PDZ domains are one of the largest families of short linear motif (or peptide) binding domains in the human proteome. These scaffolding domains are important in signaling and trafficking pathways, such as in the formation of tight junctions or in the postsynaptic density of neurons. PDZ domains are also targeted by several pathogenic viral proteins, including human papillomavirus (HPV), influenza, hepatitis, rabies, coronaviruses, among others. Previously, we investigated the specificity determinants of modulator, or non-motif, residues for two PDZ binding motifs (or PBMs) from the cystic fibrosis transmembrane conductance regulator (CFTR) and HPV16 E6 proteins to better understand differences in relative promiscuity between these similar sequences. To test whether viral PDZ-binding motifs are inherently more promiscuous than endogenous targets, we measured binding affinities for 7 endogenous and 7 viral PBMs across 8 well-studied Class I PDZ domains. Fluorescence anisotropy assays revealed no significant difference in relative binding affinities between viral and endogenous PBMs, indicating that viral versus endogenous origin alone does not explain PBM promiscuity. Our results were consistent with available data from high throughput holdup assays. Taken together, our data support previously reported sequence hotspots, whereby certain PBMs are recognized by large numbers of PDZ domains, likely due to a combination of widely favorable modulator residues.
Spatially resolved protein expression is essential for understanding tissue organization, cellular specialization, and protein function. The open-access Human Protein Atlas database (www.proteinatlas.org) has generated an extensive antibody-based tissue resource for a majority of the human protein-coding genes using conventional immunohistochemistry, enabling body-wide annotation of protein expression across normal human tissues and major cell types. However, single-marker staining often lacks the cellular and subcellular context required to resolve rare cell populations, closely related cell states, or proteins with limited functional characterization. To address this, we established a multiplex tissue resource within the Human Protein Atlas based on a large-scale multiplex immunohistochemistry workflow. The iterative workflow combines optimized antibody panels targeting established markers of cell identity, tissue organization, cellular state, and subcellular structure with candidate proteins of interest. This allows protein expression to be interpreted directly within intact tissue architecture based on expression overlap between candidate proteins and panel markers. In version 25 of the Human Protein Atlas, 1106 proteins have been analyzed using eight multiplex antibody panels across nine tissue settings, including testis, motile ciliated epithelia, salivary gland, endocrine pancreas, and kidney. These panels resolve biological contexts such as stages of spermatogenesis, Sertoli cell and ciliary subcellular compartments, salivary gland acinar and ductal structures, pancreatic endocrine cell types, and nephron segments. Here, we present the design and implementation of the multiplex tissue resource and demonstrate its utility for refining spatial protein annotation across diverse human tissue systems. By providing high-resolution spatial context for protein expression in human tissues, this publicly available resource strengthens functional protein annotation and offers a framework for generating new hypotheses about protein roles in normal tissue biology.
Alzheimer's disease affects tens of millions of people worldwide and is associated with the self-assembly of the Aβ42 peptide into amyloid aggregates. Among the species formed during this process, soluble oligomeric intermediates are the most closely linked to neurotoxicity and are therefore an attractive target for both therapeutic and diagnostic strategies. Their conformational heterogeneity and transient nature, however, have so far hindered the development of reagents that recognize them selectively, and no fully quantitative biomarker of Aβ42 oligomers is widely available. To address this problem, we use a rationally designed conformation-specific single-domain antibody, DesAbO, which binds selectively to Aβ42 oligomers. By using enzyme-linked immunosorbent assay, we show that encoding self-assembling multimerization domains in the DesAbO plasmid yields multimeric variants with increased avidity toward Aβ42 oligomers. In aggregation assays, the multimeric variants inhibited Aβ42 aggregation at concentrations at which the monomeric form was no longer effective, with the SB175 trimer performing best. These results show how multimerization can be used to enhance the recognition of Aβ42 oligomers and offer a route toward diagnostic and therapeutic agents for Alzheimer's disease and other protein misfolding disorders.
