FEBS Open Bio最新文献

{"title":"Homologous expression and purification of human HAX-1 for structural studies.","authors":"Mariana Grieben","doi":"10.1002/2211-5463.70131","DOIUrl":"https://doi.org/10.1002/2211-5463.70131","url":null,"abstract":"<p><p>The human HAX-1 protein is a ubiquitously expressed multifunctional protein that regulates various cellular processes through interactions with numerous cellular proteins and RNA. The purification protocol presented here is designed explicitly for HAX-1 expressed in mammalian cells and yields high amounts of soluble HAX-1 protein constructs N-terminally fused to a cleavable superfolder GFP. This protocol will enable structural studies of HAX-1, which is predicted to undergo posttranslational modifications and be partially disordered in the absence of a binding partner.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137072","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A general model for analysis of linear and hyperbolic enzyme inhibition mechanisms.","authors":"Rafael S Chagas, Sandro R Marana","doi":"10.1002/2211-5463.70128","DOIUrl":"https://doi.org/10.1002/2211-5463.70128","url":null,"abstract":"<p><p>The mechanisms of reversible inhibitors with a single binding site on enzymes are usually divided into two basic groups: linear and hyperbolic (or partial). Each of these two groups is subdivided into three types: competitive, non-competitive and mixed. These six mechanisms are often considered separate identities. Here, prompted by the characterization of the inhibition of the wild-type and mutant β-glucosidase Sfβgly by imidazole and 2-amino-2-(hydroxymethyl)-1,3-propanediol (i.e. Tris), we developed a unifying enzyme kinetic model that integrates these six basic inhibition mechanisms into one. From this model, we deduced a general enzyme kinetic equation that, through modulation of simple parameters (i.e. the relative inhibitor affinity for two binding sites and the reactivity of the enzyme-substrate-inhibitor complex) is converted into the particular kinetic equation of each of those six inhibition mechanisms. In short, we conclude that the six fundamental inhibition mechanisms, linear and hyperbolic, are not separate behaviors but facets of the same general kinetic model presented here.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145130377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lactoferrin treatment activates acetylcholinesterase, decreasing acetylcholine levels in non-small cell lung cancer (NSCLC) cell culture supernatants, inhibiting cell survival.","authors":"Stuti Goel, Caroline Wozniak, Aya Sabri, Ben Haddad, Brooke Lopo, Alvaro Cobos, Sarah Sarofim, Jeffrey Guthrie, Deborah Heyl, Hedeel Guy Evans","doi":"10.1002/2211-5463.70125","DOIUrl":"10.1002/2211-5463.70125","url":null,"abstract":"<p><p>Lactoferrin (Lf) is a multifunctional glycoprotein of the transferrin family which has shown to efficiently block cell migration and/or invasion in a wide range of cancer cell models. The objective of this study was to further understand how Lf targets cancer cells by examining the effect of acetylcholine (ACh) levels on Lf signaling using A549 (p53 wild-type) and H1299 (p53-null) nonsmall cell lung cancer (NSCLC) cell lines. Treatment with Lf reduced cell viability more effectively in A549 cells than in H1299 cells. The half maximal inhibitory concentration (IC₅₀) of Lf for A549 and H1299 was 8.97 ± 1.4 and 35.03 ± 4.2 mg·mL^-1, respectively. To uncover the potential molecular mechanism involved in the decreased cell viability observed in A549 cell following Lf treatment, the activity of tumor suppressor (p53), acetylcholinesterase (AChE), and ACh levels were measured. Treatment of A549 cells with Lf led to ~ 1.50-fold activation of p53, ~ 1.60-fold activation of AChE, and ~ 1.80-fold decrease in ACh levels. Vascular endothelial growth factor (VEGF) levels also decreased in cell culture supernatants upon treatment with Lf in both cell lines, and in A549 cells, the decrease occurred in a manner dependent on p53 and AChE. Given previous reports on the role of Lf in apoptosis induction, we examined AKT activity following Lf treatment and showed that AKT activity decreased ~ 1.95-fold in A549 cells and ~ 1.50-fold in H1299 cells. Furthermore, Lf-induced activation of caspase-3 was diminished by A549 cell cotreatment with siRNA targeted against p53 and/or AChE and increased by inhibiting the function of VEGF and/or AKT in both cell lines. In conclusion, this study identifies a mechanism wherein ACh concentrations in the cell culture supernatant attenuate the impact of Lf on NSCLC cell viability. These findings provide preliminary insight into the complex actions of Lf and suggest that the Lf-AChE-ACh pathway may warrant further study as a potential target in NSCLC.