The FEBS journal最新文献

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) progressing to metabolic dysfunction-associated steatohepatitis (MASH), characterized by hepatic inflammation, has significantly increased in recent years due to unhealthy dietary practices and sedentary lifestyles. Cathepsin D (CTSD), a lysosomal protease involved in lipid homeostasis, is linked to abnormal lipid metabolism and inflammation in MASH. Although primarily intracellular, CTSD can be secreted extracellularly. Our previous proteomics research has shown that inhibition of extracellular CTSD results in more anti-inflammatory effects and fewer potential side effects compared to intracellular CTSD inhibition. However, the correlation between reduced side effects and alterations in the hepatic lipid composition remains unknown. This study aims to investigate the correlation between intra- and extracellular CTSD inhibition and potential alterations in the hepatic lipid composition in MASH. Low-density lipoprotein receptor knockout (Ldlr^-/-) mice were fed a high-fat diet for 10 weeks and received subcutaneous injections every 2 days of vehicle, intracellular CTSD inhibitor (GA-12), or extracellular CTSD inhibitor (CTD-002). Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize and compare the lipid composition in liver tissues. Hepatic phosphatidylcholine remodeling was observed with both inhibitors, suggesting their therapeutic potential in treating MASH. Treatment with an intracellular CTSD inhibitor resulted in elevated levels of cardiolipin, reactive oxygen species, phosphatidylinositol, phosphatidylethanolamine, and lipids that are linked to mitochondrial dysfunction and inflammation, and induced more oxidative stress. The observed modifications in lipid composition demonstrate the clinical advantages of extracellular CTSD inhibition as a potentially beneficial therapeutic approach for MASH.

Here, we present a previously undescribed approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated that the nucleotides immediately following the start codon can significantly influence protein expression. However, the impact of these sequences is construct-specific and is not universally applicable to all proteins. Most of the previous research has been limited to selecting from a few rationally designed sequences. In contrast, we used a directed evolution-based methodology, screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increased expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) is required for diverse microglia responses in neurodegeneration, including immunometabolic plasticity, phagocytosis, and survival. We previously identified that patient iPSC-derived microglia (iPS-Mg) harboring the Alzheimer's disease (AD) TREM2^R47H hypomorph display several functional deficits linked to metabolism. To investigate whether these deficits are associated with disruptions in metabolite signaling, we generated common variant, TREM2^R47H and TREM2^-/- variant human iPS-Mg. We assessed the ability of supplementation with citrate or succinate, key metabolites and cell cycle breaking points upon microglia activation, to overcome these functional deficits with potential impact on neurons. Succinate supplementation was more effective than citrate at overcoming mitochondrial deficits in OXPHOS and did not promote a glycolytic switch. Citrate enhanced the lipid content of TREM2^R47H iPS-Mg and was more effective at overcoming Αβ phagocytic deficits, whereas succinate increased lipid content and phagocytic capacity in TREM2^-/- iPS-Mg. Microglia cytokine secretion upon pro-inflammatory activation was moderately affected by citrate or succinate showing a condition-dependent increasing trend. Neither metabolite altered basal levels of soluble TREM2 shedding. In addition, neither citrate nor succinate enhanced glycolysis; instead, drove their effects through oxidative phosphorylation. IPS-neurons exposed to conditioned medium from TREM2 variant iPS-Mg showed changes in oxidative phosphorylation, which could be ameliorated when iPS-Mg were first treated with citrate or succinate. Our data point to discrete pathway linkage between microglial metabolism and functional outcomes with implications for AD pathogenesis and treatments.

RNAs are increasingly recognized as promising therapeutic targets, susceptible to modulation by strategies that include targeting with small molecules, antisense oligonucleotides, deoxyribozymes (DNAzymes), or CRISPR/Cas13. However, while drug development for proteins follows well-established paths for rational design based on the accurate knowledge of their three-dimensional structure, RNA-targeting strategies are challenging since comprehensive RNA structures are yet scarce and challenging to acquire. Numerous methods have been developed to elucidate the secondary and three-dimensional structure of RNAs, including X-ray crystallography, cryo-electron microscopy, nuclear magnetic resonance, SHAPE, DMS, and bioinformatic methods, yet they have often revealed flexible transcripts and co-existing populations rather than single-defined structures. Thus, researchers aiming to target RNAs face a critical decision: whether to acquire the detailed structure of transcripts in advance or to adopt phenotypic screens or sequence-based approaches that are independent of the structure. Still, even in strategies that seem to rely only on the nucleotide sequence (like the design of antisense oligonucleotides), researchers may need information about the accessibility of the compounds to the folded RNA molecule. In this concise guide, we provide an overview for researchers interested in targeting RNAs: We start by revisiting current methodologies for defining secondary or three-dimensional RNA structure and then we explore RNA-targeting strategies that may or may not require an in-depth knowledge of RNA structure. We envision that complementary approaches may expedite the development of RNA-targeting molecules to combat disease.

