FEBS Open Bio最新文献

The suppression of the long noncoding RNA (lncRNA) TRIBAL in hepatocytes was recently shown to affect the expression of pivotal regulators and hundreds of poorly understood or uncharacterized transcripts. The most upregulated transcript corresponded to a predicted splice variant of the lncRNA ZFAS1. Here, we characterize and investigate the role and regulation of ZFAS1 splice variants in liver cell models. New ZFAS1 splice variants were identified, all of which were enriched in the cytoplasm of HepG2 cells. TRIBAL suppression strongly upregulated a low-abundance ZFAS1 variant in hepatocytes but not in hepatoma models. However, preventing the upregulation of the ZFAS1 splice variant did not mitigate the impact of TRIBAL suppression in hepatocytes. ZFAS1 variants were rapidly but differentially increased in response to thapsigargin, which causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Inhibition of PERK, a central sensor of the UPR, had contrasting impacts on ZFAS1 variants in response to thapsigargin. Moreover, whereas the upregulation of the main ZFAS1 form was reduced by the suppression of the UPR mediators ATF4 and NFE2L2 (also known as NRF2), the other variants were not. Lastly, ZFAS1 suppression decreased cell viability both at baseline and in response to acute thapsigargin treatment. This work identifies novel ZFAS1 variants and uncovers a link between ER stress and ZFAS1 through the UPR.

Diabetic macular oedema (DMO) is a sight-threatening complication of diabetes. Current research suggests methylglyoxal (MGO), an advanced glycation end product (AGE) and reactive oxygen species (ROS) precursor produced in states of chronic hyperglycaemia, may contribute to retinal damage in DMO. Dapagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, has shown antioxidant and anti-inflammatory properties in human brain neuronal cells. However, its protective effects in retinal cells remain unclear. This study investigates the potential protective role of current antidiabetics against MGO-induced cytotoxicity in human retinal pigment epithelial cells (ARPE-19), focussing on the NLRP3 and caspase-1 pathway. ARPE-19 cells were studied through four conditions: Control (untreated), MGO (1 nm and 1 mm), cotreatment of MGO (1 mm) with dapagliflozin (10 μm) to investigate cytotoxicity and cell viability through lactate dehydrogenase (LDH) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assays, respectively. Cells were further investigated using confocal microscopy to assess the presence and activation of NLRP3 and caspase-1 enzyme. MGO (1 mm) caused significant cytotoxicity by approximately 60%, which was reduced to 33% by dapagliflozin (10 μm), providing a significant level of protection to cells against MGO-induced cytotoxicity, in addition to a significant increase in cell viability from 60% to 83%, and reduction in NLRP3-independent caspase-1 activation and/or expression, associated with increased nuclear staining intensity reflecting potential nuclear condensation and pyroptosis. This study suggests dapagliflozin protects ARPE-19 cells from MGO-induced oxidative stress and inflammasome through reducing caspase-1 activation, underscoring its potential as a therapeutic approach for retinal inflammation and vascular dysfunction in DMO, which requires further clinical investigations.

FEBS+ meeting Exploring molecular frontiers, held in Pula, Croatia, in autumn 2024 was jointly organized by the Croatian, Finnish, and Swedish Member Societies of the Federation of European Biochemical Societies (FEBS). The congress covered a wide variety of different biochemical themes, some of the highlights of which are presented in this ‘In the Limelight’ issue of FEBS Open Bio. We hope that, in addition to being scientifically useful and interesting, these articles will give a glimpse of the excellent science presented in the FEBS3+ meeting Exploring molecular frontiers and encourage attendance to future FEBS3+ meetings.