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124611","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tryptophan metabolite atlas uncovers organ, age, and sex-specific variations.","authors":"Lizbeth Perez-Castro, Afshan F Nawas, Jessica A Kilgore, Pedro A S Nogueira, M Carmen Lafita-Navarro, Paul H Acosta, Roy Garcia, Noelle S Williams, Maralice Conacci-Sorrell","doi":"10.1002/2211-5463.70123","DOIUrl":"10.1002/2211-5463.70123","url":null,"abstract":"<p><p>Tryptophan (Trp) is the largest and most structurally complex amino acid, yet it is the least abundant in the proteome. Its distinct indole ring and high carbon content allow it to give rise to several biologically active metabolites, including serotonin, kynurenine (Kyn), and indole-3-pyruvate (I3P). Dysregulation of Trp metabolism has been implicated in a range of diseases, from depression to cancer. Investigating Trp and its metabolites in healthy tissues provides insight into how disease-associated disruptions may be targeted selectively while preserving essential physiological functions. Whereas previous studies have typically focused on individual organs or single metabolic branches, our analysis spans 12 peripheral organs, the central nervous system, and serum in male and female (C57BL/6) mice across three life stages: young (3 weeks), adult (54 weeks), and aged (74 weeks). We identified striking tissue-, sex-, and age-specific differences in Trp metabolism, including elevated levels of I3P and Kyn, both linked to tumor growth, in aging males. We also compared Trp metabolite profiles in tissues from mice fed a control defined diet versus a Trp-deficient diet for three weeks. This intervention led to a marked reduction in circulating Trp and its metabolites, with more modest effects observed in the liver and central nervous system. These findings underscore the importance of organ-specific and diet-sensitive analyses of Trp metabolism for understanding its role in both normal physiology and disease. Establishing baseline levels of Trp metabolites across tissues may also provide a foundation for identifying organ-specific metabolic reprogramming in cancer and other illnesses.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145085574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phosphomimetic mutations near active sites of proteins in Thermus thermophilus suggest a widespread regulatory mechanism.","authors":"Anzu Nishiwaki, Hiroki Okanishi, Yoshikatsu Kanai, Ryoji Masui","doi":"10.1002/2211-5463.70103","DOIUrl":"https://doi.org/10.1002/2211-5463.70103","url":null,"abstract":"<p><p>In Thermus thermophilus, an aerobic Gram-negative eubacterium used as a model organism, more than half of the phosphorylation sites identified by proteomic analysis are located near the ligand-binding site, including the active site, of the enzyme in the three-dimensional structure. We investigated the effect of these phosphorylation events on the activity of six enzymes (three nucleoside monophosphate kinases, isocitrate kinase, malate dehydrogenase and inorganic pyrophosphatase) by introducing phosphomimetic mutations, Glu, into the phosphorylation sites. All phosphomimetic mutants showed severely reduced activity compared with the wild-type, particularly in the turnover number. The proteins analyzed in this study belong to different families and have various functions. This suggests that there is a widespread mechanism by which phosphorylation of amino acid residues near the active site reduces enzyme activity independent of the protein family and function.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145063813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The impact of Hnrnpl deficiency on transcriptional patterns of developing muscle cells.","authors":"Hannah R Littel, Mekala Gunasekaran, Audrey L Daugherty, Natalya M Wells, Johnnie Turner, Christine C Bruels, Christina A Pacak, Isabelle Draper, Peter B Kang","doi":"10.1002/2211-5463.70117","DOIUrl":"https://doi.org/10.1002/2211-5463.70117","url":null,"abstract":"<p><p>Heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to RNA, regulating gene expression and splicing. HnRNP L contributes to muscle development and the pathogenesis of myotonic dystrophy. We hypothesized that hnRNP L regulates muscle expression and splicing patterns. Using nanopore long-read transcriptome sequencing and qPCR analyses, we investigated the impact of Hnrnpl knockdown on myoblasts and knockdown of the orthologous gene smooth in Drosophila. Notch signaling genes and muscle-related genes were dysregulated in both models. Several genes had altered splicing patterns, including Lamp2, Fhl1, and Dtna. The α-DB1 isoform of Dtna was downregulated, whereas the α-DB3 isoform was upregulated. Our findings indicate that hnRNP L regulates both the transcription levels and splicing patterns of genes relevant to skeletal muscle development. We demonstrate the capabilities of long-read transcriptome sequencing to study muscle development. Comparisons between nanopore long-read transcriptome sequencing and data from PCR and qPCR analyses suggest that a minimum read depth of 10 is needed on nanopore sequencing to detect splicing differences greater than 10% to 20%. Future studies could determine whether the minimum read depth that we identified in our model is valid across a broader range of genes, cell types, and conditions. There are also intriguing hints of therapeutic implications of hnRNP L regulation for muscle diseases that merit further investigation.