The Fanconi anemia complementation group M protein (FANCM)-MHF complex is required for branched DNA recognition in the Fanconi anemia pathway, but structural analysis of the intact complex has been hindered by dissociation of FANCM from the FANCM-associated histone fold (MHF) heterotetramer under crystallization conditions. Here, we used structure-guided disulfide engineering to stabilize the FANCM-MHF interface and test whether local geometry is sufficient to predict crosslinking specificity in a symmetric oligomeric assembly. Using endogenous FANCM Cys759 as an anchor, we designed two MHF2 variants, Q74C and L77C. Both supported oxidation-dependent crosslinking in the context of the FANCM-MHF complex, but with distinct outcomes. Q74C formed the intended FANCM-MHF2 disulfide, enabled crystallization of the intact heteropentamer, and preserved DNA-binding behavior under the tested conditions. In contrast, L77C favored a competing MHF2-MHF2 disulfide and yielded only the MHF heterotetramer after FANCM dissociation. Structural analysis further showed distinct crosslinking states for the two MHF tetramers in the asymmetric unit, consistent with local conformational heterogeneity at the MHF dimer-dimer interface. These results show that geometric plausibility alone does not predict crosslinking specificity in symmetric oligomers. Instead, symmetry-related competing pathways can redirect the reaction toward an alternative assembly state. This study provides a practical route to stabilizing FANCM-MHF and reveals a key design constraint for engineered disulfides in symmetric multimeric assemblies.
Accurate identification of epitope residues is essential for developing biopharmaceuticals and understanding the mechanisms of immune recognition. However, experimental approaches for residue-level epitope mapping remain time-consuming and labor-intensive, while accurate computational prediction of protein-protein interfaces remains challenging. Here, we present MAXTIA, a high-throughput kinetic screening platform that integrates cell-free protein synthesis with high-throughput surface plasmon resonance and demonstrate its application to alanine scanning-based functional epitope mapping. This workflow enables the rapid preparation and kinetic characterization of up to 384 protein variants, allowing the identification of functional epitope residues within 3 days while simultaneously providing binding affinity and kinetic parameters (KD, kon, and koff). We applied MAXTIA to map the epitope of the single-domain antibody (VHH) N1 against the pentraxin domain of neuronal pentraxin-2 (NP2 PTX). Alanine substitutions that cause substantial affinity losses clustered within a localized region on the AlphaFold3-predicted NP2 PTX structure, defining a functional epitope site. These residues closely matched the interface observed in the NP2 PTX-VHH N1 crystal structure, validating the accuracy of MAXTIA. Beyond epitope identification, MAXTIA provides a simple and versatile platform for the quantitative analysis of protein-protein interactions, including high-throughput screening of antibody variants for affinity optimization. This approach should accelerate biopharmaceutical development and facilitate mechanistic studies of molecular recognition.
Metabolic cues regulate the formation of the mitochondrial OXPHOS machinery. These regulatory processes are tightly linked to mitochondrial translation, proteolytic degradation of unassembled subunits, and the formation of supercomplexes, creating checkpoints at which nutrient availability, oxygen tension, and signaling pathways remodel OXPHOS content and activity. In particular, the cytochrome c oxidase (COX) assembly pathway is regulated at multiple steps of its biogenesis in response to cellular demands. COX consists of mitochondrially encoded catalytic core subunits and nuclear-encoded accessory subunits whose coordinated expression, cofactor insertion, and incorporation into the COX enzyme result in optimized electron transport capacity. Consequently, COX assembly depends on numerous dedicated factors and protein isoforms, many of which are expressed in a tissue-specific manner. Through these metabolically regulated processes, cells tune oxidative phosphorylation efficiency, limit reactive oxygen species production, and support context-specific metabolic programs in development, adaptation, and disease.
Retaining glycosyltransferases catalyze the formation of stereochemically conserved glycosidic bonds through mechanisms that remain debated. Using bovine α1,3-galactosyltransferase (α3GalT) as a model, we combine mutagenesis, equilibrium unfolding, kinetics, and molecular dynamics simulations to understand how donor-induced loop ordering promotes catalysis. Alanine-scanning mutagenesis of the C-terminal loop (Thr358-Val368) identified Lys359, Tyr361, and Arg365 as critical for donor binding, catalysis, and ligand-dependent stabilization. In addition, D225A and E317A were inactive and showed minimal ligand-induced stabilization, consistent with impaired metal binding and substrate stabilization, respectively. Donor binding induces an ordered conformation in the C-terminus, reducing its local flexibility by 30% and pre-organizing the active site for catalysis. MD-derived energy profiles differed markedly for the donor (UDP-Gal) and acceptor (lactose) in the ternary complex. In this context, experimental apparent Kₘ values indicate higher donor affinity than acceptor affinity. Our results show that donor binding stabilizes the C-terminal loop, assembling a competent complex for catalysis. These findings support a general coupling between conformational gating, donor stabilization, and the catalytic mechanism in retaining GT-A-fold enzymes.