Cellular RNAs are not linear single-stranded stretches of nucleic acids as depicted in textbook cartoons but fold into secondary and tertiary structures through intra- and intermolecular base-pairing. They also interact with proteins to form ribonucleoproteins (RNPs), the functional units of RNA in cells. Recent methodological developments utilising high-throughput sequencing have enabled the detailed mapping of cellular RNA-protein interactions and RNA structures. While methods for the direct determination of cellular RNP structures are still lacking, the integration of high-throughput approaches and advancements with in vitro techniques such as cryogenic electron microscopy have provided insights into the functional significance of RNP structures. In this review, we will summarise the key methods used to probe cellular RNA-protein interactions and RNA structures and then provide examples of how these approaches have led to an enhanced understanding of RNP structures in gene regulation and how this has also opened new avenues for drug development.

Primary fibroblasts are widely used in a variety of experimental and therapeutic studies. Patient-derived skin biopsies are an accessible way to generate dermal fibroblasts for wound and burn therapeutics and can be easily reprogrammed to induced pluripotent stem cells (iPSCs). Despite the increasing use and interest in skin biopsies, there is limited information regarding the culturing potential of a single biopsy and the effects of extended culture on fibroblast formation and reprogramming potential. To better understand the potential of long-term skin biopsy culture, we cultured biopsy samples for 6-16 months, resulting in 6-16 generations of explant reculturing and then analyzed subsequent generations of fibroblasts. Our results showed that fibroblast morphology and physiology are maintained over time, but although older generations remained proliferative, they did so at a decreased rate. Gene expression analyses uncovered transcriptional changes with long-term skin culture, but deep DNA sequencing did not reveal any large deletions or amplifications. Spontaneous DNA mutations in fibroblast generations appeared to be random and not enriched for any specific signaling pathways. Importantly, fibroblasts generated after 16 months and over 16 generations in explant culture retained competency for reprogramming into induced pluripotent stem cells. Taken together, our results support long-term culture of skin biopsies to generate large numbers of primary fibroblasts. These cells maintain their identity and integrity, enabling the study of fibroblast maintenance as well as rare human disorders.

Patatin-like phospholipase (PNPLA) domain-containing proteins are essential enzymes involved in lipid metabolism, membrane remodeling, and signaling pathways across various organisms. This review focuses on the structural and functional characteristics of four selected PNPLA proteins from different organisms with experimental 3D structures or biochemical data on protein–protein interactions that facilitate their co-activation mechanism, namely VipD, ExoU, PNPLA9, and PNPLA2 (also known as ATGL). VipD and ExoU, phospholipases from Legionella pneumophila and Pseudomonas aeruginosa, respectively, are multidomain proteins and utilize distinct mechanisms for host cell interaction and pathogenesis. VipD binds to Rab proteins, underscoring the critical role of Rab5 in upregulating its enzymatic activity and contributing to the pathogenicity. ExoU requires ubiquitin for activation and exhibits an inhibited structure when complexed with its chaperone SpcU. PNPLA9, a calcium-independent phospholipase A2, is predominantly expressed in the human brain, with mutations linked to neurodegenerative disorders and inflammation. The crystal structure of the Chinese hamster ortholog of PNPLA9 reveals a dimerization mechanism required for its catalytic activity, along with specific regions identified for membrane interaction and substrate binding. PNPLA2 is known for its triacylglycerol hydrolytic activity and is regulated by protein–protein interactions, particularly with the co-activator ABHD5, which is crucial for its activation. This review highlights the diversity and conserved architectural segments of PNPLA proteins, reflecting their varied biological roles and regulatory mechanisms. Understanding the diverse protein–protein interactions that activate these enzymes is crucial for elucidating their roles in physiological and pathological contexts.

The malaria parasite P. falciparum exports a large number of proteins to the human host cell, including members of the J-domain protein (JDP) family. In other systems, JDPs stimulate the ATPase activity of HSP70 and direct client protein interactions. Three exported PfJDP are highly homologous yet appear to have divergent roles. We examined the ability of isolated J-domains from these proteins to stimulate the ATPase activity of PfHSP70-X and human HsHSP70 and HsHSC70. In vitro assays demonstrate that all domains stimulate the ATPase activity of each HSP70 tested. While overall stimulation was broadly comparable, differences were observed between individual PfJDP-HSP70 pairings. Our findings support a model in which the parasite systematically exports JDPs to exploit the host's chaperone power.

