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bioRxiv2026

Subchronic Toxicity Study of Nitric Oxide Nano Bubbles Injection in Sprague-Dawley Rats

Following the success of hydrogen nanobubble technology, development has expanded to include similar products with other gases, such as nitric oxide (NO). In previous studies, NO donors have been frequently used for both therapeutic and diagnostic purposes. However, the use of NO in nanoformulations has received little attention thus far. Thus, 90 days of subchronic toxicity testing in Sprague-Dawley rats were used in this study to assess potential adverse effects of intravenous administration of NO nanobubbles (NONB). Hematological factors, serum biochemistry, and histology of the liver, kidney, heart, lung, and spleen were investigated. The findings of the intravenous NONB injection trial in rats, using graded doses of 0.01 mL, 0.04 mL, and 0.06 mL, demonstrated no deaths during the 90-day treatment period. Biochemical parameters for liver function, lipid profile, kidney function, and serum electrolyte levels remain within acceptable limits, with slight variations in sodium and potassium levels. This condition was also supported by histological findings in the liver, kidney, and spleen, indicating mild to moderate damage, while the heart and lungs were normal. Thus, the intravenous administration of NONB at a dose of up to 0.06 mL in this subchronic toxicity test remains safe. Further studies with adjustments to the test formulation are highly encouraged until the clinical trial stage in humans.

pharmacology and toxicology↗

bioRxiv2026

Decreased tRNA abundance contributes to decreased translation elongation rate in a prolonged mitosis

Cells undergo dramatic structural rearrangements upon entering mitosis. In addition, the biochemistry of mitotic cells is dramatically altered, including a significant decrease in protein synthesis. The majority of studies of mitotic translation have used cells synchronized by cell cycle altering drugs in transformed cells and much less is known about mitotic translation in primary cells under native conditions. Previous work has found that mitosis activates the integrated stress response (ISR) to trigger eIF2 phosphorylation, but little is known about the input for this response. In this study, we focus on mitotic translational regulation in an immortalized, non-transformed cell line. We confirm decreased mitotic protein synthesis in primary cells and under native conditions. Additionally, we confirm activation of the ISR by phosphorylation of eIF2 during both normal and prolonged mitosis. Interestingly, we also find that decreased translational elongation during mitosis, as evidenced by increased eEF2 phosphorylation and a slower elongation rate. Analysis of mitotic ribosome profiling data revealed an increase in pausing at Alanine-GCG codons during mitosis and a decreased abundance of its cognate tRNA-AlaCGC by northern blotting. Decreased tRNA-AlaCGC is likely sustained by the inability to synthesize additional tRNA due to RNAPol III inhibition in mitosis, yielding an stronger effect with an increased time in mitosis. These results suggest that decreased translation elongation in mitosis triggers inhibition of initiation to decrease global protein synthesis.

cell biology↗

bioRxiv2026

Type IV Secretion System Drives Lipid Mixing

Type IV secretion systems (T4SSs) are versatile molecular machines used by bacteria to secrete protein effectors into host cells, promoting pathogenesis, and to transfer DNA between bacteria through conjugation, driving horizontal gene transfer. Most, like Dot/Icm of the pathogen Legionella pneumophila (L. pneumophila) or Escherichia coli (E. coli) RK2, are primed for substrate delivery only upon contact with a target membrane, but mechanisms are unknown. A pilus could bind a receptor to initiate priming, but many T4SSs, especially those that deliver effectors, lack a pilus. Here, we present evidence that T4SSs are primed by direct contact with target membrane lipids. Combining fluorescence assays with genetics and biochemistry, we found that Dot/Icm drives lipid exchange between bacterial cells and between bacteria and synthetic membranes containing only lipids. Lipid exchange requires membrane contact but does not require ATP hydrolysis or even full complex assembly. Minimally, the outer membrane core complex protein DotG needs to be present in at least one of the apposed membranes. We similarly observed lipid mixing with the simpler E. coli RK2 T4SS, where we could follow lipid mixing and plasmid transfer simultaneously. We found that lipid mixing always preceded or accompanied plasmid transfer, suggesting it may be part of the contact-dependent priming mechanism. Lipid mixing was inhibited or promoted by lipids that inhibit or promote membrane fusion, respectively. Lipids inhibiting lipid mixing also inhibited substrate transfer. Together, our results suggest that initial contact between DotG outer segments and target membrane lipids promotes lipid mixing as part of the mechanism that primes T4SS for substrate translocation. HighlightsO_LIThe Legionella pneumophila Dot/Icm secretion system requires contact with a target membrane for effector translocation, but how membrane contact primes the machinery for this is unknown. C_LIO_LIWe found that Dot/Icm drives contact-dependent lipid mixing between bacteria or between bacteria and liposomes, showing that Dot/Icm priming does not require a protein receptor. C_LIO_LILipid mixing also occurs with the simpler Type IVA system (Escherichia coli RK2) during conjugation, suggesting lipid mixing is a general feature of Type IV Secretion System function. C_LIO_LIThe core complex protein DotG is sufficient to drive lipid mixing, suggesting DotG-target membrane interactions may destabilize the target membrane. C_LI

