FEBS Open BioPub Date : 2025-06-13DOI: 10.1002/2211-5463.70063
Satoshi Fudo, Marina Verkhovskaya, Coralie Di Scala, Claudio Rivera, Tommi Kajander
{"title":"Biophysical characterization and ion transport with cell-based and proteoliposome reconstitution assays of invertebrate K<sup>+</sup>-Cl<sup>-</sup> cotransporters.","authors":"Satoshi Fudo, Marina Verkhovskaya, Coralie Di Scala, Claudio Rivera, Tommi Kajander","doi":"10.1002/2211-5463.70063","DOIUrl":"https://doi.org/10.1002/2211-5463.70063","url":null,"abstract":"<p><p>The cation-chloride cotransporter (CCC) family includes ion symporters that cotransport monovalent cations and Cl<sup>-</sup>, playing a crucial role in controlling cytoplasmic ion content. K<sup>+</sup>-Cl<sup>-</sup> cotransporters (KCCs) facilitate the symport of ions across the plasma membrane. The CCCs participate in various physiological processes, such as transepithelial ion transport and regulation of cell volume. Among KCCs, KCC2 has unique and essential functions in the central nervous system. KCC from Drosophila melanogaster (DmKCC) is an ortholog of mammalian KCCs. Its critical role in neuronal transmission has been demonstrated. Also, the cnidarian Hydra vulgaris has a functional KCC (HvKCC). Comparative analyses of these transporters with vertebrate counterparts can provide insights into the mechanism of KCC ion transport, regulation, and evolution. Thus, here we purified DmKCC and HvKCC and characterized their biophysical properties using differential scanning fluorimetry and light scattering. We evaluated their functionality in cells and developed a method to study ion transport with flame photometry. Further, a fluorescence-based assay for DmKCC reconstituted into proteoliposomes was developed. The activity of DmKCC was found to be dependent on Ca<sup>2+</sup>, which is reminiscent of some other chloride transport protein families and potentially important for the KCC protein family overall.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144283091","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}
FEBS Open BioPub Date : 2025-06-13DOI: 10.1002/2211-5463.70062
Cecilia Giulivi, Richard Kotz
{"title":"Earthing effects on mitochondrial function: ATP production and ROS generation.","authors":"Cecilia Giulivi, Richard Kotz","doi":"10.1002/2211-5463.70062","DOIUrl":"https://doi.org/10.1002/2211-5463.70062","url":null,"abstract":"<p><p>Mitochondria are central to cellular energy production and the regulation of oxidative stress. Traditional methods for assessing mitochondrial ATP and reactive oxygen species (ROS) rely on metal probes, which unintentionally ground the system, confounding results. To investigate the impact of grounding on mitochondrial function, we utilized fluorescence-based experiments to assess these mitochondrial outcomes under three conditions: wired (grounded), sham, and naïve. Mitochondria under grounded conditions produced significantly more ATP (by 5-11%), reduced ROS production (by 22-33%), and decreased mitochondrial membrane potential (by 5-6%) than sham and naïve. These findings indicate that grounding improves mitochondrial bioenergetics by reducing oxidative stress. Future research should explore the broader implications of grounding over time on mitochondrial health and its potential therapeutic applications.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144289410","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":"Analysis of the regulation of undecaprenyl diphosphate dephosphorylation in Escherichia coli.","authors":"Tomotaka Jitsukawa, Yasushi Shigeri, Shingo Fujisaki","doi":"10.1002/2211-5463.70066","DOIUrl":"https://doi.org/10.1002/2211-5463.70066","url":null,"abstract":"<p><p>Undecaprenyl phosphate (C<sub>55</sub>P) is an essential sugar carrier for bacterial cell wall synthesis, which has gained importance in recent years as a promising target for new antibiotic development. In Escherichia coli, C<sub>55</sub>P is produced by dephosphorylation of undecaprenyl diphosphate (C<sub>55</sub>PP) by BacA and two type 2 phosphatidic acid phosphatase (PAP2) family enzymes, PgpB and YbjG, in the periplasmic space. To clarify the regulatory mechanism of C<sub>55</sub>PP dephosphorylation, we quantified C<sub>55</sub>P and C<sub>55</sub>PP using a new high-performance liquid chromatography method, conducted susceptibility tests against bacitracin, and analyzed the gene expression of bacA, pgpB, and ybjG in E. coli single- and double-disruption strains of those genes. C<sub>55</sub>P levels were similar in all strains, but C<sub>55</sub>PP levels increased only in the bacA, ybjG double-disruption strain. The double-disruption strains containing bacA disruption and the bacA single-disruption strain were more susceptible to bacitracin than the other strains. In the double-disruption strains containing bacA disruption, the expression of the remaining genes pgpB and ybjG increased. These results indicate that the transcription of the PAP2 family enzyme genes, pgpB and ybjG, was activated under conditions where C<sub>55</sub>PP dephosphorylation activity in cells was reduced. This transcriptional regulation might contribute to the maintenance of C<sub>55</sub>P levels in cells.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144283090","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}
