Biochemical and biophysical research communications最新文献

Bispecific antibodies (bsAbs) that direct immune cells toward tumor cells have substantial therapeutic potential. However, bsAbs produced by tandemly linking single-domain antibodies (VHHs) often exhibit reduced activity because the N-terminal region of each VHH, which contains the complementarity-determining regions (CDR) loops, becomes sterically hindered by the adjacent domain. To address this issue, we developed a branched VHH complex architecture, termed Dendrobody, in which VHHs are joined through their C-terminal sides using orthogonal SpyCatcher/SpyTag and SnoopCatcher/SnoopTag systems. This design enables site-specific covalent assembly of multiple VHHs while preserving accessibility of their antigen-binding regions. We generated a bispecific anti-CD3/anti-EGFR Dendrobody (Dendrobody-CD3/EGFR) through a one-pot ligation of three recombinant components. The purified complex displayed correct folding with a thermal transition temperature (Tm) of 56 °C. Biolayer interferometry showed strong EGFR binding, and the Dendrobody triggered selective T-cell-mediated cytotoxicity toward EGFR-positive cancer cells. No cytotoxicity was detected in EGFR-negative cells, confirming antigen specificity. In addition, the platform allowed straightforward production of another variant, Dendrobody-CD16/EGFR, demonstrating its modularity. The Dendrobody framework thus offers an alternative strategy for constructing bispecific and multispecific VHH-based therapeutics with minimal steric interference and high functional adaptability.

Introductions: Oral squamous cell carcinoma (OSCC) has been increasingly associated with dysbiosis of the oral microbiome. Among oral pathogens, Prevotella intermedia (P. intermedia) is frequently enriched in patients with OSCC; however, the role of its virulence factors-particularly its deoxyribonuclease (DNase) activity-remains poorly understood.

Methods: We compared the effects of culture supernatants from wild-type P. intermedia OMA14 and DNase-deficient mutant strains (nucA, nucD and nucA nucD) on the migration and invasion of the OSCC cell line SAS, using wound healing and Matrigel invasion assays. Transcriptomic profiling of SAS cells exposed to bacterial supernatants was performed using RNA sequencing (RNA-seq), followed by differential gene expression and pathway enrichment analyses.

Results: Supernatants from the nucA nucD mutant strain significantly enhanced SAS cells migration and invasion compared with those from the OMA14 strain. RNA-seq revealed marked transcriptomic reprogramming, including upregulation of genes related to extracellular matrix degradation, epithelial-mesenchymal transition (EMT), and inflammatory signaling. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses confirmed the enrichment of EMT, cytokine signaling, and tumor-promoting pathways.

Conclusions: Our findings demonstrate a dual role of bacterial DNase activity in SAS cell. Although DNases contribute to immune evasion via neutrophil extracellular trap degradation, their absence enhances tumor invasion by promoting proinflammatory and EMT-related transcriptional programs. These results highlight the complex interplay between microbial nucleases, extracellular DNA, and host signaling, providing novel insights into the contribution of the oral microbiome to OSCC pathogenesis.

Cells maintain proteostasis by sequestering misfolded proteins into deposition sites. Aggregation-prone endoplasmic reticulum (ER) proteins form membrane-bound nuclear compartments that are cleared during cell division, yet the mechanisms underlying their spatial organization remain unclear. Here, using transcriptomic and proteomic analyses, we identified the ER-localized Hsp70 chaperone BiP as a key player. Genetic depletion or chemical inhibition of BiP prevented nuclear aggregate formation, while manipulating BiP regulators perturbed the aggregate formation. BiP-driven aggregation precedes the inner nuclear membrane synthesis that encapsulated the aggregates. Under proteostatic stress, nuclear aggregates localized adjacent to ER-derived aggregates. Our findings demonstrate that BiP is essential for organizing ER-derived aggregates in the nucleus, which further regulate nuclear proteostasis through spatial interactions with nuclear aggregates.

RALY, a heterogeneous nuclear ribonucleoprotein, binds to nascent RNA and participates in multiple aspects of RNA metabolism, including transport, splicing, transcription, and translation. Recent studies have revealed that RALY is overexpressed in various cancers, such as breast, uterine, and liver cancers. This overexpression has been associated with poor patient survival and uncontrolled carcinoma cell proliferation. In this study, we demonstrate that RALY functions as a key regulator of cell proliferation, migration, and invasion in the hepatocellular carcinoma (HCC) cell lines Hep3B and HepG2. Mechanistically, RALY promotes epithelial-mesenchymal transition (EMT) through regulation of the transcription factor Snail. RALY directly binds to Snail mRNA, thereby enhancing its stability. In addition, RALY modulates the TGF-β signaling pathway to promote Snail transcription. Together, our findings establish a functional link between RALY and EMT and reveal a previously unrecognized role of RALY in cancer cell metastasis. Accumulating evidence, including the results presented here, suggests that RALY represents a potential therapeutic target for cancer treatment.