Intervening proteins (inteins) interrupt host protein sequences and are removed by a self-mediated protein splicing reaction. Inteins are abundant in the microbial world and are often found within essential genes involved in DNA replication, recombination, and repair. Compelling examples of conditional protein splicing, where intein removal and subsequent host protein activation are highly dependent on environmental cues, suggest that some inteins serve as post-translational regulatory elements. Within the cellular context, however, the factors influencing protein splicing are still largely unknown. In this work, we demonstrate that the splicing of an intein from Mycobacterium smegmatis DnaB, an essential helicase, is temperature sensitive. Using two artificial extein systems-a kanamycin intein splicing reporter (KISR) and an in vitro splicing reporter-we show that splicing is inhibited at elevated, yet physiological, temperatures. In accordance with our results from M. smegmatis, we find that overexpression of GroEL in Escherichia coli expressing the KISR system drastically increased antibiotic resistance in a temperature-dependent manner. Under heat stress, we find that GroEL dramatically increases the levels of unspliced precursor protein. Mechanistically, GroEL does not appear to directly promote protein splicing, but rather increases the pool of unspliced precursor, which in turn results in more overall splicing. These findings represent the first description of a chaperone promoting protein splicing, demonstrating the ability of cellular factors to influence intein excision. From a physiological perspective, we hypothesize that a M. smegmatis DnaB intein may act as a switch to inhibit DNA replication under conditions of heat stress. Extending past this, our findings have broad implications for improving the efficiency of intein-based protein engineering technologies.
Protein cages that adopt multiple assembly states provide tractable systems for probing the mechanisms that govern icosahedral shell formation. The Myxococcus xanthus encapsulin shell protein assembles into either a T = 1 shell (60 subunits) or a T = 3 shell (180 subunits), making it an ideal model to investigate how solution conditions bias competing assembly pathways. Here, we examined the reassembly of M. xanthus encapsulin following complete urea-mediated disassembly, systematically varying buffer identity (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], Tris, or phosphate) and ionic strength (0-1M NaCl) at pH 7.5. Assembly products were analyzed by dynamic light scattering and size-exclusion chromatography, with selected fractions characterized by transmission electron microscopy. Buffer identity and salt concentration reproducibly determined the T = 1 versus T = 3 distribution: phosphate buffer strongly favored T = 1 under all conditions, Tris at low ionic strength promoted T = 3 formation, and increasing NaCl progressively shifted outcomes toward T = 1 or unassembled subunits depending on the buffer. Assembled populations remained fixed upon shell closure and did not interconvert upon buffer exchange, demonstrating kinetic trapping. Interface analysis of both assembly states reveals that T = 3 formation requires reversible sampling of multiple weak contacts following formation of one dominant interface, whereas T = 1 relies on repeated formation of a single hydrophobic interface. Together, these results show that M. xanthus encapsulin assembly proceeds through competing kinetic pathways, with the outcome determined by how solution conditions modulate the reversibility and selectivity of early subunit encounters.
Protein-bound uremic toxins (PBUTs) such as 4-ethylphenyl sulfate (4-EPS) challenge kidney failure management due to strong binding to human serum albumin (HSA), thus limiting dialysis clearance. This study examined 4-EPS-HSA interactions using saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), isothermal titration calorimetry (ITC), in silico analysis via molecular docking, molecular dynamics (MD), and molecular mechanics Generalized Born surface area (MM/GBSA) simulations. STD-NMR qualitatively showed competitive binding with the site-specific ligands, warfarin and ibuprofen, indicating interaction with both Sudlow Sites I and II; competitive ITC results also showed competition between these molecules. ITC analysis using a two-component model indicated two distinct binding interactions with association constants Ka1 = 6.62 × 105 M-1 (entropically unfavorable) and Ka2 = 2.71 × 104 M-1 (enthalpically favorable), respectively. Docking models were used to analyze representative binding poses at Sudlow Sites I and II, while MD revealed hydrophobic stabilization at Site I and polar interactions at Site II. MM/GBSA per-residue decomposition identified key stabilizing residues. Circular dichroism measurements showed that the observed binding effects are not accompanied by gross protein unfolding. Comparisons with other PBUTs, including indoxyl sulfate and p-cresyl sulfate, indicated the role of electrostatic and hydrophobic forces involved in binding. These findings advance understanding of PBUT-HSA interactions and inform strategies for improved toxin removal in kidney failure treatments.