FEBS Open BioPub Date : 2025-06-09DOI: 10.1002/2211-5463.70064
Shweta Singh, Gagan D Gupta
{"title":"An efficient strategy for producing RNA-free Nucleocapsid protein of SARS-CoV-2 for biochemical and structural investigations.","authors":"Shweta Singh, Gagan D Gupta","doi":"10.1002/2211-5463.70064","DOIUrl":"https://doi.org/10.1002/2211-5463.70064","url":null,"abstract":"<p><p>The SARS-CoV-2 Nucleocapsid (N) protein plays a crucial role in genome packaging, replication, transcription, and pathogenesis, making it a promising target for antiviral drug development. However, its large intrinsically disordered regions and propensity to form RNA condensates pose significant challenges for recombinant expression and purification. In this study, we successfully expressed and purified full-length N protein with a cleavable N-terminal Thioredoxin (Trx) fusion to enhance solubility and stability. The acidic Trx tag helped in the efficient binding of basic N protein to an anion-exchange column, enabling complete removal of bound RNA. Through a four-step process-immobilized metal affinity chromatography (IMAC), anion exchange, TEV protease-mediated tag cleavage followed by a second IMAC to remove cleaved fragments, and final polishing by size-exclusion chromatography (SEC)-we obtained highly homogeneous, RNA-free N protein. A single well-defined peak on SEC and dynamic light scattering confirmed the homogeneity of the purified protein. Electrophoretic mobility shift assays revealed strong RNA-binding activity, as a nearly complete RNA shift was observed at N protein concentrations as low as 0.25 μm. Fluorescence polarization assays further quantified RNA-binding affinity, yielding a dissociation constant of ~28 nm. These results establish an effective strategy for obtaining nucleic acid-free N protein suitable for biochemical and structural studies. Ultimately, this work provides a foundation for high-resolution structural investigations and the development of novel antiviral therapeutics targeting the N protein to combat COVID-19.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144257686","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":"Construction of hyperthermostable d-allulose 3-epimerase from Arthrobacter globiformis M30 using the sequence information from Arthrobacter psychrolactophilus.","authors":"Kensaku Shimada, Kouhei Ohtani, Pushpa Kiran Gullapalli, Kazuhiko Ishikawa","doi":"10.1002/2211-5463.70060","DOIUrl":"https://doi.org/10.1002/2211-5463.70060","url":null,"abstract":"<p><p>d-Allulose is one of the rare monosaccharides and is considered as a safe ingredient in foods. It can be enzymatically produced from d-fructose by the enzyme d-allulose 3-epimerase. More stable enzymes can operate effectively for longer durations, reducing the need for frequent replacements and thereby lowering costs. In addition, the preparation of the recombinant Arthrobacter globiformis M30 (AgDAE) enzyme requires heat treatment at 60-70 °C to remove host cell debris and potential microbial contaminants. Therefore, to address the need for more thermostable enzymes in d-allulose production, we aimed to create thermostable mutants of AgDAE using the protein engineering method. We cloned d-allulose identified from A. globiformis M30 and, using sequence homology, we constructed thermostable mutants by protein engineering. Each effect of the five mutations used was independent and additive. By integrating positive mutations, we succeeded in the construction of a chimeric enzyme exhibiting hyperthermostability without loss of enzymatic activity. The constructed chimera mutant was highly functional above 95 °C and remained stable under 80 °C. Our approach using structural information for the chimeric construction experiments also suggested that incorporating mutations from other homologous enzymes can impart advantages in enzymes in a simple and effective manner.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144257687","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}
FEBS Open BioPub Date : 2025-05-27DOI: 10.1002/2211-5463.70031
Kaustuv Ghosh, Dayang Asyiqin
