, favoring the creation of the Pd-Co interface. Both experimental and theoretical calculations demonstrate that the electron transfer from Pd to Co at the interface results in the downshift of the d-band center of Co atoms, accompanied by the destabilization of H and furan ring adsorption on the Co surface, respectively. The former improves the furfural hydrogenation with TOF on Co elevating from 0.20 to 0.62 s-1, and the latter facilitates the desorption of formed furfuryl alcohol from the Co surface for subsequently hydrogenative rearrangement of the furan ring to cyclopentanone on acid sites. The resultant Pd/Co3O4-6 catalyst delivers superior activity with a 99% furfural conversion and 85% overall selectivity toward cyclopentanone/cyclopentanol. We anticipate that such a concept of tailoring the d-band center of Co via interface engineering provides novel insight and feasible approach for the design of highly efficient catalysts for furfural hydroconversion and beyond.
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Unfolded protein response (UPR) maintains the endoplasmic reticulum (ER) homeostasis, survival, and physiological function of mammalian cells. However, how cells adapt to ER stress under physiological or disease settings remains largely unclear. Here by a genome-wide CRISPR screen, we identified that RBBP8, an endonuclease involved in DNA damage repair, is required for ATF4 activation under ER stress in vitro. RNA-seq analysis suggested that RBBP8 deletion led to impaired cell cycle progression, retarded proliferation, attenuated ATF4 activation, and reduced global protein synthesis under ER stress. Mouse tissue analysis revealed that RBBP8 was highly expressed in the liver, and its expression is responsive to ER stress by tunicamycin intraperitoneal injection. Hepatocytes with RBBP8 inhibition by adenovirus-mediated shRNA were resistant to tunicamycin (Tm)-induced liver damage, cell death, and ER stress response. To study the pathological role of RBBP8 in regulating ATF4 activity, we illustrated that both RBBP8 and ATF4 were highly expressed in liver cancer tissues compared with healthy controls and highly expressed in Ki67-positive proliferating cells within the tumors. Interestingly, overexpression of RBBP8 in vitro promoted ATF4 activation under ER stress, and RBBP8 expression showed a positive correlation with ATF4 expression in liver cancer tissues by co-immunostaining. Our findings provide new insights into the mechanism of how cells adapt to ER stress through the crosstalk between the nucleus and ER and how tumor cells survive under chemotherapy or other anticancer treatments, which suggests potential therapeutic strategies against liver disease by targeting DNA damage repair, UPR or protein synthesis.
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias Hepáticas , Animais , Camundongos , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas , Neoplasias Hepáticas/genética , MamíferosRESUMO
OBJECTIVE: Obesity hypoventilation syndrome is associated with diaphragmatic dysfunction. This study aimed to explore the role of endoplasmic reticulum (ER) stress in mediating obesity-induced diaphragmatic dysfunction. METHODS: A pulmonary function test and ultrasound were applied to evaluate diaphragmatic function and magnetic resonance imaging was applied to measure diaphragmatic lipid deposition in human patients. For the mechanistic study, obese mice were introduced to a high-fat diet for 24 weeks, followed by diaphragmatic ultrasound measurement, transcriptomic sequencing, and respective biochemical analysis. Automatic force mapping was applied to measure the mechanical properties of C2C12 myotubes. RESULTS: People with obesity showed significant diaphragm weakness and lipid accumulation, which was further confirmed in obese mice. Consistently, diaphragms from obese mice showed altered gene expression profile in lipid metabolism and activation of ER stress response, indicated by elevated protein kinase R-like ER kinase (PERK) and c-Jun NH2 -terminal kinase (JNK) activation. In C2C12 myotubes, inhibition of PERK or JNK signaling abrogated lipotoxicity-induced intracellular lipid deposition and insulin resistance. Inhibition of JNK signaling reversed lipotoxicity-induced impairment of elasticity in C2C12 myotubes. CONCLUSIONS: These data suggest that ectopic lipid deposition impairs the diaphragmatic function of people with obesity. Activation of PERK/JNK signaling is involved in the pathogenesis of lipotoxicity-induced diaphragm weakness in obesity hypoventilation syndrome.
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Síndrome de Hipoventilação por Obesidade , Transdução de Sinais , Camundongos , Animais , Humanos , Transdução de Sinais/fisiologia , Diafragma/metabolismo , Síndrome de Hipoventilação por Obesidade/complicações , Camundongos Obesos , Estresse do Retículo Endoplasmático/fisiologia , Obesidade/genética , LipídeosRESUMO
Dysregulated production of peptide hormones is the key pathogenic factor of various endocrine diseases. Endoplasmic reticulum (ER) associated degradation (ERAD) is a critical machinery in maintaining ER proteostasis in mammalian cells by degrading misfolded proteins. Dysfunction of ERAD leads to maturation defect of many peptide hormones, such as provasopressin (proAVP), which results in the occurrence of Central Diabetes Insipidus. However, drugs targeting ERAD to regulate the production of peptide hormones are very limited. Herbal products provide not only nutritional sources, but also alternative therapeutics for chronic diseases. Virtual screening provides an effective and high-throughput strategy for identifying protein structure-based interacting compounds extracted from a variety of dietary or herbal sources, which could be served as (pro)drugs for preventing or treating endocrine diseases. Here, we performed a virtual screening by directly targeting SEL1L of the most conserved SEL1L-HRD1 ERAD machinery. Further, we analyzed 58 top-ranked compounds and demonstrated that Cryptochlorogenic acid (CCA) showed strong affinity with the binding pocket of SEL1L with HRD1. Through structure-based docking, protein expression assays, and FACS analysis, we revealed that CCA enhanced ERAD activity and promoted the degradation of misfolded proAVP, thus facilitated the secretion of well-folded proAVP. These results provide us with insights into drug discovery strategies targeting ER protein homeostasis, as well as candidate compounds for treating hormone-related diseases.
