Bioluminescence has independently evolved multiple times during animal evolution, yet the biochemical mechanisms underlying light production in true flies (Diptera) remain poorly understood. Here we describe the identification, cloning and functional characterization of the first dipteran luciferase from the fungus gnat Keroplatus testaceus. The 76‑kDa enzyme, KerLuc, is active when expressed heterologously in yeast, yielding a blue emission spectrum indistinguishable from native larvae. Sequence and domain analyses place the protein within the hemocyanin/hexamerin superfamily, but intriguingly, the protein lacks the canonical copper‑binding histidines. Structure prediction points to a hydrophobic cavity consistent with binding of the known Keroplatus oxyluciferin (3‑hydroxykynurenic acid), suggesting a novel catalytic mechanism. The luciferase is encoded by a single‑exon gene adjacent to a closely related paralogue within the conserved Enhancer of Split complex locus, indicating recent duplication and possible neofunctionalisation. Phylogenetic comparisons highlight proximity to Orfelia homologues and support independent origins of bioluminescence within Keroplatidae. Our results provide compelling evidence for a novel evolutionary origin for a luciferase from a storage protein, thereby closing a long-standing gap in understanding the molecular mechanisms of bioluminescence in Diptera.

Sponges are capable of rebuilding entire functional organisms from dissociated somatic cells. This feat demands radical cytoskeletal reprogramming whose molecular logic remains obscure. Using the Arctic demosponge Halisarca dujardinii, we mapped molecular signatures across free-swimming larvae, sessile adults, and early cell aggregates. Integrating RNA-seq, single-cell deconvolution, DIA-LC-MS proteomics, native complex fractionation, live-cell proteasome imaging, and immunofluorescence, we identified stage-associated profiles of cytoskeletal proteins and post-translational modifications (PTMs). Larvae enrich non-classical myosins (Myosin-9/15) and dynamic microtubules for motility; adults deploy spectrin scaffolds and deacetylated microtubules for stability; cell aggregates shift toward microtubule-based transport with targeted proteolysis and stage-specific PTMs on divergent actin HdA6 and ferritin. Live imaging shows ubiquitin-independent 20S proteasome activity at cell contacts in forming aggregates, while mass spectrometry reveals coordinated methionine oxidation and ubiquitination events coinciding with cytoskeletal remodeling. Although bulk data cannot fully separate cell-type composition from intracellular reprogramming, the multi-layered signatures suggest a tightly regulated, stage-specific program. Together, these findings establish a correlative framework linking cytoskeletal architecture, redox-sensitive PTMs, and proteostasis within sponge development and early reaggregation stage. We propose that conserved structural networks are chemically tuned to enable morphological plasticity in early-branching metazoans - a foundation for future functional studies of multicellular morphogenesis.

Non-invasive radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression can guide patient stratification for EGFR-targeted therapies. The designed ankyrin repeat protein (DARPin) E01, which binds EGFR ectodomain III with sub-nanomolar affinity, is a promising scaffold for single-photon emission computed tomography (SPECT) imaging probes. In the present study, we compared site-specific radioiodination of DARPin E01 using the bifunctional prosthetic group (4-hydroxyphenyl)ethyl maleimide (HPEM) with site-unspecific radioiodination via [123I]I-para-iodobenzoate (PIB). [123I]I-HPEM was conjugated to the C-terminus of DARPin E01 via Glu-Glu-Glu-Cys ([123I]I-E01-E3C-HPEM) or Gly-Gly-Gly-Cys ([123I]I-E01-G3C-HPEM) linkers. Radiolabelling yields were 6 ± 2% and 13 ± 5%, respectively. Size-exclusion purification provided radiochemical purity > 98%. Both HPEM conjugates retained nanomolar EGFR-binding affinity (KD: 3.2 ± 0.6 and 4.8 ± 0.9 nM) and demonstrated EGFR-specific tumour accumulation in A-431 xenografts. Cellular processing was characterised by rapid binding, slow internalisation, and non-residualising behaviour of all variants. Kidney uptake was lower for the site-specifically labelled variants. However, site-specific labelling evidently elevated hepatobiliary excretion and uptake in Na/I-symporter-expressing organs compared to [123I]I-(HE)3-E01-PIB, while linker composition (E3C vs. G3C) did not significantly alter biodistribution. Site-unspecific radioiodination with [123I]I-PIB remains the preferred approach for clinical SPECT imaging of EGFR expression with DARPin E01.

