The FEBS journal最新文献

The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is widely used for the generation of a variety of gene products, including proteins, vaccines, and gene therapy vectors; however, it has some limitations, including a constrained host range and low protein yields. In a previous study, we established the RIRI-PA1 cell line, which was derived from Periplaneta americana. This cell line is susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection, which results in a higher yield production of recombinant protein within a short post-infection period of 24-48 h compared to the commonly used engineered cell line Sf21. To elucidate the basis for this phenomenon, we used RNA sequencing and transcriptome analysis of RIRI-PA1 and Sf21 cells infected with AcMNPV-GFP at 24, 72, and 168 h post-infection. Differentially expressed genes (DEGs) were identified in both cell lines. GO, eggNOG, and KEGG annotation analyses were used to identify DEGs and select candidate genes that could regulate recombinant protein expression. The results indicated a significant link between ribosomal pathway regulation and recombinant protein expression. After 24 h of AcMNPV-GFP infection, relatively high levels of protein were produced in RIRI-PA1 cells compared to Sf21 cells, which exhibited lesser enrichment of ribosomal protein-related DEGs (7 : 12). Moreover, a correlation was observed in the gene expression patterns between AcMNPV-GFP infection and recombinant protein synthesis, including genes associated with the ribosome, Toll and Imd signaling, and the cytochrome P450 pathway. Overall, our findings suggested that the ribosomal pathway might be more involved in regulation of protein expression during the early stages of RIRI-PA1 infection. The mechanisms underlying this process could have potential future applications in engineering cell modifications to reduce production time for recombinant proteins and to promote the use of IC-BEVS.

Discovered two decades ago, PIWI-interacting RNAs (piRNAs) are crucial for silencing transposable elements (TEs) in animal gonads, thereby protecting the germline genome from harmful transposition, and ensuring species continuity. Silencing of TEs is achieved through transcriptional and post-transcriptional suppression by piRNAs and the PIWI clade of Argonaute proteins within non-membrane structured organelle. These structures are composed of proteins involved in piRNA processing, including PIWIs and other proteins by distinct functional motifs such as the Tudor domain, LOTUS, and intrinsic disordered regions (IDRs). This review highlights recent advances in understanding the roles of these conserved proteins and structural motifs in piRNA biogenesis. We explore the molecular mechanisms of piRNA biogenesis, with a primary focus on Drosophila as a model organism, identifying common themes and species-specific variations. Additionally, we extend the discussion to the roles of these components in nongonadal tissues.

Critical limb ischemia (CLI) is the most advanced stage of peripheral arterial disease, posing a high risk of mortality. Sphingomyelin, a sphingolipid synthesized by sphingomyelin synthases (SMSs) 1 and 2, plays an essential role in signal transduction as a component of lipid rafts. However, the role of sphingomyelin in the inflammation of ischemic skeletal muscles remains unclear. In this study, we analyzed the roles of sphingomyelin and SMSs in CLI-induced myopathy using a mouse hindlimb ischemia model. We observed that hypoxia after CLI triggered an increase in SMS2 levels, thereby elevating sphingomyelin concentrations in ischemic skeletal muscles. The expression of SMS2 and sphingomyelin was induced by hypoxia in C2C12 myotubes and regulated by the prolyl hydroxylase domain enzyme. Additionally, SMS2 deficiency suppressed skeletal muscle inflammation after CLI, attenuated the phosphorylation of inhibitor of κBα (IκBα), and reduced the nuclear translocation of nuclear factor κB (NFκB) p65. Meanwhile, the administration of sphingomyelin hampered skeletal muscle inflammation by inhibiting IκBα phosphorylation and NFκB p65 nuclear translocation and extending inflammation post-CLI. Our results suggest that hypoxia-induced enhancement in SMS2 levels and the consequent increase in sphingomyelin expression levels promote inflammation in ischemic muscle tissues via the NFκB pathway and propose sphingomyelin as a potential therapeutic target in patients with CLI and other hypoxia-related inflammatory diseases.

Co-chaperones are key elements of cellular protein quality control. They cooperate with the major heat shock proteins Hsp70 and Hsp90 in folding proteins and preventing the toxic accumulation of misfolded proteins upon exposure to stress. Hsp90 interacts with the co-chaperone stress-inducible phosphoprotein 1 (Sti1/Stip1/Hop) and activator of Hsp90 ATPase protein 1 (Aha1) among many others. Sti1 and Aha1 control the ATPase activity of Hsp90, but Sti1 also facilitates the transfer of client proteins from Hsp70 to Hsp90, thus connecting these two major branches of protein quality control. We find that misbalanced expression of Sti1 and Aha1 in yeast and mammalian cells causes severe growth defects. Also, deletion of STI1 causes an accumulation of soluble misfolded ubiquitinated proteins and a strong activation of the heat shock response. We discover that, during proteostatic stress, Sti1 forms cytoplasmic inclusions in yeast and mammalian cells that overlap with misfolded proteins. Our work indicates a key role of Sti1 in proteostasis independent of its Hsp90 ATPase regulatory functions by sequestering misfolded proteins during stress.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) can transfer the methyl group from S-adenosyl-l-methionine (SAM) to one of the hydroxyl groups of a catechol substrate in the presence of Mg²⁺. However, there is no consensus view of the kinetic mechanism of COMT involving multiple substrates. Further progress requires the development of methods for determining enzyme kinetic behavior and the binding mode of ligands to the protein. Here, we establish a multisubstrate kinetic system covering the fluorescence and mass spectrometry techniques to quantify the products in a COMT-catalyzed reaction. The catechol substrate, 3-BTD, can be methylated by COMT to form a single product, 3-BTMD, with a sensitive fluorescence response and the conversion of SAM to S-adenosyl-l-homocysteine (SAH) was monitored by LC-MS/MS. The kinetic assays suggested that the reaction occurred via an ordered sequential mechanism, in which SAM first bound to COMT, followed by the addition of Mg²⁺ and 3-BTD. The chemical step involved a quaternary complex of COMT-SAM-Mg²⁺-3-BTD, followed by the ordered dissociation of 3-BTMD, Mg²⁺, and SAH. In cooperation with molecular dynamics simulation, the binding of COMT to Mg²⁺ induced a shape change in the catechol-binding site, which accommodated 3-BTD binding and facilitated catalysis. These findings provide new insights into the kinetic mechanism of COMT, contributing to the development of previously undescribed functional COMT ligands.

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.