microbiology↗

bioRxiv2026

Point-Cloud Enhancement and Structural Interpretation Framework for Cryo-EM Maps

Cryo-Electron Microscopy (cryo-EM) has become a cornerstone of modern structural biochemistry, enabling the reconstruction of 3D protein maps at near-atomic resolution. Despite its transformative impact, interpreting maps remains challenging. Structural heterogeneity and molecular flexibility often produce low-resolution densities, which post-processing can only sharpen in well-ordered regions. This leaves flexible areas either poorly resolved or lost. Recent deep-learning approaches have demonstrated strong potential for enhancing cryo-EM maps, enabling an extended interpretation of cryo-EM densities. Most of these methods operate on small volumetric blocks, which restricts the receptive field of the model and prevents it from leveraging the broader structural context of the protein. To address this limitation, we introduce CryoPC, a model that enriches local block-based processing with a compact point-cloud representation of the entire map. By conditioning the network on this global representation, CryoPC is able to capture both local and distant structural features, allowing it to make more globally consistent predictions. We demonstrate that incorporating this global context consistently improves enhancement quality across a variety of samples. Furthermore, CryoPC achieves great performance compared to existing methods of similar speed, offering a practical and scalable solution for cryo-EM map enhancement. Finally, we present a framework for assesing a quality of enhanced maps using metrics from the Phenix package.

bioinformatics↗

bioRxiv2026

PySteMoDA: An Open Source Python Package for the Analysis of Steered Molecular Dynamics Simulations Data

Molecular dynamics simulation is a powerful computational technique used for predicting and understanding the dynamic behavior of biomolecular systems. Steered molecular dynamics (SMD) simulations enable the study of force-induced processes in biomolecules, effectively mimicking single-molecule force spectroscopy experiments probing protein unfolding and receptor-ligand unbinding. Given the stochastic nature of these mechanical events, accurately exploring the dynamic behavior of biomolecules and extracting accurate physical information requires several in-silico experiments. This includes performing many pulling simulations at different velocities or force loading rates. The large amount of data obtained from these simulation sets requires efficient automated data processing tools. We present PySteMoDA, a novel Python package with a user-friendly graphical interface specifically designed for constant-velocity SMD data analysis. The automated force peak detection methods reduce user bias, improve accuracy, and accelerate data analysis. The package also allows identification of residues involved in mechanical events through computation of the time-dependent mechanical work and correlation factors between residue pairs. This package not only addresses automated data processing in SMD simulations and accurate parameter extraction, but also significantly enhances accessibility and usability. Through PySteMoDA, users can efficiently analyze simulation data without the barrier of coding, facilitating a wider range of investigations and insights in the field of computational biochemistry and biophysics.

biophysics↗

bioRxiv2026

Microtubule occupancy at kinetochores links checkpoint silencing with mitotic memory

The spindle assembly checkpoint (SAC) promotes faithful chromosome segregation by delaying mitosis until all kinetochores attach to spindle microtubules. Paradoxically, a p53-dependent memory mechanism--the mitotic stopwatch" --blocks daughter cell proliferation after unusually prolonged mitoses. How the SAC coordinates with the mitotic stopwatch remains unknown. Here, we found that microtubule occupancy at kinetochores is a cornerstone linking SAC silencing with mitotic memory. By combining live-cell with super-resolution microscopy, FRAP, laser microsurgery, biochemistry and molecular perturbations in Indian muntjac fibroblasts, we show that SAC silencing at kinetochores is gradual, non-uniform and confined to highly-localized microtubule attachments. Augmin promotes timely SAC silencing with high microtubule occupancy at kinetochores, whereas MPS1/CDK1 inhibition bypasses this requirement. Conversely, low microtubule occupancy delays SAC silencing, increases segregation errors and blocks daughter cell proliferation due to mitotic stopwatch surveillance. Thus, timely SAC silencing with high microtubule occupancy avoids "bad memories" of mitosis to allow daughter cell proliferation.