Premature accumulation of senescent cells results in tissue destruction, and it is one of the potential primary mechanisms underlying the accelerated progression of diabetes and periodontitis. However, whether this characterized phenomenon could account for periodontal pathogenesis under hyperglycemic conditions remains unclear. In this study, we assessed the senescent phenotypic changes in experimental periodontitis under hyperglycemic conditions. Next, we investigated the mitochondrial function and the potential mitophagy pathways in cellular senescence in vitro and in vivo. Our findings showed that significant senescence occurred in the gingival tissues of diabetic periodontitis mice with increased expression of senescence-related protein p21^Cip1 and the senescence-associated secretory phenotype response as well as the decreased expression of NIP3-like protein X (NIX), a mitochondrial receptor. Likewise, we showed that mitochondrial dysfunction (e.g., reduction of mitochondrial membrane potential and accumulation of reactive oxygen species) was attributed to cellular senescence in: human periodontal ligament cells (hPDLCs) through hyperglycemia-induced and Porphyromonas gingivalis lipopolysaccharide (P.g-LPS)-induced oxidative stresses. Notably, the resulting reduced NIX expression was reversed by the use of the mitochondrial reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC), thus correcting the mitochondrial dysfunction. We further verified the expression of inflammatory mediators and senescence-related factors in mice gingival tissues and identified the possible regulatory pathways. Taken together, our work demonstrates the critical role of cellular senescence and mitochondrial dysfunction in periodontal pathogenesis under hyperglycemic conditions. Hence, restoration of mitochondrial function may be a potential novel therapeutic approach to tackling periodontitis in diabetic patients.

Photoreceptors control cellular processes in response to light. Most photoreceptors sense blue or red light, but the recent discovery of the cobalamin-dependent photoreceptor, CarH, has expanded the wavelength range of photoreception to other regions of the electromagnetic spectrum to include the green light region. Further identification of cobalamin-dependent green light-sensitive photoreceptors has been hampered owing to poor annotation of the light responsiveness of cobalamin-binding domains (CBDs) in public databases. Here we report a computational workflow, SignatureFinder, that uses a combination of sequence and structural analyses to identify new light-responsive CBD-containing proteins. The light response of exemplar proteins containing the proposed signature were confirmed experimentally. A structural analysis of these new photoreceptors, including the crystal structure of a new CBD domain, highlights how the signature elements interact with the cobalamin chromophore to sense light. Database mining of 128 000 CBD-containing sequences using the identified signature revealed more diverse CBD-containing photoreceptors, thereby expanding the family of green-light photoreceptors. A SignatureFinder web server is available (https://enzymeevolver.com) for wider applications, including the identification of signature sequences of other biological ligands of interest.

Hypoxia is a predominant risk factor at high altitudes, and evidence suggests that high-altitude hypoxia alters the gut microbiota, which plays an essential regulatory role in memory function. However, the causal relationship between the gut microbiota and memory impairment under hypoxic conditions remains unclear. In this study, we employed a high-altitude hypoxia model combined with fecal microbiota transplantation (FMT) approach in mice to explore the effects of the gut microbiota on memory impairment in a hypoxic environment. We observed that high-altitude hypoxia exposure reduced short- and long-term memory and hippocampus-dependent fear memory abilities, along with decreased relative abundance of Ligilactobacillus and Muribaculum. Moreover, hypoxic conditions increased intestinal and blood-brain barrier permeability. FMT from hypoxia-exposed mice into naïve antibiotic-treated mice resulted in similar memory impairments, Ligilactobacillus and Muribaculum abundance changes, and increased intestinal/blood-brain barrier permeability. Correlation analysis showed a robust positive association between Ligilactobacillus and Muribaculum with hippocampus-dependent contextual fear memory. Likewise, Ligilactobacillus was positively correlated with short-term memory. Therefore, Ligilactobacillus and Muribaculum may be key microbes in reducing memory ability in hypoxia, with the intestinal and blood-brain barriers as primary pathways. Our findings provide further evidence for the potential regulatory mechanism by which gut microbiota dysbiosis may contribute to memory impairment in a high-altitude environment.

