Molecular and Cellular Biology最新文献

Cellular senescence is a complex biological response to sublethal damage. The RNA-binding protein HNRNPK was previously found to decrease prominently during senescence in human diploid fibroblasts. Here, analysis of the mechanisms leading to reduced HNRNPK abundance revealed that in cells undergoing senescence, HNRNPK mRNA levels declined transcriptionally and full-length HNRNPK protein was progressively lost, while the abundance of a truncated HNRNPK increased. The ensuing loss of full-length HNRNPK enhanced cell cycle arrest along with increased DNA damage. Analysis of the RNAs enriched after HNRNPK ribonucleoprotein immunoprecipitation (RIP) revealed a prominent target of HNRNPK, CDC20 mRNA, encoding a protein critical for progression through the G2/M phase of the cell division cycle. Silencing HNRNPK markedly decreased the levels of CDC20 mRNA via reduced transcription and stability of CDC20 mRNA, leading to lower CDC20 protein levels; conversely, overexpressing HNRNPK increased CDC20 production. Depletion of either HNRNPK or CDC20 impaired cell proliferation, with a concomitant reduction in the levels of CDK1, a key kinase for progression through G2/M. Given that overexpressing CDC20 in HNRNPK-silenced cells partly alleviated growth arrest, we propose that the reduction in HNRNPK levels in senescent cells contributed to inhibiting proliferation at least in part by suppressing CDC20 production.

RNA modifications are highly conserved across all domains of life, suggesting an early emergence and a fundamental role in cellular processes. The modification 3-(3-amino-3-carboxypropyl)uridine (acp³U) is found in tRNAs of eukaryotes and prokaryotes, and in the 16S rRNA of archaea. In eukaryotic rRNA, a complex modification containing the acp group, m¹acp³Ψ is present at the analogous position. Although this modification was first identified in tRNA in 1969, only recently have the enzymes responsible for the synthesis of this modification on tRNA been identified. Despite its deep evolutionary conservation, the biological role of acp³U on tRNAs remains elusive. In Escherichia coli, it may contribute to genomic stability, while in human cells, loss of both tRNA acp³U-modifying enzymes impairs cell growth, though the underlying mechanisms are not yet understood. The conservation and multifunctionality of acp³U highlight the broader challenges of elucidating the roles of tRNA modifications in cellular homeostasis.

Acute lung injury (ALI) is a major cause of death in bacterial sepsis due to endothelial inflammation and endothelial permeability defects. Mitochondrial dysfunction is recognized as a key mediator in the pathogenesis of sepsis-induced ALI. Sirtuin 3 (SIRT3) is a histone protein deacetylase involved in preservation of mitochondrial function, which has been demonstrated in our previous study. Here, we investigated the effects of SIRT3 deficiency on impaired mitophagy to promote lung endothelial cells (ECs) pyroptosis during sepsis-induced ALI. We found that 3-TYP aggravated sepsis-induced ALI with increased lung ECs pyroptosis and enhanced NLRP3 activation. Mitochondrial reactive oxygen species (mtROS) and extracellular mitochondrial DNA (mtDNA) released from damaged mitochondria could be exacerbated in SIRT3 deficiency, which further elicit NLRP3 inflammasome activation in lung ECs during sepsis-induced ALI. Furthermore, Knockdown of SIRT3 contributed to impaired mitophagy via downregulating Parkin, which resulted in mitochondrial dysfunction. Moreover, pharmacological inhibition NLRP3 or restoration of SIRT3 attenuates sepsis-induced ALI and sepsis severity in vivo. Taken together, our results demonstrated SIRT3 deficiency facilitated mtROS production and cytosolic release of mtDNA by impaired Parkin-dependent mitophagy, promoting to lung ECs pyroptosis through the NLRP3 inflammasome activation, which providing potential therapeutic targets for sepsis-induced ALI.

During mammalian development, production sites of the erythroid growth factor erythropoietin (EPO) shift from the neural tissues to the liver in embryos and to the kidneys in adults. Embryonic neural EPO-producing (NEP) cells, a subpopulation of neuroepithelial and neural crest cells, express the Epo gene between embryonic day (E) 8.5 and E11.5 to promote primitive erythropoiesis in mice. While Epo gene expression in the liver and kidneys is induced under hypoxic conditions through hypoxia-inducible transcription factors (HIFs), the Epo gene regulatory mechanisms in NEP cells remain to be elucidated. Here, we confirmed the presence of cells co-expressing EPO and HIFs in mouse neural tubes, where the hypoxic microenvironment activates HIFs. Chemical activation and inhibition of HIFs demonstrated the hypoxic regulation of EPO expression in human fetal neural progenitors and mouse embryonic neural tissues. In addition, we found that histone deacetylase inhibitors can reactivate EPO production in cell lines derived from NEP cells and human neuroblastoma, as well as in mouse primary neural crest cells, while rejuvenating these cells. Furthermore, the ability of the rejuvenated cells to produce EPO was maintained in hypoxia. Thus, EPO production is controlled by epigenetic mechanisms and hypoxia signaling in the immature state of hypoxic NEP cells.