cell biology↗

bioRxiv2026

Bacterial exo-α-sialidases subvert the complement system through desialylation

The complement system is a central pillar of innate immunity, yet how bacterial glycan-modifying enzymes subvert complement function remains poorly understood. Bacterial sialidases remove terminal sialic acids from host sialoglycans, but their direct impact on complement immunity is unclear. Here, we investigate the impact of six sialidases from five human pathogens on complement immunity using an integrated approach combining genetics, biochemistry, glycobiology, mass spectrometry, and structural biology. We demonstrate that major complement components (IgG, C1q, C4, and C5) and regulators (Factor I, Factor H, and C4bp) are sialylated, and that bacterial sialidase-mediated desialylation suppresses complement activation and surface deposition, thereby enabling complement evasion. Despite extensive sequence diversity, biochemical and structural analyses reveal that all examined sialidases share a conserved six-bladed {beta}-propeller catalytic domain and cleave both N-acetylneuraminic and N-glycolylneuraminic acids, the predominant mammalian sialic acids. Together, these findings uncover a conserved mechanism by which diverse bacterial pathogens disable complement immunity through desialylation.

microbiology↗

bioRxiv2026

Pioneer-factor activity requires stable chromatin occupancy mediated by both sequence-specific binding and disordered protein domains

Pioneer transcription factors overcome the restrictive barrier imposed by chromatin to drive cell-fate specification, yet how their domains collectively support this activity remains unclear. Here, we use the deeply conserved pioneer factor Grainy head to define the protein-intrinsic features that govern pioneering activity. By integrating biochemistry, genomics and quantitative live-cell imaging, we determined that both the conserved DNA-binding domain and the extended, intrinsically disordered N-terminus are required for the stable chromatin occupancy that supports access to closed chromatin and the induction of chromatin accessibility. The disordered N-terminus supports pioneer activity through interactions that do not rely on strict amino acid sequence but instead overall composition. While our results show that pioneering activity depends on the combinatorial contributions of structured and disordered domains, mitotic retention depends solely on sequence-specific DNA binding. These results support stable chromatin occupancy mediated by multiple protein domains as necessary for pioneering function and that this is separable from the mechanisms required for mitotic retention.

molecular biology↗

bioRxiv2026

HDAC11 Regulates RNA Splicing via De-Fatty Acylation of SF3B2

Histone deacetylase 11 (HDAC11) is a lysine de-fatty acylase whose cellular substrates and mechanisms remain incompletely defined. Here, using metabolic labeling, mass spectrometry, click chemistry, and standard molecular biology, we show that SF3B2 is modified by lysine myristoylation at K10 and that HDAC11 efficiently removes this modification in cells, establishing SF3B2 as a direct enzymatic substrate. A de-myristoylation mimetic mutant (SF3B2 K10R) exhibits altered pre-mRNA binding activity in a context-dependent manner. In HCC cells, loss of SF3B2 lysine myristoylation enhances SF3B2 association with androgen receptor (AR) splice variant loci and promotes alternative splicing towards the AR-v7 variant. Consistently, HDAC11 overexpression increases, and HDAC11 knockdown decreases, the AR-v7/AR-FL splice isoform ratio in HCC cells in a manner requiring HDAC11 catalytic activity and recapitulated by SF3B2 K10R. In contrast, modulation of HDAC11 does not alter AR splicing in prostate cancer cells, indicating cell type specific regulation. Together, these findings establish lysine myristoylation as a reversible regulatory modification on a spliceosomal component and reveal HDAC11-catalyzed de-myristoylation of SF3B2 as a mechanism that can tune alternative splicing in liver cancer cells. In BriefClements et al. utilize metabolic labelling, mass spectrometry, click chemistry, and protein and RNA biochemistry to establish that a histone deacetylase enzyme, HDAC11, can influence RNA splicing through de-fatty acylation of the RNA splicing factor SF3B2. De-fatty acylation of SF3B2 at K10 by HDAC11 modulates SF3B2s pre-mRNA binding to AR splice variant loci, thereby driving alternative splicing of the AR-v7 variant in a cell type dependent manner. This work provides direct mechanistic evidence linking an HDAC to RNA splicing, identifies a reversible lipid modification on SF3B2, and expands current understanding of post-translational regulation of spliceosomal proteins and HDAC11 de-fatty acylation substrates. HighlightsO_LIHDAC11 de-fatty-acylates SF3B2 at K10, revealing a previously unrecognized modification on SF3B2. C_LIO_LISF3B2 de-fatty acylation enhances alternative splice-site binding in liver cancer cells. C_LIO_LIHDAC11 regulates RNA splicing through enzymatic de-fatty acylation of a spliceosomal protein. C_LI