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145052600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interplay between RNA-protein interactions and RNA structures in gene regulation.","authors":"Jenni Rapakko, Mauro Scaravilli, Minna-Liisa Änkö","doi":"10.1002/2211-5463.70122","DOIUrl":"https://doi.org/10.1002/2211-5463.70122","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145039514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural and functional analysis of Bacillus sarcosine oxidase and its activity toward cyclic imino acids.","authors":"Yuqi Zhang, Yoshitaka Nakajima, Masae Kurobe, Tsutomu Nakamura, Tomoki Himiyama, Yoshiaki Nishiya","doi":"10.1002/2211-5463.70119","DOIUrl":"https://doi.org/10.1002/2211-5463.70119","url":null,"abstract":"<p><p>This study investigated the reactivity of sarcosine oxidase (Sox) toward minor substrates through kinetic and structural analyses, along with mutational engineering to elucidate their reaction mechanisms. Sarcosine oxidase from Bacillus sp. (SoxB) recognizes the cyclic imino acids l-proline (l-Pro), d-proline (d-Pro), and l-thioproline (l-Tpr) as minor substrates. The reaction behavior varied depending on the substrates; notably, the absorption spectrum of l-Tpr exhibited charge transfer, which was characteristic of substrate inhibition. Crystal structures of the enzyme-substrate complexes suggested that Tyr254 causes spatial interference with cyclic imino acids at the active site. The Tyr254Ala and Tyr254Gly mutants exhibited enhanced reactivity toward cyclic imino acids by eliminating this spatial interference. Crystallographic analysis of the mutants revealed an enlarged active site, which facilitated reactions with five-membered cyclic imino acids. These mutations disrupted the electron delocalization associated with l-Tpr, thereby eliminating charge transfer and substrate inhibition. A water network was also identified near the enzyme's active site, interacting with the side chain of Tyr254. These findings provide valuable insights into substrate specificity and may facilitate the development of enzymes with broader substrate scope and enhanced catalytic activity.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145033174","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Berberine-cinnamic acid co-crystal effect in ameliorating hyperlipidemia might be regulated through the PI3K/AKT/mTOR/SREBP-1 signaling pathway.","authors":"Wenheng Gao, Yunlong Li, Lihua Chen, Wenshuo Yang, Yong He, Ye Yang, Dengke Yin, Song Tan","doi":"10.1002/2211-5463.70115","DOIUrl":"https://doi.org/10.1002/2211-5463.70115","url":null,"abstract":"<p><p>Hyperlipidemia is a common chronic disease characterized by elevated levels of lipids in the blood. There is some evidence that suggests that berberine (BBR) might be beneficial for the treatment of hyperlipidemia. However, its low intestinal bioavailability limits its potential therapeutic action. In the present study, we explored the effect and the underlying mechanism of berberine-cinnamic acid co-crystal (BBR-CA), which is self-assembled from CA and BBR and displays a high intestinal bioavailability. In mice, BBR-CA showed the ability to decrease body weight gain and hepatic lipid accumulation in animals fed a high-fat diet. To further characterize the molecular basis of this effect, we established a hyperlipidemia cell model by treating human hepatocellular carcinoma cells (HepG2) with free fatty acids. Similarly to our in vivo experiments, lipid accumulation in free fatty acids-induced HepG2 cells was also reduced by BBR-CA. We hypothesized that BBR-CA might act through the regulation of sterol regulatory element-binding proteins-1 (SREBP-1), a key factor regulating lipid synthesis, and, indeed, SREBP-1 protein expression was inhibited by BBR-CA treatment, resulting in the decreased expression of its downstream proteins stearoyl-CoA desaturase 1 and acetyl-CoA carboxylase. Furthermore, the phosphorylation of phosphatidylinositol 3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) was inhibited by BBR-CA, contributing to decreased active SREBP-1 in the nucleus, and was reversed and enhanced by the PI3K agonist recilisib and inhibitor LY294002, respectively. Taken together, our results suggest that BBR-CA could function by modulating the PI3K/AKT/mTOR signaling pathway, resulting in decreased nuclear expression of SREBP-1, as well as reduced expression of stearoyl-CoA desaturase 1 and acetyl-CoA carboxylase, thus alleviating hyperlipidemia. Further experimental validation is required to confirm these results.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145029208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of amino acids essential for angulin-1/3 binding of the tricellular tight junction binder, angubindin-1.","authors":"Taiki Kuzu, Yumi Iwashita, Keisuke Tachibana, Itsuki Nishino, Yuki Niwa, Atsuko Uyeda, Kazuki Matsuo, Masahiro Nagahama, Masuo Kondoh","doi":"10.1002/2211-5463.70113","DOIUrl":"https://doi.org/10.1002/2211-5463.70113","url":null,"abstract":"<p><p>Tight junctions (TJs) are formed where two or three cells meet and are therefore categorized, respectively, into bicellular TJs (bTJs) and tricellular TJs (tTJs). Angubindin-1 is the first tTJ modulator enhancing intestinal macromolecule permeation via binding to the key tTJ proteins, angulin-1 and angulin-3. It is a fragment (amino acids 421-664) derived from domain IV of Clostridium perfringens iota toxin. Here, we identified critical residues (L562, L598, E638, V640, Y643, K644) of angubindin-1 to be essential for binding to angulins by alanine scanning. Mutants substituting these amino acids with alanine exhibited reduced binding to angulin-expressing cells. Simultaneous substitution of all these amino acids lost binding to angulins and resulted in the loss of tTJ-modulating functions of angubindin-1. These insights highlight crucial residues for the tTJ-modulating activity of angubindin-1, which may hold promise in the design of noninvasive, targeted therapeutics using angubindin-1 as a prototype tTJ modulator to enhance the permeation of drugs.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145029233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}