Biochemical and biophysical research communications最新文献

Uterine leiomyosarcoma (Ut-LMS) is a rare and aggressive gynecologic malignancy with limited effective therapeutic options. In this study, we investigated the cytotoxic effects and underlying mechanisms of bortezomib in Ut-LMS cell lines SK-LMS-1 and SK-UT-1B. Bortezomib treatment significantly reduced cell viability and increased lactate dehydrogenase release, indicating pronounced cytotoxicity. Apoptotic cell death was induced, as evidenced by increased Annexin V-positive cell populations. Bortezomib also suppressed proliferative activity, reflected by reduced Ki67 expression, and induced G2/M cell cycle arrest in SK-LMS-1 cells, whereas SK-UT-1B cells exhibited minimal alterations in cell cycle distribution. In addition, bortezomib increased reactive oxygen species production in SK-UT-1B cells and induced mitochondrial membrane depolarization in both cell lines, while antioxidant treatment attenuated bortezomib-induced apoptosis in SK-UT-1B cells, indicating partial involvement of oxidative stress. Western blot analysis further revealed enhanced cleavage of poly(ADP-ribose) polymerase and caspase-3, along with modulation of cell cycle regulatory proteins, including upregulation of p21 and differential regulation of p53 between the two cell lines. Finally, autophagy-related analyses demonstrated increased LC3B-II levels accompanied by p62 accumulation, suggesting altered autophagic processing rather than simple activation of autophagy. Collectively, these findings demonstrate that bortezomib exerts cytotoxic effects in Ut-LMS cells through coordinated regulation of proteasome inhibition-associated apoptosis, cell cycle control, mitochondrial dysfunction, and autophagy-related signaling, with cell line-specific differences in stress response pathways.

Phosphotransferase system regulatory domain-containing virulence regulators (PCVRs), including Enterococcus faecalis MafR and Streptococcus pneumoniae Mga-like protein (MgaSpn), are transcription factors in pathogenic bacteria that control the transcription of virulence genes by binding dsDNA in promoter regions. Although PCVRs play critical roles in bacterial pathogenesis, the structural and molecular basis by which PCVRs recognize dsDNA for transcriptional regulation has remained unclear. Here, we present the unique structure of MafR and demonstrate the critical role of its two N-terminal helix-turn-helix (HTH) domains in dsDNA binding. MafR adopts a five-domain architecture with an open-ring conformation and dimerizes into a table-like structure, in which the C-terminal domain mediates dimerization in the middle of the dimer structure, and the two N-terminal HTH domains project from one face of the dimer. Our extensive modeling, biochemical, and mutational analyses of the MgaSpn-dsDNA interaction reveal that the tandem HTH domains are required for dsDNA binding by inserting their recognition helices into the major groove of dsDNA. Our findings highlight a unique mode of DNA recognition by PCVRs, in which two HTH domains are simultaneously employed for DNA binding, rather than the single HTH domain typically used by conventional HTH-containing transcriptional regulators.

Microgravity exposure or mechanical unloading profoundly disrupts skeletal homeostasis, leading to rapid bone loss that poses a major challenge during spaceflight and long-term bed rest. Despite existing countermeasures, effective pharmacological strategies with minimal side effects remain elusive. Here, we identify curcumin, a naturally polyphenolic compound with pleiotropic bioactivity, as a promising candidate unloading-induced osteoporosis. Daily administration of curcumin (200 mg/kg) markedly mitigates the reduction of trabecular bone mass and microarchitecture under hindlimb unloading (HU) mouse model, as evidenced by increased BV/TV, Tb.N, and Tb.Th, and reduced Tb.Sp. Curcumin significantly suppressed osteoclast function and bone resorption, reducing TRAP⁺ multinucleated cells and downregulating Ctsk, Mmp9, Trap, Nfatc1, and NFAT2 expression, while exerted minimal influence on cortical bone strength or key osteogenic markers (Alp, Col1a1, Bglap). Mechanistically, curcumin inhibited the NF-κB signaling cascade, specifically through attenuated phosphorylation of p65 and IκBα, which restrained osteoclastogenesis. Collectively, these findings demonstrated the potential roel of curcumin in preventing microgravity- or disuse-related bone loss.

Objective: The study aimed to explore the impact of sleep deprivation on hepatic steatosis in metabolic dysfunction-associated fatty liver disease (MASLD) and its possible mechanisms.