molecular biology↗

bioRxiv2026

The Yersinia pestis virulence effector YopM binds to a key regulatory site on the human pyrin death domain to inhibit inflammasome activation and effector-triggered immunity

Bacteria in the genus Yersinia use a type III secretion system to inject several effectors into host cells to disrupt multiple signaling processes. Two effectors disrupt host signaling by inactivating RhoA GTPases. Inactivation of RhoA triggers pyrin inflammasome assembly in infected phagocytes leading to a protective immune response. A third protein, YopM, is an essential virulence factor that counteracts effector-triggered immunity by inactivating pyrin. YopM has a leucine-rich repeat (LRR) domain and hijacks host kinases to inactivate pyrin by phosphorylation. Previously, the LRR domain of Yersinia pestis YopM was implicated in binding to an N-terminal region of human pyrin, which includes the eponymous pyrin domain (PYD), a member of the death domain family. However, the interaction mechanism was undefined. Using a bacterial two-hybrid assay, protein biochemistry and X-ray crystallography, we determined that the concave surface of the YopM LRR domain binds to the PYD. Identification of critical residues in the interaction revealed that YopM binds PYD R42, an amino acid important for positive and negative regulation of pyrin. YopM binding to the PYD was reduced by R42W, a codon change associated with the autoinflammatory disease Familial Mediterranean Fever (FMF). Furthermore, we show that YopM codon substitution variants defective for PYD binding fail to inhibit the pyrin inflammasome in human monocytes infected with Yersinia. In addition, we found that PYD binding is dispensable for YopM to inhibit the inflammasome in murine macrophages, suggesting this effector uses a distinct mechanism to target mouse pyrin. These results define how Y. pestis YopM binds the human PYD and provide insights into how this interaction likely selected for pyrin gain of function variants resulting in FMF. SIGNIFICANCEPyrin is an inflammasome sensor encoded by the MEFV gene that is notable for its role in the autoinflammatory disease Familial Mediterranean Fever (FMF) and immunity to the plague agent Yersinia pestis. Y. pestis normally prevents inflammasome activation using the virulence factor YopM which binds pyrin and inhibits effector-triggered immunity. Gain of function mutations in MEFV that cause FMF were likely selected during historic plague pandemics to counteract YopM. Here, we defined the molecular mechanism of the YopM-pyrin interaction, which revealed that the effector binds to a key site of positive and negative regulation on the N-terminal death domain. These findings have important implications for understanding how YopM promotes pathogenesis and likely selected for gain of function mutations in MEFV.

microbiology↗

bioRxiv2026

A sterol reductase responsible for the unusual 8(14)-unsaturation in bacterial sterol production and degradation

Sterols are a class of lipids that play a crucial role in human health through their essential physiological roles and as a point of interaction between commensal and pathogenic bacteria. The biosynthesis and modification of these lipids is a well-characterized process in many eukaryotes and increasingly in bacteria. However, the proteins responsible for formation of the unusual 8(14)-unsaturation found in the sterols produced by aerobic methanotrophs, dinoflagellates, nematodes, and marine sponges, remains unknown. Here, we utilize a heterologous expression system to identify a bacterial 8,14-sterol reductase (8,14-Bsr) responsible for generating the 8(14)-unsaturation in the aerobic methanotroph Methylococcus capsulatus. This enzyme modifies the direct product of C-14 demethylation, reducing one double bond in the nuclear core structure and isomerizing the other to produce an 8(14)-sterol. We subsequently tested the requirement of putative active site residues for catalysis through site directed mutagenesis, identifying residues likely involved in interacting with the sterol substrate and directly catalyzing this reaction. Bioinformatic analysis of the distribution of 8,14-Bsr reveals it is unique to the bacterial domain, found primarily in the Methylococcaceae family, the Mycobacteriales order, and yet uncultured members of the Myxococcota phylum. Further phylogenetic analysis of 8,14-Bsr suggests it shares an evolutionary history with the C-14 demethylase in these organisms and that these two enzymes were likely inherited together. These results provide insight into novel sterol biochemistry, further delimiting sterol biosynthesis in the bacterial domain from eukaryotes and illustrating the importance of molecular characterization to identify bacterial proteins that interact with sterols. ImportanceMany of the eukaryotic proteins required for the biosynthesis of sterols, such as cholesterol, have been characterized. However, the pathways governing analogous processes in the bacterial domain are less characterized. Here, we identify an 8,14-sterol reductase in aerobic methanotrophs. This enzyme carries out a unique biochemical reaction, saturating and isomerizing double bonds in the nuclear core structure to produce an 8(14)-sterol. This reductase is restricted to the bacterial domain, further separating the evolution of bacterial sterol production from eukaryotes. Additionally, we find this reductase is prevalent in members of the sterol degrading order Mycobacteriales, highlighting a potential role for this protein in the remodeling of host sterol production by these pathogens.

