Aug 2026· Toxicon· pp.
109273
· 0 citations· 54 references
Medicine
TL;DR
Although CD59 contains linear segments that recognize CB, this interaction is not sufficient to reproduce the PLA2-inhibitory function of CNF, suggesting that any functional interplay between these Ly6/uPAR proteins is likely to depend on more complex structural determinants.
Abstract
Structural motifs can be conserved among proteins with low primary-sequence identity, potentially supporting distinct yet related molecular functions. Endogenous phospholipase A2 inhibitors from snake plasma (sbPLIs) include the γ-type inhibitor from Crotalus durissus terrificus, known as Crotalus neutralizing factor (CNF), which protects the snake by selectively inhibiting the lethal and PLA2 activities of crotoxin (CTx). CD59, a complement regulator and single-LU-domain member of the Ly6/uPAR superfamily, shares the three-finger fold with CNF, raising the possibility of structural and functional convergence. Here, CD59 linear peptide arrays produced by SPOT synthesis were probed with CB, the PLA2 subunit of CTx, to identify CD59 regions capable of binding CB. Two reactive clusters were mapped, and derived peptides were synthesized and tested for their ability to inhibit the PLA2 activity of CTx and CB in vitro. Four CD59-derived peptides interacted with CB on the arrays; however, none inhibited PLA2 activity, in contrast to native CNF used as a positive control. These findings indicate that, although CD59 contains linear segments that recognize CB, this interaction is not sufficient to reproduce the PLA2-inhibitory function of CNF, suggesting that any functional interplay between these Ly6/uPAR proteins is likely to depend on more complex structural determinants.
Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor existing in two isoforms, TFPI-1 and TFPI-2. Peptides derived from the TFPI C-terminus exhibit potent antimicrobial activity. In this study, a TFPI-1 homolog from Sebastes schlegelii (SsTFPI-1) was identified, and its sequence characteristics, immune response profile, and the antimicrobial activity of its C-terminal peptide TS20 were systematically characterized. The open reading frame of SsTFPI-1 is 867 bp in length, encoding 288 amino acids that comprise a signal peptide, three Kunitz domains, and a low-complexity region. SsTFPI-1 was ubiquitously expressed across all examined tissues, with the highest expression in blood, and its transcript levels were significantly upregulated in the spleen or head kidney following stimulation with Listonella anguillarum, Edwardsiella piscicida, or poly(I:C). Subcellular localization revealed that SsTFPI-1 predominantly localizes in the cytoplasm of EPC cells, and its C-terminus undergoes endogenous cleavage. Antimicrobial assays demonstrated that TS20 exerts pronounced antibacterial activity against Micrococcus luteus with favorable thermal stability. Mechanistically, TS20 disrupts the bacterial cell membrane, enters the cells, binds to DNA, and progressively degrades it. Furthermore, TS20 significantly inhibits SVCV infection in EPC cells by targeting both viral particles and host cells, thereby reducing viral adhesion and entry, while simultaneously downregulating the expression of type I interferon (IFN-I) related genes. In vivo experiments confirmed that TS20 significantly reduced viral loads of SVCV and RBIV-C1 in common carp and turbot, respectively. Collectively, these findings elucidate the immunological role of SsTFPI-1 in S. schlegelii and underscore the potential of TS20 as a promising antimicrobial agent.
Zhi-shu Zhu, Zi-yue Chen, Guang-hua Wang et al.· Fish and Shellfish Immunolog...· 0 citations
Simple Summary Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes substantial economic losses to the global swine industry. PRRSV invades host target cells via receptor-mediated endocytosis, and CD163 is the most important receptor for PRRSV entry. Among the CD163 domains, the scavenger receptor cysteine-rich 5 (SRCR5) domain is essential for PRRSV infection. Therefore, CD163 and its SRCR5 are commonly selected as targets for developing anti-PRRSV strategies, including for the generation of CD163-SRCR5 knockout pigs. In addition, CD163 divergence also serves as a barrier to cross-species cellular infection by arteriviruses. Nevertheless, CD163 monoclonal antibodies (mAbs) with defined cross-species reactivity and anti-PRRSV activities have rarely been characterized. Here, we developed two mAbs against porcine CD163. Epitope mapping demonstrated that mAb 5A recognizes a novel epitope within the long loop 5-6 of the SRCR5 domain, while mAb 11D targets a conserved epitope within the SRCR4 domain. Functional validation revealed that mAb 5A can inhibit PRRSV infection, while mAb 11D cannot. Remarkably, mAb 11D exhibits broader cross-species reactivity than mAb 5A. Overall, this study provides a solid foundation for elucidating CD163-domain-dependent biological functions.
Yifan Meng, Shuai Yang, Jun Jiao et al.· Veterinary Sciences· 0 citations
The Hom family and canilysin are defined as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.