Complex metabolic diseases due to overnutrition such as obesity, type 2 diabetes, and fatty liver disease are a major burden on the healthcare system worldwide. Current research primarily focuses on disease endpoints and trying to understand underlying mechanisms at relatively late stages of the diseases, when irreversible damage is already done. However, complex interactions between physiological systems during disease development create a problem regarding how to build cause-and-effect relationships. Therefore, it is essential to understand the early pathophysiological effects of overnutrition, which can help us understand the origin of the disease and to design better treatment strategies. Here, we focus on early metabolic events in response to high-fat diets (HFD) in rodents. Interestingly, insulin resistance, fatty liver, and obesity-promoting systemic inflammatory responses are evident within a week when mice are given consecutive HFD meals. However, as shown in human studies, these effects are usually not visible after a single meal. Overall, these results suggest that sustained HFD-intake within days can create a hyperlipidemic environment, globally remodeling metabolism in all affected organs and resembling some of the important disease features.

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) constitute members of the dual-specificity family of protein phosphatases that dephosphorylate the MAPKs. MKP-5 dephosphorylates the stress-responsive MAPKs, p38 MAPK and JNK, and has been shown to promote tissue fibrosis. Here, we provide insight into how MKP-5 regulates the transforming growth factor-β (TGF-β) pathway, a well-established driver of fibrosis. We show that MKP-5-deficient fibroblasts in response to TGF-β are impaired in SMAD2 phosphorylation at canonical and non-canonical sites, nuclear translocation, and transcriptional activation of fibrogenic genes. Consistent with this, pharmacological inhibition of MKP-5 is sufficient to block TGF-β signaling, and that this regulation occurs through a JNK-dependent pathway. By utilizing RNA sequencing and transcriptomic analysis, we identify TGF-β signaling activators regulated by MKP-5 in a JNK-dependent manner, providing mechanistic insight into how MKP-5 promotes TGF-β signaling. This study elucidates a novel mechanism whereby MKP-5-mediated JNK inactivation is required for TGF-β signaling and provides insight into the role of MKP-5 in tissue fibrosis.

Med15 is a general transcriptional regulator and tail module subunit within the RNA Pol II mediator complex. The Saccharomyces cerevisiae Med15 protein has a well-structured N-terminal KIX domain, three activator binding domains (ABDs) and several naturally variable polyglutamine (poly-Q) tracts (Q1, Q2, Q3) embedded in an intrinsically disordered central region, and a C-terminal mediator association domain (MAD). We investigated how the presence of ABDs and changes in length and composition of poly-Q tracts influences Med15 activity using phenotypic, gene expression, transcription factor interaction and phase separation assays of truncation, deletion, and synthetic alleles. We found that individual Med15 activities were influenced by the number of activator binding domains (ABDs) and adjacent polyglutamine tract composition. Robust Med15 activity required at least the Q1 tract and the length of that tract modulated activity in a context-dependent manner. Reduced Msn2-dependent transcriptional activation due to Med15 Q1 tract variation correlated with reduced Msn2:Med15 interaction strength, but interaction strength did not always mirror phase separation propensity. We also observed that distant glutamine tracts and Med15 phosphorylation affected the activities of the KIX domain, and interaction studies revealed that intramolecular interactions may affect some Med15-transcription factor interactions.

In esophageal squamous cell carcinoma, genetic activation of NRF2 increases resistance to chemotherapy and radiotherapy, which results in a significantly worse prognosis for patients. Therefore NRF2-activated cancers create an urgent clinical need to identify new therapeutic options. In this context, we previously identified the geldanamycin family of HSP90 inhibitors, which includes 17DMAG, to be synthetic lethal with NRF2 activity. As the first-generation of geldanamycin-derivative drugs were withdrawn from clinical trials due to hepatotoxicity, we designed second-generation compounds with C19-substituted structures in order to inhibit glutathione conjugation-mediated hepatotoxicity. In this study, using a variety of in vitro and in vivo cancer models, we found that C19-substituted 17DMAG compounds maintain their enhanced toxicity profile and synthetic lethal interaction with NRF2-NQO1-activated cancer cells. Importantly, using a xenograft mouse tumor model, we found that C19-substituted 17DMAG displayed significant anticancer efficacy against NRF2-NQO1-activated cancer cells without causing hepatotoxicity. These results clearly demonstrate the improved clinical potential for this new class of HSP90 inhibitor anticancer drugs, and suggest that patients with NRF2-NQO1-activated esophageal carcinoma may benefit from this novel therapeutic approach.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG triplet repeat expansion within the 3' untranslated region of the DMPK gene. Expression of the expanded allele generates RNA containing long tracts of CUG repeats (CUGexp RNA) that form hairpin structures and accumulate in nuclear RNA foci; however, the factors that control DMPK expression and the formation of CUGexp RNA foci remain largely unknown. We performed an unbiased small molecule screen in an immortalized human DM1 skeletal muscle myoblast cell line and identified HSP90 as a modifier of endogenous RNA foci. Small molecule inhibition of HSP90 leads to enhancement of RNA foci and upregulation of DMPK mRNA levels. Knockdown and overexpression of HSP90 in undifferentiated DM1 myoblasts validated the impact of HSP90 with upregulation and downregulation of DMPK mRNA, respectively. Furthermore, we identified p-STAT3 as a downstream mediator of HSP90 impacting levels of DMPK mRNA and RNA foci. Interestingly, differentiated cells exhibited an opposite effect of HSP90 inhibition displaying downregulation of DMPK mRNA through a mechanism independent of p-STAT3 involvement. This study has revealed a novel mediator for DMPK mRNA and foci regulation in DM1 cells with the potential to identify targets for future therapeutic intervention.