Tropomyosin (Tpm) is an actin-binding protein that, together with troponin (Tn), mediates Ca2+-regulation of cardiac muscle contraction. Tpm coiled-coil dimers bind each other through overlap junctions between their N- and C-termini, forming a continuous strand along the actin filament. Among the many TPM1 mutations identified in cardiac Tpm (Tpm1.1), few substitute canonical residues with proline, the amino acid most disruptive to coiled-coil structure. We examined properties of recombinant cardiac Tpm with L43P or L57P substitutions in both chains and compared them with wild-type (WT) Tpm using differential scanning calorimetry, viscometry, molecular dynamics (MD) simulations, and an in vitro motility assay. Both mutations markedly destabilized the N-terminal part of the Tpm molecule. In MD simulations, Pro43 and Pro57 disrupted nearby backbone hydrogen bonds, and Pro43 promoted N-terminal unfolding, demonstrating a long-range effect within supercoiled Tpm molecules. Both substitutions strongly reduce Tpm's affinity for F-actin in the absence of Tn. In addition, viscometry showed that the L43P Tpm variant polymerizes less efficiently than WT and L57P Tpm, resulting in a reduced ability to assemble into a continuous strand along an actin filament. The L57P substitution increases maximum sliding velocity of thin filaments in in vitro motility assay and enhances Ca2+ sensitivity of actin-myosin interaction, a feature commonly associated with hypertrophic cardiomyopathy. In contrast, the L43P substitution hinders the formation of fully regulatory-competent thin filaments and severely impairs the Ca2+-regulatory function of reconstructed thin filaments in vitro. Our study reveals distinct mechanisms of pathogenic effects for these two largely similar amino acid substitutions.

Snake venom vascular endothelial growth factors (svVEGFs) are homodimeric proteins comprising two equal subunits bound by two intermolecular disulfide bonds. The main biological effects of svVEGFs are local vasodilatation, increasing of vascular permeability, and hypotension. Barietin, a svVEGF purified from the venom of puff adder Bitis arietans, differs from other representatives of this toxin family by its shortened C-terminal amino acid sequence. No data about barietin effects on hemodynamics are available. Studying those in rats we found that barietin at a dose of 1 mg/kg significantly increased heart rate, exerting only a minor influence on blood pressure. In this respect, it differs from other svVEGFs having a significant effect on blood pressure. We aimed to find the barietin active fragment which may be responsible for its tachycardic activity. Because in literature the fragments corresponding to the so-called loop 1 of VEGF-A (a mammalian homologue of barietin) manifested some affinity to VEGF receptors, we synthesized a peptide homologous to this loop comprising barietin fragment 24-36, with two Cys residues added at the N- and C-termini to form a disulfide. At intravenous injection at a dose of 5 mg/kg, it showed a tendency to increase heart rate without any significant effect on blood pressure, thus demonstrated a trend to reproduce the effect of barietin. So, barietin is the first svVEGF which increases the heart rate without substantial effect on the blood pressure, with its fragment demonstrating a trend for a similar activity.

The search for new regulators of the immune response is an important task of modern immunology. In this work, we have found that Mts1 binds with high specificity to the innate immunity receptor TREM-1 and forms a stable complex with it. The same complex was found on the cell surface of macrophages. The appearance of soluble sTREM-1 in a conditioned medium after Mts1 interaction is considered the starting point of receptor activation. PCR analysis indicates activation of proinflammatory genes IL6, IL1β, and TNF after Mts1 administration. Based on these results, Mts1 can be considered a novel TREM-1 ligand. Using limited trypsinolisis, we have identified the epitopes of Mts1 responsible for TREM-1 activation. Similar to the full-length protein Mts1, the 17aa М7 peptide (41ELPSFLGKRTDEAAFQK57) of the Mts1 protein activates the TREM-1 receptor. Incubation of human lymphocytes with the Mts1 protein of its M7 peptide results in the appearance of cytotoxic subpopulations of NK cells and T lymphocytes, able to lyse HLA-deficient cancer cells via apoptosis or necroptosis. Activated lymphocytes induce apoptosis and necroptosis in HLA-negative tumor cells. The new regulatory peptide may be potentially used for the regulation of inflammatory processes and activation of antitumor immunity.

Protein complexes, assembled by scaffold proteins, act as molecular machines driving development. The mechanosensitive adapter protein Zyxin is a key example, integrating actin cytoskeleton dynamics with gene expression. However, the developmental regulation of its interactions and post-translational modifications remains poorly understood. Here, we characterize the dynamic Zyxin interactome across three early developmental stages of Xenopus laevis (from gastrulation to neurulation) using co-immunoprecipitation coupled with quantitative mass spectrometry (DDA and DIA). We identify stage-specific changes in Zyxin's association with core focal adhesion components, transcriptional regulators and kinases. Furthermore, we uncover developmentally regulated phosphorylation events on isoforms, suggesting dynamic post-translational control of its interactions. Our work provides a comprehensive resource that positions Zyxin as a central orchestrator of cell adhesion, survival, and gene regulatory programs during morphogenesis. These findings underscore the role of Zyxin as a multifaceted regulatory hub, with important implications for understanding tissue homeostasis and related pathologies.