{"title":"FEBS-IUBMB-ENABLE Conference crosses seven seas: Report on the 3rd International Molecular Biosciences PhD and Postdoc Conference—“Artificial intelligence: reshaping biomedical and healthcare research”","authors":"Kaustuv Ghosh, Dayang Asyiqin","doi":"10.1002/2211-5463.70031","DOIUrl":"https://doi.org/10.1002/2211-5463.70031","url":null,"abstract":"<p>The 3<sup>rd</sup> FEBS-IUBMB-ENABLE International Molecular Biosciences PhD and Postdoc Conference was held for the first time outside Europe, in South East Asia. On 4–6 December 2024, the Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, hosted over 260 participants from all around the world. The theme of this year's conference was “Artificial Intelligence – Reshaping biomedical and healthcare research”. The conference included a 2-day scientific symposium subdivided into four sessions: “AI in metabolic diseases, population and global health”, “AI in neuroscience and mental health”, “AI in respiratory and infectious diseases” and “AI in skin diseases and wound repair”. Ten global experts working at the forefront of expanding these horizons using AI were hosted. Through over 40 talks and 80 poster presentations, the participants at the conference, mostly young scientists at the doctoral and postdoctoral levels, had the chance to exhibit their research, fostering scientific exchange and collaboration. The last day of the conference included a career day dedicated to workshops, career sharing sessions and a panel discussion, where the focus was on career development, personal well-being, and translating academic research into industry innovations and startups. Pre-conference activities aimed towards fostering camaraderie and creating lasting memories made it a ‘must-to-attend’ event. The legacy of this conference will be carried forward by the next edition of FEBS-IUBMB-ENABLE titled “Bridging minds – Interdisciplinary research for the future of life sciences”, that will be hosted by Cancer Research UK (CRUK) Scotland Institute in Glasgow, UK, on 10–12 September 2025.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":"15 S1","pages":"3-13"},"PeriodicalIF":2.8,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/2211-5463.70031","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144148268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEBS Open BioPub Date : 2025-05-27DOI: 10.1002/2211-5463.70057
Edyta Skurska, Mariusz Olczak
{"title":"GDP-fucose transporter SLC35C1: a potential regulatory role in cytosolic GDP-fucose and fucosylated glycan synthesis.","authors":"Edyta Skurska, Mariusz Olczak","doi":"10.1002/2211-5463.70057","DOIUrl":"https://doi.org/10.1002/2211-5463.70057","url":null,"abstract":"<p><p>Glycosylation occurs mainly in the Golgi apparatus, whereas the synthesis of nucleotide sugars occurs in the cytoplasm or nucleus. GDP-fucose in mammalian cells could be produced via de novo and salvage pathways in the cytoplasm; the first one is responsible for about 90% of GDP-fucose in the total pool of this nucleotide sugar in the cell. SLC35C1 (C1) is the primary transporter of GDP-fucose to the Golgi apparatus. In the absence of this transporter, it was proposed that nucleotide sugar could still reach the Golgi apparatus via a SLC35C2, the homologue of SLC35C1. However, simultaneous inactivation of the two transporters did not influence GDP-fucose transport across the Golgi apparatus membranes after external fucose supplementation. In this study, we combined the inactivation of SLC35C1 and enzymes of the GDP-fucose biosynthesis pathways (FCSK, GMDS and TSTA3) to study the impact of double inactivation on the production of nucleotide sugar and fucosylated glycans. We found that a lack of SLC35C1 changed the level of enzymes of both de novo and salvage pathways. Upon fucose supplementation, stimulation of the salvage pathway was remarkably high in the absence of the TSTA3 protein, and the concentration of GDP-fucose increased to millimolar values. In this work, we discovered that simultaneous deficiency of the SLC35C1 protein and TSTA3 enzyme increased GDP-fucose production via the salvage pathway to an even higher level. Finally, we found that nucleotide sugar still accessed the Golgi apparatus and had differential effects on N- and O-glycans.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144149996","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}
FEBS Open BioPub Date : 2025-05-22DOI: 10.1002/2211-5463.70059
Emily Feng, Eric Feng, Tracy Berg, Isabella S Nguyen, Lilac G Nguyen, William Chen, Meng Zhang, David Quigley, Marina Sharifi, Haolong Li, Ilsa Coleman, Peter S Nelson, Martin Sjöström, Shuang G Zhao