Obesity-related liver disease remains a critical global health challenge, underscoring the need to elucidate the molecular mechanisms underlying hepatic inflammation and metabolic dysfunction, and to identify novel therapeutic targets. This study aimed to determine whether erythropoietin (EPO) modulates soluble epoxide hydrolase (sEH) and lipid mediator pathways to ameliorate hepatic inflammation and metabolic dysfunction in high-fat diet (HFD)-induced obese mice. Male C57BL/6 mice were fed HFD with or without EPO treatment, and metabolic phenotyping, including glucose tolerance testing and homeostasis model assessment of insulin resistance, was performed. Hepatic histology and quantitative real-time polymerase chain reaction were conducted, together with flow cytometry to assess macrophage polarization, western blotting for sEH, and targeted liquid chromatography-tandem mass spectrometry profiling of cytochrome P450 epoxygenase-derived epoxides. EPO treatment improved glucose metabolism, reduced hepatic steatosis, lowered Ccr2, Mcp1, and Tnfα expression, promoted a shift of hepatic macrophages toward an M2 (anti-inflammatory) phenotype, downregulated hepatic sEH protein levels, and increased both hepatic and plasma concentrations of epoxygenase-derived epoxides. These findings indicate that EPO suppresses hepatic sEH and favors pro-resolving lipid mediator signaling, suggesting a potential therapeutic avenue for obesity-related hepatic inflammation.

Ubiquitination serves as a key regulatory mechanism in nearly all cellular processes, though it was originally identified as a post-translational modification that targets proteins for degradation. Accordingly, these two molecular events have since been tightly linked, and changes in protein abundance are frequently interpreted as indirect indicators of ubiquitination dynamics. Nevertheless, the relationship between protein abundance and ubiquitination has not been systematically examined across distinct biological systems. Here, we conducted a comprehensive meta-analysis to assess the correlation between protein abundance and ubiquitination levels determined by mass spectrometry in mammals, plants, and yeast. Quantitative proteomics and diGly-ubiquitin peptides data from 19 independent studies encompassing over 50 experimental conditions were analyzed. The findings indicate that alterations in protein abundance cannot reliably be used to infer ubiquitination events - and vice versa - highlighting the need for caution when interpreting proteomic data as a proxy for ubiquitin-mediated regulation.

Retraction: J. Cai, J. Xia, J. Zou, Q. Wang, Q. Ma, R. Sun, H. Liao, L. Xu, D. Wang, and X. Guo, "The PI3K/mTOR Dual Inhibitor NVP-BEZ235 Stimulates Mutant p53 Degradation to Exert Anti-Tumor Effects on Triple-Negative Breast Cancer Cells," FEBS Open Bio 10, no. 4 (2020): 535-545, https://doi.org/10.1002/2211-5463.12806. The above article, published online on 06 February 2020 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Miguel De la Rosa; the Federation of European Biochemical Societies; and John Wiley & Sons Ltd. The retraction has been agreed upon following an investigation into concerns raised by a third party. The investigation found multiple duplications of western blot bands among Figures 2A, 2B, 3A, 4A, 4B, 4C, 5A, and 5C. An overlap was also detected between the MDA-MB-468 0h CON image and the MDA-MB-468 0 h BEZ235 image shown in Figure 1B. The authors did not respond to address the concerns raised. Given the extent of the identified issues, the editors have lost confidence in the data presented and consider the conclusions of this manuscript substantially compromised. As a result, the Editor-in-Chief, FEBS Press, and John Wiley and Sons Ltd. have determined that a retraction is necessary. The authors were informed of the decision to retract but did not respond to requests for comment.