Molecular mechanisms underlying the green insect camouflage have puzzled researchers for over a century. Here, we isolated and identified a green water-soluble protein from the integument of bush-cricket Tettigonia cantans. De novo sequencing and cloning revealed a severely fragmented form of vitellogenins, ubiquitous and multifunctional, but still largely enigmatic glycolipoproteins essential for embryonic development and lacking structural characterization. The distinctive color of the identified chromoprotein results from binding of a remarkable combination of farnesylated bilins (recently identified, tentative heme A catabolites) and xanthophylls, which commensurably absorb light in the 600 to 700 nm and 400 to 550 nm spectral regions and thereby produce a hue that perfectly mimics foliage. The high-resolution crystal structure of this unique ~80 kDa dichromophoric protein, which we named "dibilinoxanthinin" (DBXN), revealed two DBXN protomers, each consisting of three polypeptides, with a novel fold enclosing a large hydrophobic cavity that accommodates two bilins, two luteins, and four phosphatidylcholines, all anchored by hydrogen bonds and giving DBXN unique biochemical and optical properties. Among the green insects tested, some contained yellow and blue chromophores in separate fractions, while others had green proteins similar to DBXN, although not necessarily of the same size. Surprisingly, we isolated and identified a larger vitellogenin proteoform with DBXN-like absorption, from the green huntsman spider Micrommata virescens. These data illustrate striking variations in the DBXN-related pigmentation mechanism among different green arthropods and suggest that vitellogenins may have undergone neofunctionalization, reflecting their potential for functional diversification.

Characteristics of adult patients with high-grade gliomas.

Motility of Hsp70-positive cells emerging from a piece of tumor during cultivation for three days.

Motility of Hsp70-positive cells in a GBM tumor sample detected using laser scanning confocal microscopy during observation for 1 h.

Analysis of the mass-spectrometry data from isolated lipid rafts.

Grade content and co-localization of biomarkers (Hsp70, Nestin, SOX2) on histological preparations of human GBM.

Motility of Hsp70-positive cells in a tumor sample.

Confocal microscopy images of primary glioblastoma cells from patients stained for mHsp70 and Nestin.

African swine fever virus (ASFV) is a large DNA virus that causes a highly lethal disease in pigs and currently has no effective vaccines or antiviral treatments available. We designed a protein switch that combines the DNase domain of colicin E9 (DNase E9) and its inhibitor Im9 with the viral protease cleavage site. The complex is only destroyed in the presence of an ASFV pS273R protease, which releases DNase activity. Several Im9 variants were constructed by inserting the pS273R protease cleavage sequence into different exposed loops. From these, we identified an optimized variant (Im9-1.4) that remains highly stable and tightly bound to DNase E9, suppressing its activity in the absence of protease. Exposure to the ASFV protease results in cleavage of Im9-1.4, rendering it unable to inhibit DNase E9 activity. In vitro assays confirmed that the DNase E9/Im9-1.4 complex becomes catalytically active upon proteolytic digestion with pS273R protease. This virus-triggered 'kill switch' is designed to render pig cells nonpermissive to ASFV by aborting infection via viral DNA degradation. Our study offers a generalizable synthetic biology strategy that uses virus-encoded proteases to trigger dormant effectors, exemplified by this protease-sensing DNase. This synthetic restriction system might be used to develop ASFV-resistant pigs.

Characteristics of patients with brain metastases.

Gating of mHsp70High cells (field R2) and mHsp70Low cells (field R3) by FACS.

Effect of Hsp70 inhibitors PES and JG-98 on cell viability analyzed using the MTT assay.

Virus-neutralizing peptides (VNPs) emerged as promising antiviral drug candidates with unprecedented specificity and cost-effectiveness during the recent COVID-19 pandemic. However, limited avidity, lack of effector functions, short circulatory half-life, and restricted administration routes make them inferior compared to neutralizing antibodies. To address these constraints, a potent VNP that targets the SARS-CoV-2 S protein is combined with Barnase, a highly active RNA-cleaving enzyme from Bacillus amyloliquefaciens. The resulting LCB1-Barnase (LCB1-Bn) chimera retains strong binding affinity for the SARS-CoV-2 S protein and demonstrates a fourfold reduction in IC50 compared to the LCB1 peptide alone in competitive ELISA and in in vitro neutralization tests. In transgenic CAG-hACE2 mice infected with wild-type SARS-CoV-2, intranasal administration of LCB1-Bn significantly improves survival and reduces viral load by 29-fold. To extend circulation life and allow systemic intravenous administration, an albumin-binding domain (ABD) from Streptococcus protein G is added to LCB1-Bn, producing LCB1-ABD-Bn fusion protein which displays a 95-fold increase in serum half-life. LCB1-ABD-Bn exhibits good tolerability at doses below 10 mg/kg and provides protection of SARS-CoV-2-infected CAG-hACE2 animals in 24-hour post-infection intraperitoneal treatment. Cryo-EM reveals the LCB1-ABD-Bn’s tight interaction with S protein RBD domains, highlighting its potential as a promising drug candidate against SARS-CoV-2.