Peroxynitrite (PN), generated by the reaction of nitric oxide with superoxide, is a potent reactive nitrogen species (RNS) implicated in nitroxidative protein damage in cardiovascular and neurodegenerative diseases. Hemoglobin (Hb), the principal oxygen-transport protein of erythrocytes, is a key intravascular scavenger and target of PN; however, how controlled PN exposure translates into structural remodeling and aggregation of Hb remains poorly understood. Purified human Hb was exposed to PN across sub-stoichiometric to moderate oxidant excess and characterized using intrinsic fluorescence, UV-visible (UV-vis) spectroscopy, 2,4-dinitrophenylhydrazine (DNPH) carbonyl assay, Thioflavin T (ThT), 8-anilino-1-naphthalenesulfonic acid (ANS), Congo Red (CR), Fourier-transform infrared (FTIR) spectroscopy with Amide I deconvolution, dynamic light scattering (DLS) with zeta potential, x-ray powder diffraction (XRPD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and fluorescence microscopy. At low oxidant loads, PN caused concentration-dependent fluorescence quenching, carbonyl accumulation, and graded UV-visible absorbance increases, consistent with heme-centered oxidation and limited aromatic residue modification. At higher concentrations, Amide I deconvolution revealed collapse of α-helical content from 66.41% to 8.81% and β-sheet enrichment to 71.18%, accompanied by surface charge neutralization, increased hydrodynamic diameter, and enhanced nanoscale roughness. Despite β-sheet accumulation and ThT/CR-positive aggregate formation, XRPD, AFM, SEM, and fluorescence microscopy showed no long-range crystalline order or fibrillar morphology, demonstrating that PN drives Hb toward amorphous and oligomeric assemblies rather than canonical amyloid fibrils. Time-dependent assays at 37°C confirmed that PN modification lowers the nucleation barrier for thermally driven aggregation. These findings define physicochemical thresholds separating protective scavenging from structural destabilization and offer a framework for distinguishing nitroxidative protein aggregation from classical amyloid fibrillation.
Malic enzymes (ME) regulate central carbon metabolism and cellular redox balance, and the mitochondrial isoform ME2 is frequently upregulated in aggressive cancers to support metabolic flexibility and stress resistance. Isoform-selective inhibition has remained out of reach because the catalytic machinery is essentially invariant across the three human enzymes (ME1-3), suggesting that selectivity must arise elsewhere than the active site. Here, we define matched kinetic and regulatory profiles for all three isoforms, highlighting key differences in substrate and cofactor dependence and metabolic regulation. Our x-ray crystal structures show that the active-site inhibitor 3',6'-dihydroxy-4,4″-dimethoxy-[1,1':4',1″-terphenyl]-2',5'-dione (NPD-389) occupies a conserved, metal-coordinating pose in all three isoforms, explaining its non-selective inhibition observed in enzyme assays. We further identify a cryptic pocket adjacent to the active site that is engaged by our probe molecule, flavianic acid (FLA), and accessible only in the mitochondrial enzymes ME2 and ME3. FLA binding locks an open, inactive enzyme conformation in place, with kinetic studies revealing isoform-specific allosteric responses and suggesting that this pocket may be a native regulatory site sensitive to the mitochondrial metabolic state. Our cellular viability assays suggest that molecules exploiting this cryptic pocket reduce proliferation in cancer cell models with elevated ME2 expression. Conformational dynamics, rather than sequence divergence at the catalytic center, can therefore generate isoform-specific regulatory and inhibitory mechanisms within a conserved enzyme family.
Protein aggregation is increasingly recognized as a biologically relevant process in viral proteins, yet the molecular determinants governing such phenomena remain poorly understood. Intrinsically disordered proteins (IDPs) are ubiquitous in viral proteomes, where conformational plasticity not only enables functional diversity but also permits aberrant self-assembly. Members of the Paramyxoviridae family, including henipaviruses such as the Nipah and Hendra viruses (two biosafety level-4 pathogens), encode the P, V, and W proteins that share a common intrinsically disordered N-terminal domain (NTD). V and W are virulence factors that undergo fibrillation. Here, we explored the aggregation landscape of seven homologous PNT1 subdomains from the NTD that harbors a conserved cryptic amyloidogenic region (CAR). By integrating Taylor dispersion analysis (TDA), Raman spectroscopy, and all-atom molecular dynamics (MD) simulations, we unraveled both early assembly kinetics and structural outcomes. Despite the conserved CAR motif, the PNT1 homologs exhibit strikingly divergent aggregation behaviors. TDA revealed distinct oligomerization pathways with variations in nucleation, growth kinetics, and monomer recruitment, indicating virus-specific pathways. Raman spectroscopy unveiled substantial variations in β-sheet content, side-chain packing, and aromatic residue environments, while MD simulations showed that conformational heterogeneity in the CAR flanking regions modulates hydrogen-bond networks and structural stability. These findings indicate that the sequence context beyond a conserved amyloidogenic core governs the aggregation landscape of Henipavirus P, V, and W proteins. Evolutionary diversification modulates conformational heterogeneity and assembly pathways, giving rise to distinct structural outcomes. This work sheds light on the molecular grammar underlying viral protein fibrillation with potential implications for viral pathogenicity.