Methods: Forced exercise was used to establish sleep deprivation(SD) models in Sprague-Dawley rats. After 8 weeks of modeling, lipid profile, liver function, pathological feature, inflammatory cytokines, oxidative stress markers, and gut microbiota were determined.

Results: Sleep deprivation exacerbated hepatic steatosis in MASLD rats, as evidenced by significant alteration in morphological analysis and pathological features, accompanied by more severe metabolic disorders and liver injury. Moreover, sleep deprivation dramatically enhanced the secretion of pro-inflammatory cytokines and oxidative stress damage in the liver of MASLD rats. The results of 16S rRNA analysis confirmed a novel causal role of gut microbiota dysbiosis in driving the development of MASLD. Furthermore, sleep deprivation exacerbated gut microbiota dysbiosis in MASLD rats, especially reducing beneficial bacteria including s_roseburia hominis, s_Bacteroides vulgatus, and s_Akkermansia muciniphila. Interestingly, fecal microbiota transplantation (FMT) had demonstrated potential to restore gut microbiota dysbiosis induced by the synergism of high-fat diet (HFD) and sleep deprivation. After partially counteracting the impact of the synergistic effects on gut microbial homeostasis by FMT, hepatic steatosis, hepatic inflammation, and oxidative stress damage in rats of the HFD + SD group were substantially improved.

Conclusions: These results reveal that sleep deprivation exacerbates hepatic steatosis in MASLD by disrupting gut microbial homeostasis, thereby aggravating hepatic inflammation and oxidative stress, providing novel insights into the potential therapeutic strategies for MASLD and other sleep deprivation-related disorders.

Cytotoxic T lymphocytes (CTLs) regenerated from induced pluripotent stem cells (iPSCs) have emerged as an ideal cellular material for introduction of T-cell receptor (TCR) genes specific for defined tumor antigens, as they can be expanded virtually indefinitely while retaining potent cytolytic activity. Indeed, we previously demonstrated that introducing tumor antigen-specific TCR genes into iPSC-derived CTLs using retroviral or lentiviral vectors confers effective antitumor activity. Nonetheless, viral vector-mediated transfer results in random, multicopy genomic integration, which may yield heterogeneous TCR expression and oncogenic transformation depending on the insertion site. In this study we thus attempted to introduce distinct tumor antigen-specific TCR genes into the endogenous T-cell receptor α constant (TRAC) locus of iPSC-derived CTLs by genome editing. The engineered CTLs displayed robust antigen-specific expansion enabled by stable expression of the introduced TCRs under native regulatory control. Moreover, they produced IFN-γ and upregulated the degranulation marker CD107a in an antigen-specific manner, and exhibited strong cytotoxic activity against target cells expressing the cognate peptide. Collectively, these results provide the first demonstration, to our knowledge, that successful TCR replacement was achieved in iPSC-derived CTLs via genome editing and that the introduced TCRs are functionally competent.

The retinoic acid-related orphan receptor α (RORα) is a potential drug target for cancer, inflammation, and metabolic diseases. Structure-guided ligand optimization can facilitate the development of selective RORα modulators. However, structural studies of the RORα ligand-binding domain (LBD) have been limited due to difficulties in purifying recombinant protein suitable for crystallographic analysis. Here, we engineered a chimeric RORα LBD C-terminally fused to the LXXLL motif of the coactivator RIP-140. This fusion improved the stability and solubility of the RORα LBD, enabling high-yield purification using the E. coli expression system. We determined the crystal structure of the chimeric RORα LBD in complex with cholesterol at 2.7 Å resolution. The cholesterol-bound RORα LBD adopted an active conformation of helix 12 that accommodates coactivator binding. The coactivator peptide appears to stabilize the RORα LBD by shielding the hydrophobic surface of the AF-2 region. Although monomeric in solution, the RORα LBD-LXXLL fusion formed a dimer in the crystal lattice through extensive interactions involving the LXXLL motifs and α3 helices, which may represent a physiological homodimer. Fusion of a coactivator motif to the LBD is expected to facilitate structural studies of RORα and may be broadly applicable to other nuclear receptor LBDs for generating stable recombinant proteins.