Transglutaminase 2 (TG2) is a uniquely versatile protein with diverse catalytic activities, such as transglutaminase, protein disulfide isomerase, GTPase and protein kinase, and participates in several biological processes. According to information available in the RBP2GO database, TG2 can act as an RNA-binding protein (RBP). RBPs participate in posttranscriptional gene expression regulation, therefore influencing the function of RNA, whereas RNA molecules can also modulate the biological activity of RBPs. The present study aimed to confirm this novel characteristic of TG2 in human umbilical cord vein endothelial cells (HUVEC), which physiologically express TG2. First, UV cross-linked RNA-protein complexes were isolated from immortalised HUVECs using orthogonal organic phase separation. Compared with the RBP2GO database, mass spectrometry identified 392 potential RBPs, including TG2 and 20 previously undescribed, endothelium-related RBPs. Recombinant human TG2 was also pulled down by magnetic bead-immobilised total RNA from HUVEC. Complex formation between TG2 and a 43-mer RNA molecule with a secondary structure as well as a homo-oligomeric single-stranded poly(dG), but not poly(dA), could be observed in magnetic RNA-protein pull-down experiments. Experiments with TG2 inhibitors NC9 and GTPγS, which stabilise its open and closed conformation, respectively, revealed that the open conformation of the enzyme favoured RNA-binding. Biolayer interferometry revealed a high binding affinity between TG2 and RNA with a K_D value of 88 nm. Based on modelling and site-directed mutagenesis studies, we propose that superficial residues on the catalytic core domain (173-177 amino acids), present in a hidden position in the closed TG2 conformation, are involved in RNA binding. The present study demonstrates the previously uncharacterised RNA-binding ability of TG2, opening new avenues for understanding its multifunctionality.

Hypoxia is a critical microenvironmental factor that induces tumorigenesis and cancer progression, including metastasis. The highly dynamic nature of the extracellular matrix (ECM) plays a crucial role in metastasis. Collagens are the predominant component of structural proteins embedded within the ECM. The biosynthesis of collagen typically undergoes a series of posttranslational modifications, such as hydroxylation of lysine and proline residues by procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) and prolyl 4-hydroxylases (P4Hs), respectively. Collagen hydroxylation is critical for ECM remodeling and maintenance. We recently investigated hypoxia-induced translation in human colon cancer HCT116 cells and identified several collagen-modifying enzymes, including procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and prolyl 4-hydroxylase subunit alpha 1 (P4HA1). Although the translation of bulk mRNAs is repressed in hypoxia, specific mRNAs remain efficiently translated under such conditions. We have found that PLOD2 and P4HA1 are significantly upregulated in hypoxic HCT116 cells compared to normoxic cells. HIF-1 is known to induce the transcription of PLOD2 and P4HA1 during hypoxia. However, the molecular mechanisms of hypoxia-induced translation of PLOD2 and P4HA1 remain largely unclear. We provide evidence that RBM4 and eIF4E2 are required for hypoxia-induced translation of PLOD2 and P4HA1 mRNAs. The 3' UTRs of PLOD2 and P4HA1 mRNAs are involved in translational control during hypoxia in HCT116 cells.

Japanese encephalitis virus (JEV) is the leading causative agent of viral encephalitis in India and contributes to a significant disease burden in South Asian countries. However, no antiviral treatment is available against JEV-induced encephalitis, highlighting the urgent need for novel therapeutic approaches. Repurposing or repositioning drugs was found to be more economical and practical in the current drug development scenario. The present study aimed to develop a host-directed strategy through a computational drug repurposing approach. As part of the strategy, we first generated a dynamic signature of differentially expressed JEV infection-associated proteins in mice brains through a semiquantitative proteomics approach. With the help of the Connectivity Map (CMap) analysis, we narrowed down the lists of drugs with a high negative CMap score (-70 or lower). Based on the CMap score, we chose the top three compounds (Tipifarnib, Ly303511 and MDL11939) with CMap scores of -91.83, -88.18 and -91.15, respectively. The antiviral potential of these three compounds was further compared in both JEV-infected mouse neuroblastoma cells and C57BL/6 mice. Oral administration of Ly303511 and MDL11939, alone or in combination, showed improved outcomes (e.g. delayed death, increased survival, and less viral load than Tipifarnib alone or combined). The JEV-infected mice survived upon drug treatment, effectively reducing viral load and reversing the antiviral signature. Our results highlight Ly303511 and MDL11939 as promising host-targeted inhibitors of JEV infection and pathogenesis. Moreover, our results favor the combination of Ly303511 and MDL11939 therapy to improve clinical symptoms and reduce JEV-induced damage, thus warranting inclusion in clinical studies.