The SARS-CoV-2 main protease (Mpro) is essential for viral replication and functions as a homodimer, with dimerization being critical for catalytic activity. Mpro contains an unusually high number of cysteine residues. Among these, C117 and the catalytic nucleophile C145 can form a reversible disulfide bond under oxidative conditions. To investigate the structural and functional consequences of irreversible oxidation of these residues, we generated oxidation mimics by substituting these residues with aspartate (C117D and C145D), to mimic the sulfinic acid oxidation state. Kinetic assays revealed that both variants are catalytically inactive, with C117D exhibiting at least 100-fold lower activity than wild-type (WT) Mpro. Small-angle X-ray scattering (SAXS) and differential scanning fluorimetry (DSF) demonstrated that C117D adopts a monomeric, destabilized state in solution, whereas C145D retains a dimeric conformation similar to WT. Crystallographic analysis of C117D revealed a dramatic rearrangement of domain III, involving a ~40° rotation relative to domains I and II, and disorder in the N- and C-terminal regions, disrupting the canonical dimerization interface. Local structural changes propagated from the C117D site to the active site, including an unwound oxyanion loop that provides structural evidence for the observed inactivity. Rescue of the stable, dimeric state for C117D was achieved through formation of the covalent C117D-GC376 complex. These findings establish a high-resolution structure of monomeric full-length SARS-CoV-2 Mpro and underscore a critical role of C117 in maintaining dimerization and enzymatic function. Furthermore, the unique monomeric domain II-III interface present in the monomeric form may offer opportunities for allosteric inhibitor design targeting Mpro dimerization.
Neutron crystallography was used to determine a 2.5-Å resolution all-atom structure of macrophage migration inhibitory factor (MIF) interacting with 3-(4-hydroxyphenyl)-pyruvate (HPP). MIF is a pro-inflammatory, pro-tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF-catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction-derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine-32 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N-terminal Pro-1 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room-temperature synchrotron x-ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation-induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.
It has been known since at least the 1980's that the structure and chemistry of membranes and membrane proteins are matched. Exploiting this fact, a graph neural network model of proteins was trained on experimentally determined membrane protein structures to predict the native membrane environment of transmembrane domains from their structure. The algorithm, "GPSforTMDs," learns to generalize about membrane protein structure, obtains overall performance that is competitive with sequence-based methods, and obtains exceptional performance for some categories of membrane environment, even when training examples are few. Other categories it finds more challenging, in some cases for clear reasons (for example, compatibility of TMDs with membranes along the secretory pathway), and in other cases that are mysterious (mistaking archaeal TMDs for bacterial, and vice versa). The results motivate the need for high quality databases reporting TMD localization, and suggest that peering inside the algorithm will reveal new "rules" for membrane proteins. The code and associated database is available at https://github.com/bivekpok/GPSforTMDs.
Somatic mutations and antibody clone selection occur in B-cell hyper-evolution over extremely brief periods of time. Herein, we developed a technique for antibody screening by immunizing alpacas with antigens and observing antibody sequence transitions over time, a method we named Tracking the Evolution of Antibodies over time. This technique allows the observation of sequence transitions of somatic mutations in antibody populations originating from the same genome. The accumulation of somatic mutations was significantly associated with enhanced antigen-binding capacity and reduced stability in clusters. In comparison with the first clone to emerge in this cluster, numerous clones exhibited a decline in thermal stability, with a maximum variation of 21°C. Somatic mutations within the clusters demonstrating high similarity were observed to be concentrated in CDR-1, CDR-2, and FR-3. This suggests that these mutations have a significant impact on binding capacity and stability. However, the correlation between antigen binding capacity and stability was insignificant and weak. The thermal stability of antibodies correlated with acid and alkali resistance, with clones exhibiting lower thermal stability demonstrating higher acid and alkali resistance in antigen-antibody complexes. The results indicate that in the antibody selection process, the strength of antigen binding involves optimizing stability, with antibodies with more flexible structures being selected.