{"title":"Identifying prognostic targets in metastatic prostate cancer beyond AR.","authors":"Emily Feng, Eric Feng, Tracy Berg, Isabella S Nguyen, Lilac G Nguyen, William Chen, Meng Zhang, David Quigley, Marina Sharifi, Haolong Li, Ilsa Coleman, Peter S Nelson, Martin Sjöström, Shuang G Zhao","doi":"10.1002/2211-5463.70059","DOIUrl":"https://doi.org/10.1002/2211-5463.70059","url":null,"abstract":"<p><p>Genome-wide screens using CRISPR/RNAi can identify new therapeutic vulnerabilities in prostate cancer. In this study, we combine DepMap functional screen data with a large gene expression database (N = 1012) and clinical outcomes to identify potentially druggable targets. Eight genes (CYC, CYP51A1, DHFR, EBP, KIF15, PPM1D, SQLE, and UMPS) demonstrated strong dependency in cell lines and were also associated with worse prognosis clinically, representing potential therapeutic targets in metastatic prostate cancer. Four of these (DHFR, EBP, KIF15, and PPM1D) demonstrated higher expression in neuroendocrine prostate cancer. Furthermore, all but one (KIF15) were not significantly decreased from pretreatment to posttreatment, suggesting that they may remain targetable postabiraterone therapy. All eight genes showed evidence of protein expression in prostate cancers or cell lines. These potentially druggable targets associated with prostate cancer cell line dependency and worse clinical outcomes have also demonstrated literature support as potential targets, supporting further research into their potential clinical relevance as therapeutic targets in prostate cancer.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126815","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}
FEBS Open BioPub Date : 2025-05-22DOI: 10.1002/2211-5463.70058
Heng Wang, Xiaofang Tan, Deyu Chen
{"title":"Short peptide perturbs spermatogenesis via immune microenvironment dysregulation and mitochondrial imbalance.","authors":"Heng Wang, Xiaofang Tan, Deyu Chen","doi":"10.1002/2211-5463.70058","DOIUrl":"https://doi.org/10.1002/2211-5463.70058","url":null,"abstract":"<p><p>A short peptide derived from the occludin protein regulates tight junctions (TJ) of the blood-testis barrier and impairs germ cell development. However, the mechanism behind how this peptide regulates TJ and induces cell apoptosis remains unclear. In the present study, an animal model with induced TJ disruption via the short peptide was used to evaluate its impact on spermatogenesis. Here, we demonstrate that the short peptide promoted the infiltration of immune cells into the testicular interstitial tissue, accompanied by upregulation expression of the pro-inflammatory factors interleukin-6 and tumor necrosis factor-α. Moreover, mitochondrial fragmentation and mitophagy were upregulated in Sertoli cells and Leydig cells. Consistently, terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed extensive apoptosis in the testes during spermatogenesis. Notably, the severity of these disruptions began to attenuate after 27 days, although full functional recovery was not observed. Our findings reveal a novel mechanism wherein peptide-induced immune dysregulation and mitochondrial dysfunction synergistically impair spermatogenesis, potentially via microenvironmental perturbation of the TJ. Overall, these findings could hold valuable insights for the development of non-hormonal male contraceptives.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126853","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":"FEM1B enhances TRAIL-induced apoptosis in T lymphocytes and monocytes.","authors":"Chenbo Yang, Wenhui Yu, Cui Dang, Jingjing Zhang, Jiahan Lu, Jing Xue","doi":"10.1002/2211-5463.70056","DOIUrl":"https://doi.org/10.1002/2211-5463.70056","url":null,"abstract":"<p><p>FEM1B is recognized for its significant pro-apoptotic function in colorectal cancer; however, its influence and mechanisms regarding apoptosis in immune cells remain inadequately elucidated. In this study, we demonstrated that FEM1B enhances TRAIL-induced apoptosis in Molt-4, Jurkat, THP-1, and U937 cell lines. Notably, the knockdown of FEM1B in transfected cells resulted in a reversal of the observed increase in cell apoptosis. Our findings indicate that FEM1B activates caspase-3 and caspase-8, but not caspase-9, in response to TRAIL stimulation, suggesting its involvement in the extrinsic caspase-dependent apoptotic pathway. Furthermore, we found that FEM1B interacted with TRAF2 and downregulates its expression in Molt-4 and Jurkat cells, thereby diminishing TRAF2's inhibitory effect on caspase-8. In THP-1 and U937 cells, FEM1B was found to upregulate TRAIL-R2, thereby promoting TRAIL-induced apoptosis. Knockout studies in murine models further corroborated that FEM1B facilitates TRAIL-induced apoptosis. These results demonstrate that FEM1B enhances TRAIL-induced apoptosis in T lymphocytes and monocytes through a caspase-dependent mechanism involving TRAF2 or TRAIL receptors.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144110206","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}