microbiology↗

bioRxiv2026

Expanded stoichiometric model of chondrocyte metabolism: response to cyclical shear and compressive loading

Cartilage deterioration is a hallmark osteoarthritis, and there is substantial interest in developing strategies for cartilage repair. Cyclical mechanical stimulation has been known for decades to drive synthesis of cartilage matrix proteins. Matrix synthesis requires activation of central metabolism for producing precursors to non-essential amino acids required for protein translation. However, there are gaps in knowledge regarding how mechanical stimuli affect chondrocyte central metabolism. Here, we find that cyclical shear and compression drive differences in chondrocyte central metabolism in a sex-dependent manner. Based on established biochemistry, we developed and tested a stoichiometric model containing 139 metabolites and 172 reactions from central metabolism that includes production of key cartilage matrix proteins. We then used experimental metabolomics data from shear and compressive stimulation of osteoarthritic chondrocytes to constrain this model and ran multiple simulations examining the potential for producing matrix proteins and ATP. Our results show that both shear and compression can stimulate osteoarthritic chondrocyte metabolism in a manner consistent with production of cartilage matrix proteins, with notable differences between male and female chondrocytes. Additionally, and importantly, our simulation results suggest that nitrogen availability is a key limitation to chondrocyte synthesis of matrix proteins. These results are a starting point for using central metabolism of chondrocytes to optimize synthesis of matrix proteins for cartilage repair. For example, increasing glutamine levels in the presence of cyclical compression has potential to increase production of both types II and VI collagen. These strategies have potential for improving cartilage tissue engineering and repair.

bioengineering↗

bioRxiv2026

ENHANCING TUMOR PERFUSION AND NANOMEDICINE DELIVERY VIA ENDOGENOUS NITRIC OXIDE RELEASE BY METHYL PALMITATE NANOPARTICLES

Despite a few clinical successes, the efficacy of cancer nanomedicines remains limited by rapid clearance by the mononuclear phagocytic system and poor permeation across the abnormal tumor vasculature. We previously showed that methyl palmitate nanoparticles (MPN) can safely and reversibly inhibit the phagocytic activity of immune cells for several hours, thereby improving tumor accumulation and the efficacy of systemically administered nanomedicines. Here, we demonstrate that, on a shorter time scale, MPN can induce vasodilation, introducing an additional mechanism to enhance the accumulation of therapeutic agents within the malignant tissue. Upon internalization by macrophages and endothelial cells, MPN trigger the release of endogenous nitric oxide (NO), a key mediator of vasodilation, in a concentration-, and time-dependent manner. Following MPN administration, raster-scanning optoacoustic mesoscopy (RSOM) revealed vasodilation across multiple tissues, with the strongest effect observed in tumors. To assess enhanced tumor accumulation, we injected 70 kDa fluorescent dextran and demonstrated via histology a markedly increased fluorescence signal exclusively in MPN-treated tumors compared to controls 24 hours later. In addition, positron emission tomography (PET) imaging of 89Zr-labeled clinical iron oxide nanoparticles (Feraheme) showed significantly greater tumor accumulation after a 15-minute MPN pretreatment. Finally, general serum biochemistry panels and histological analyses of major organs in healthy mice revealed no toxicity following either single or repeated MPN dosing. Overall, this study demonstrates that MPN-induced vasodilation occurring within minutes enhances intra-tumoral deposition of macromolecules and small nanoparticles. Together with their longer-term effects on phagocytosis inhibition, these findings indicate that MPN can improve therapeutic delivery through complementary, time-dependent mechanisms that increase tumor perfusion and vascular permeability.