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Degradação Associada com o Retículo Endoplasmático , Hormônios Peptídicos , Animais , Retículo Endoplasmático/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas/metabolismo , Hormônios Peptídicos/metabolismo , Mamíferos/metabolismoRESUMO
Diabetic retinopathy (DR) is one of the most prevalent microvascular complications caused by diabetes mellitus. Previous studies demonstrate that microvascular endothelial inflammation caused by chronic hyperglycemia and hyperlipidemia plays a key role in the pathogenesis of DR. However, the detailed mechanisms on how endothelial inflammation contributes to DR are not fully understood. The STING pathway is an important innate immune signaling pathway. Although STING has been implicated in multiple autoimmune and metabolic diseases, it is not clear whether STING is involved in the pathogenesis of DR. Thus, re-analysis of the public single cell RNA sequencing (sc-RNAseq) data demonstrated that STING was highly expressed in mouse retinal vessels. Moreover, our results demonstrated that STING and p-TBK1 protein levels in retinal endothelial cells are significantly increased in mice fed with high fat diet compared with chow diet. In vitro, palmitic acid treatment on HRVECs induced mitochondrial DNA leakage into the cytosol, and augmented p-TBK1 protein and IFN-ß mRNA levels. As STING is localized to the ER, we analyzed the relation between STING activation and ER stress. In HRVECs, STING pathway was shown to be activated under chemical-induced ER stress, but attenuated when IRE1α was abolished by genetic deletion or pharmacological inhibition. Taken together, our findings revealed that STING signaling plays an important role in mediating lipotoxicity-induced endothelial inflammatory and injury, and IRE1α-XBP1 signaling potentiated STING signaling. Thus, targeting the IRE1α or STING pathways to alleviate endothelial inflammation provides candidate therapeutic target for treating DR as well as other microvascular complications.
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Retinopatia Diabética , Hiperlipidemias , Camundongos , Animais , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Células Endoteliais/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Hiperlipidemias/metabolismo , Retinopatia Diabética/genética , Inflamação/metabolismoRESUMO
Disturbed endoplasmic reticulum (ER) stress response driven by the excessive lipid accumulation in the liver is a characteristic feature in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Restoring metabolic homeostasis by targeting ER stress is a potentially therapeutic strategy for NAFLD. Here we aim to identify novel proteins or pathways involved in regulating ER stress response and therapeutic targets for alleviating NAFLD. Proteomic and transcriptomic analysis demonstrated that major urinary proteins (MUPs) were significantly reduced in the livers from NAFLD mouse models. Then we confirmed that MUP1, the major secreted form of MUPs, was reduced at mRNA and protein expression levels in hepatocytes both in vivo and in vitro under ER stress. We further illustrated that MUP1 protein levels in the urine were reduced in mice with NAFLD, which was reversed by GLP-1 receptor agonist treatment. To study the relationship between ER stress and MUP1 biology, our analysis demonstrated that MUP1 was misfolded and trapped in the ER under ER stress in vivo. Interestingly, we discovered that recombinant MUP1 treatment in hepatocytes increased calcium efflux from the ER, which resulted in transient ER stress response, including reduced protein synthesis. These responses facilitated the alleviation of chemical induced ER stress in hepatocytes, which was suggested as "pre-adaptive ER stress". Besides, recombinant MUP1 pretreatment also improved ER stress-induced insulin resistance in hepatocytes. Our findings revealed a novel and critical role of MUP1, and recombinant MUP1 or its potential derivates may serve as a promising therapeutic target for alleviating NAFLD.
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Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Estresse do Retículo Endoplasmático , Hepatócitos , Metabolismo dos Lipídeos , Fígado , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , ProteômicaRESUMO
The selection of suitable rice varieties is the key to achieve high and stable yields, and the correct identification of rice varieties is the prerequisite for seed selection. In this paper, with Kenjing No.5, No.6, and No.9 as the subjects, the effectiveness of near-infrared spectroscopy (NIRS) combined with soft independent modeling of class analogy (SIMCA) in the rapid identification of rice varieties was explored. The modeling sets of Kenjing No.5, No.6, and No.9 samples were respectively used to establish a SIMCA classification model based on principal component analysis (PCA). The accuracies of the model in classifying the rice samples in the modeling set were 100, 100, and 97.5%, respectively. Then, the established SIMCA model was used to identify the rice samples in the test set. According to the experimental findings, the SIMCA analytical method achieved 100% prediction accuracy for the Kenjing No.5, Kenjing No.6, and Hongyu 001-1 samples. For the Kenjing No.9 sample, the accuracy rate was 90% with a 10% sample of Kenjing No.9 misidentified as Kenjing No.6. Therefore, the analytical method of NIRS combined with SIMCA could effectively identify the rice varieties, providing a new approach for the correct selection of planting varieties.