A. Rodríguez-Banqueri, T. Goulas, Marina Girbal-González et al.· Journal of Molecular Biology· 0 citations
Avian influenza viruses (AIVs) are RNA viruses endemic to wild birds, with highly pathogenic strains such as H5N1 causing major outbreaks. Hemagglutinin (HA) and neuraminidase (NA) proteins are key mediators of viral entry and spread. Cyclotides, plant-derived peptides with stable structures and potent bioactivity, have emerged as novel antiviral candidates. This study explored cyclotide-based drug design against H5N1 using computational approaches. HA and NA protein sequences were retrieved from NCBI and their physicochemical properties analyzed using ProtParam. Cyclotides from CyBase were modeled and refined in PyMOL, followed by molecular docking via ClusPro. Binding energies were further assessed with molecular mechanics generalized born surface area, while toxicity was predicted using ToxinPred. Molecular dynamics simulations supported the stability analyses. Results revealed that Psyle B and Psyle C exhibited strong interactions with HA and NA. Psyle B achieved a docking score of -1001.3 kcal/mol, and Psyle C -1031.6 kcal/mol, both predicted as non-toxic. These findings highlight Psyle B and Psyle C as promising antiviral leads. Nonetheless, in vitro and in vivo validation remains essential to confirm their therapeutic potential.
S. Altaf, Kainat Sarwar, Rao Zahid Abbas et al.· Journal of Biomolecular Stru...· 0 citations
Tumor necrosis factor receptor-associated factor 7 (TRAF7) is an atypical member of the TRAF family, with well-established roles in modulating vertebrate innate immunity, apoptosis, and intracellular signal transduction. In contrast, its functional repertoire in invertebrates, particularly among marine mollusks, remains poorly characterized. In this study, a TRAF7 ortholog from the Zhikong scallop (Chlamys farreri) was identified and designated as CfTRAF7. The cloned CfTRAF7 cDNA comprises a 1953-bp open reading frame encoding a 650-amino-acid polypeptide. Function domain prediction confirmed that the N-terminal RING finger domain and the C-terminal WD40 repeat region exhibit significant sequence homology with TRAF7 orthologs across metazoans. Phylogenetic analysis indicated that CfTRAF7 clusters with TRAF7 homologs from other mollusks. Quantitative real-time PCR assays demonstrated that CfTRAF7 mRNA was widely expressed in all tested scallop tissues, with the highest expression in hemocytes. After stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid, and peptidoglycan, the mRNA expression of CfTRAF7 was significantly upregulated, with temporal differences in response to various pathogen-associated molecular patterns. Co-immunoprecipitation confirmed that CfTRAF7 interacted with C. farreri transforming growth factor beta-activated kinase 1 (CfTAK1). Western blot analysis revealed that overexpression of CfTRAF7 in HEK293T cells significantly enhanced the phosphorylation of mitogen-activated protein kinase (MAPK) proteins (e.g. JNK and Erk1/2), whereas RNA interference (RNAi) of CfTRAF7 suppressed MAPK phosphorylation levels in vivo following LPS challenge, but had no obvious effect on p38 phosphorylation. RNAi assays demonstrated that silencing of CfTRAF7 significantly altered the expression of several immune-related genes, including CfTAK1, CfRel, CfIL17A, CfIL17D, CfTNF-like, and CfSOD, following LPS challenge. Dual-luciferase reporter assay showed that overexpression of CfTRAF7 could activate immune-related reporter genes such as interleukin-6 (IL-6), interferon α/γ (IFNα/γ), activator protein-1 (AP-1), nuclear factor kappa B (NF-κB), and tumor necrosis factor α (TNFα), in a dose-dependent manner. This study not only enriches the functional research on TRAF7 in invertebrates but also provides a new perspective for understanding the evolutionary conservation and species specificity of molluscan innate immune signaling pathways. Additionally, it also offers important theoretical support for further comprehension of the molecular mechanism of scallop immune defense and promoting the green control of aquatic diseases in marine farming.
Xiaoxue Lin, Zihao Zhang, Xiaolong Chu et al.· Fish and Shellfish Immunolog...· 0 citations
In silico approaches are becoming increasingly important for enhancing the affinity of antibodies and antibody fragments, including single-chain variable fragments (scFvs). In this study, we present an in silico affinity-maturation workflow designed to improve the affinity of an scFv directed against
Neisseria meningitidis
factor H-binding protein (fHbp) by integrating in silico maturation, molecular dynamics (MD) simulations, and experimental validation. Approximately 300 single-point substitutions were designed at scFv residues predicted to interact with fHbp, and the resulting V3 (R99D) scFv–fHbp complexes were analyzed through all-atom MD simulations. Guided by bioinformatic analyses, the top V3 (R99D) variant was cloned and expressed, and its binding activity was validated by ELISA.
MD analyses identified five variants, Y35H, L31R, R99D, R99H, and A100K, that exhibited markedly enhanced complex stability. In all variants, the scFv secondary structure remained intact, whereas fHbp showed shrinkage relative to the scFv in the V3 (R99D)–fHbp complexes during the simulations. Hydrogen-bonding patterns and inter-residue distances further supported the improved affinity of the variants relative to the native form. The R99D variant showed the highest binding affinity, with an experimentally measured value of 67.93 × 10⁹ M⁻¹.
Collectively, these findings underscore affinity maturation as a powerful strategy for engineering high-affinity antibody fragments.
Bijan Ghazanfari, Zahra Ebrahimi, F. Yarian et al.· BMC Biotechnology· 0 citations