Glucagon is a well-established therapeutic peptide, widely used to treat hypoglycemia. Like many peptide drugs, it offers advantages such as specificity, biocompatibility, and high affinity for receptor targets, but suffers from limited physical and chemical stability. In particular, improper conditions can promote the formation of amyloid fibrils, leading to loss of biological activity and, in some cases, cytotoxicity. Understanding the conditions that modulate the structural behavior of glucagon is therefore crucial. Currently, two injectable formulations are available on the market: a lyophilized vial of glucagon with lactose at acidic pH, and a more recent auto-injector ready-to-use (RTU) formulation containing glucagon in dimethyl sulfoxide (DMSO) with trehalose. In this study, we investigated the conformational properties of glucagon in these two marketed formulations and compared them with glucagon dissolved in aqueous solution at pH 3.5, a metastable condition prone to aggregation. Preliminary circular dichroism and fluorescence spectroscopy were used to confirm glucagon stability over time; subsequently, NMR analysis showed that structural destabilization consistently begins at the C-terminal region, while the 11Ser-Leu14 segment remains structured across all environments. These findings highlight two key determinants of glucagon stability and aggregation. Strategies that preserve the integrity of the C-terminal region while stabilizing the 11Ser-Leu14 motif may improve peptide solubility and extend shelf life, providing a rational basis for the design of next-generation glucagon formulations for emergency use.
Snake envenomation remains a critical global public health challenge. In Brazil, the genus Bothrops is responsible for most accidents, typically resulting in coagulopathy, hemorrhage, and renal failure. Despite its clinical prevalence, the molecular mechanisms driving plasma protein disturbances remain poorly understood. In this study, we applied mass spectrometry to characterize the plasma proteome and peptidome of mice following a sub-lethal dose of Bothrops jararaca venom and subsequent single dose antivenom treatment at timepoints 3, 6 and 24 h. From a total of 781 identified proteins, quantification of 590 revealed systemic pathological signatures such as the lower abundance of key coagulation and complement proteins, indicating coagulopathy and immune disruption. Simultaneously, an increase in tissue damage markers, innate immune response proteins, and Damage-Associated Molecular Patterns was detected. In the acute phase, the biological response was highly conserved regardless of antivenom administration, while by 24 h the group treated only with venom exhibited prolonged inflammation and metabolic stress, whereas the venom + antivenom group transitioned to cellular recovery and detoxification. The peptidomic analysis identified a large degradome of 1964 unique peptides, showing proteolysis of fibrinogen, apolipoprotein A-I, complement factors and protease inhibitors, among others. The analysis of cleavage sites implicated the activity of cysteine-, metallo-, and serine proteases in this process. Our results demonstrate that B. jararaca venom induced a rapid, dramatic systemic imbalance and proteostasis disruption that was only partially mitigated by antivenom. These findings underscore the necessity of developing complementary therapies that target both the primary toxins and secondary host-driven pathology.
Understanding the link between phase separation (PS) of disease-linked proteins to form liquid-like condensates, and their aggregation, requires insights into the conformational changes that the proteins undergo inside the condensates as they age and become solid-like. In this study, the structural changes undergone by the mouse prion protein (moPrP) inside condensate induced by PS have been characterized. Hydrogen-deuterium exchange in conjunction with mass spectrometry reveals that the N-terminal region (NTR), which is unstructured in monomeric native moPrP, gains significant stable structure as the condensate ages. The structured C-terminal domain remains native-like, albeit with higher stability, but subtle changes are seen. Conformational change initiates in the native monomer inside the condensate, with different regions undergoing rapid, slow, or no conformational change as it ages. The β1-α1 loop undergoes rapid conformational change to lose stability, while the NTR gains structure slowly concomitantly with conformational change at the C-terminal end of α3. Infrared (IR) spectroscopy shows that β-structure forms, IR and circular dichroism spectroscopy indicate that secondary structure becomes heterogeneous, and dynamic light scattering measurements reveal that the protein forms oligomeric nanoscale assemblies as the condensate ages. The formation of the nanoscale assemblies inside the condensate is responsible for the fraction of protein present as mobile monomer decreasing with time of aging, when fluorescence recovery after photobleaching is quantified. Such assembly and the resultant conformational change in the protein appear to be responsible for a change in its material properties of the condensate, which manifests itself as a liquid-like to solid-like transition.