Albumin nanoparticles have emerged as a promising drug delivery platform for targeted breast cancer therapy, offering advantages such as biocompatibility, non-toxicity, and the ability to overcome drug resistance. This comprehensive review critically synthesizes mechanistic evidence, formulation strategies, representative case studies, and clinical outcomes to evaluate functionalized albumin nanoparticles as drug-delivery platforms for conventional and resistant breast cancer. The passive and active targeting capabilities of albumin nanoparticles are discussed, along with various surface modification strategies using ligands like folic acid, transferrin, and monoclonal antibodies to enhance tumor specificity. Various techniques for preparing albumin nanoparticles, such as desolvation, emulsification, thermal gelation, and self-assembly, are also discussed. The application of albumin nanoparticles loaded with different drugs, such as doxorubicin, paclitaxel, and curcumin, in breast cancer therapy is extensively explored. Notably, the review highlights the potential of albumin nanoparticles to overcome drug resistance mechanisms in breast cancer, such as drug efflux, hypoxia, and apoptosis resistance, through co-delivery of chemotherapeutic agents and resistance modulators. Recent research studies and clinical outcomes are presented, showcasing the efficacy of albumin-based nano-formulations in breast cancer treatment. FDA-approved nab-paclitaxel (Abraxane) demonstrated effectiveness by enhancing progression metrics and survival versus solvent-based paclitaxel, while nab-docetaxel (ABI-008) showed limited benefits. The challenges and future directions in this field are also discussed, emphasizing the need for further optimization and clinical translation of albumin-based nanomedicines. Overall, this review provides a comprehensive overview of the mechanistic approach and therapeutic potential of functionalized albumin nanoparticles for breast cancer therapy, underlining their promise as a targeted drug delivery platform.

Gaucher disease (GD) is a rare autosomal-recessive lysosomal storage disorder caused by mutations in the GBA1 gene encoding the lysosomal hydrolase glucocerebrosidase (GCase). Mutations in GCase lead to glucosylceramide accumulation within macrophages. Current treatments, including enzyme replacement therapy (ERT) and substrate reduction therapy (SRT), alleviate symptoms but are limited by high cost, frequent dosing, and adverse effects, highlighting the need for novel strategies. To achieve higher expression levels and improved stability, we designed and optimized a series of hGBA1-mRNA by changing untranslated regions (UTRs), codon usage, and poly(A) tails, and evaluated their performance in vitro and in vivo. Optimized constructs achieved >6-fold higher GCase activity compared with the least efficient variants 24 h post-transfection in HEK293T and RAW264.7 cells, with an average half-life exceeding 54 h. The expressed enzyme localized to lysosomes and restored normal morphology and substrate accumulation in GBA1-knockout (KO) HEK293T cells. Following a single administration of hGBA1-mRNA encapsulated in lipid nanoparticles (LNPs) in wild-type FVB mice, GCase activity was detectable in the liver and spleen within 72 h. Our results demonstrate that optimized hGBA1-mRNA-LNPs can deliver functional human GCase in vivo, providing a promising and efficient mRNA-based therapeutic approach for GD.

Mechanical loading induces skeletal muscle hypertrophy, however the underlying molecular mechanisms remain incompletely understood. In rodents, synergist ablation (SA) is widely used to induce hypertrophy, yet most studies focus on fast-twitch muscles, such as the plantaris (PL), because traditional ablation causes severe inflammation in slow-twitch muscles like the soleus (SOL), complicating data interpretation. Recent studies have highlighted the distinct responses of slow- and fast-twitch muscles to mechanical overload. In this study, we developed a refined rodent model of mechanical overload-induced hypertrophy targeting both slow (SOL) and fast (PL) muscles. We compared this modified partial synergist ablation (PSA) method with conventional SA and tenotomy (TT) regarding muscle hypertrophy, fiber type composition, inflammation, and regeneration. C57BL6/J male mice (16 weeks old, n = 18) were assigned to one of three surgical groups: SA, TT, and PSA. In all groups, the relative SOL weight in overloaded legs significantly increased compared to the contralateral control legs (SA: 137%; TT: 118%; PSA: 120%). Similarly, PL weight increased (SA: 145%; TT: 134%; PSA: 115%). Notably, inflammation and muscle regeneration were observed in the SOL of SA and TT groups but not in PSA group. The ratio of central nuclei to subsarcolemmal nuclei among EdU⁺/PCM1⁺ nuclei were much higher in SOL compared to those in PL among three models, suggesting distinct modality of hypertrophy in slow- and fast-muscles. These findings indicate that PSA provides a minimal inflammatory and effective approach for inducing hypertrophy in both fast and slow muscles, making it a valuable model for studying muscle adaptation.