bioengineering↗

bioRxiv2026

An A-rich linker between dengue virus tandem xrRNAs facilitates functional coordination

Orthoflaviviruses use programmed resistance to host 5' to 3' exoribonucleases to produce subgenomic flaviviral RNAs (sfRNAs) during infection. This resistance is conferred by exoribonuclease-resistant RNA (xrRNA) structures that often occur in tandem and whose function can be coupled. In dengue virus serotype 2 (DENV2) this coupling results in changing patterns of sfRNA identity and abundance linked to the ability of the virus to adapt to host vs. vector infections. The physical basis of this coupling was unknown. Using a combination of virology, biochemistry, bioinformatics, structural biology, and biophysics, we explored the structural and sequence determinants of tandem xrRNA coupling in DENV2. We discovered that the spatial proximity, order, and structural integrity of the tandem xrRNAs are all important for coupling. Furthermore, an unpaired A-rich linker that lies between the two xrRNAs is essential in stabilizing a specific structure that correlates to coupling. This A-rich sequence likely forms tertiary contacts with an adjacent stem-loop structure to form a physical bridge between the two xrRNAs, a finding that is supported by a mid-resolution cryoEM map of the DENV2 tandem xrRNAs. Disruption of the structure of this bridge by mutation changes the relative orientation or spacing between the tandem xrRNAs, which is correlated to their functional coupling. These findings provide an explanation for the coupling between tandem xrRNAs and suggests new mechanistic hypotheses. IMPORTANCEDengue virus (DENV) generates non-coding subgenomic flaviviral RNAs (sfRNAs) that affect several cellular pathways and are important for successful infection. These sfRNAs are formed by structured RNA elements in the viral genome called exoribonuclease-resistant RNAs (xrRNAs), which fold into a distinct three-dimensional topology to block degradation by host cell exoribonucleases and often occur in tandem. Specific patterns of sfRNAs made during infection are important for host vs. vector fitness, and in DENV2 this pattern depends on functional coupling between tandem xrRNAs. However, the source of this functional coupling was unknown. We determined that an unpaired A-rich linker between the tandem xrRNAs is necessary for creating a structural bridge between the tandem xrRNAs. This bridge appears to favor a specific orientation between the tandem xrRNAs that is correlated to coupling and therefore to the patterns and relative abundance of sfRNAs produced during infection.

microbiology↗

bioRxiv2026

In silico analysis reveals the structural basis of TomEP specificity, a tomato extensin peroxidase

BackgroundExtensin peroxidases (EPs) are class III plant peroxidases and are responsible for intermolecular covalent crosslinking of extensin (EXT) monomers to create scaffolds within plant cell walls. The formation of these scaffolds impacts plant development, mechanical wounding, and response to pathogen attacks. Therefore, elucidating the molecular mechanism controlling covalent crosslinking of EXT monomers is crucial for understanding cell wall deposition and potentially improving plant growth and adaptation. The focus of this work is to use in silico analysis to determine the structural characteristics of an EP from tomato (TomEP) to elucidate its specificity for crosslinking of EXT monomers. ResultsIn this study the two-dimensional (2D) and three-dimensional (3D) structures of TomEP were determined using several advanced bioinformatics tools and compared to two other peroxidases: GvEP1 (a known EP) and HRP-C (having a low affinity for EXT substrates). The results revealed that TomEP is a stable and hydrophilic protein with high thermal stability. The heme binding pockets of TomEP and GvEP1 have more hydrophobic residues and larger volume and pocket area compared to HRP-C. Molecular docking at the active site, which includes a heme heteroatom, showed that the ligands consisting of the hydrophobic Tyrosine-X-Tyrosine [-Y-X-Y-] motifs (i.e., [-Y-K-Y-], [-Y-V-Y-], and [-Y-Y-Y-] found in EXTs, and their derivatives, Isodityrosine (IDT), Pulcherosine (Pul), Di-Isodytirosine (diIDT), bind perfectly to the active site of TomEP with dominant interactions of Val54, Ser94, Ala96 and Phe196 residues. Pulcherosine had the highest binding affinity, while [-Y-K-Y-] showed the lowest binding affinity. Molecular dynamics simulations showed that [-Y-X-Y-] motifs (and the derivative substrate ligands) remain bound to the active site of TomEP throughout the 100 ns long simulation. Furthermore, the binding of these substrates stabilized the protein structure. ConclusionThese results may explain why TomEP is particularly well-suited for EXT crosslinking and will have significant implications on biochemistry, biotechnology, and the potential use of these EPs in crops improvement.

plant biology↗

bioRxiv2026

The Cell Surface Proteome of Malignant Peripheral Nerve Sheath Tumors Reveals Therapeutic Targets

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas and the most common cause of disease-associated death for Neurofibromatosis Type 1 (NF1) patients. In the context of NF1, MPSNTs develop from benign premalignant precursors. The transition to malignancy is usually accompanied by loss of the polycomb repressive complex 2 (PRC2), leading to aberrant upregulation of many genes. The specific mechanisms disrupted by PRC2 loss remain incompletely understood. There is a significant gap in our knowledge of which cell-surface targets become derepressed and therapeutically actionable following PRC2 loss, contributing to the current lack of effective targeted therapies for MPNSTs. This study aims to address this gap by using cell-surface capture technology with mass spectrometry to profile MPNST models. In doing so, we define PRC2-dependent effects on the cell surface proteome, including specific biological pathways that are enhanced or suppressed at the cell surface protein level. We also create an MPNST cell-surface protein compendium comprised of proteins that are highly expressed across a variety of well-defined MPNST models. We prioritized proteins that are preferentially expressed in MPNST or other cancers and for which FDA-approved therapies already exist. Specific proteins from this compendium were molecularly targeted with antibody-drug conjugates in these models to surmise their therapeutic efficacy. Results reveal PTK7 as a novel and promising target for MPNST. In total, these efforts represent a step toward addressing the knowledge gap in MPNST genesis and identifying new therapeutic targets for further testing. Additionally, this data serves as a resource for other researchers wishing to characterize specific molecular targets. KEY POINTSPRC2 modulates key MPNST signaling pathways through the cell surface proteome Cell surface proteomics identifies a plethora of therapeutic targets for MPNST targeted therapy Antibody-drug conjugates targeting PTK7 show enhanced efficacy in reducing MPNST viability IMPORTANCE OF THE STUDYThis study utilizes advances in biochemistry to profile the surface proteome of malignant peripheral nerve sheath tumors. In doing so, it identifies many proteins whose presence is abundant on the cell surface of MPNST cells. Pre-clinical drug testing shows that use of antibody-drug conjugates may be effective in killing MPNST cells when targeted to epitopes identified in our MPNST cell surface proteome compendium. This study is a departure from more commonly used transcriptomic methods to identify cell surface proteins by using direct surface capture and mass spectrometry, providing a more direct measurement of cell surface protein abundance. Additionally, it identifies a handful of proteins which can be directly targeted pharmaceutically and one in particular, PTK7, whose targeting is highly effective in killing MPNST cells.

cancer biology↗

bioRxiv2026

Survey of the human proteostasis network: the ubiquitin-proteasome system

Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1430 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3150 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

bioinformatics↗

bioRxiv2026

Physiologically relevant media are associated with overlapping metabolic responses in primary human hepatocytes and Huh7 cells

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is challenging to study in vivo in humans and in vitro models are limited. Although primary human hepatocytes (PHHs) are considered the gold-standard, immortalized hepatic cell lines are utilised due to scalability. This study compared the metabolic responses of PHHs with our Huh7-based model cultured in physiologically-relevant fatty acid (FA) mixtures. MethodsPHH and Huh7 cells were treated with 2% human serum, sugars and FAs enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 4 or 7 days, respectively. Stable isotope tracers investigated basal metabolic changes in response to treatment. Cell viability, media biochemistry, intracellular metabolism, lipid droplet morphology and gene expression were quantified. ResultsHuh7 cells had greater viability than PHHs, while NEFA uptake and triglyceride secretion were similar. OPLA and POLA increased large lipid droplets in Huh7 cells, whereas only OPLA produced comparable effects in PHHs. Despite higher baseline TG in PHHs, both models showed similar lipid composition, de novo lipogenic responses, and glycogen levels. Compared to Huh7 cells, PHHs exhibited higher 3-hydroxybutyrate, lower lactate, reduced glucose uptake, and donor-dependent transcriptomic variability. ConclusionsHuh7 cells are metabolically adaptable and when cultured in physiologically-relevant media, produce metabolic readouts similar PHH cells.

cell biology↗