Deregulated inflammatory signaling via STAT family transcription factors, particularly STAT1, underlies a variety of immune-related diseases, including inflammatory bowel disease. Whereas activation of STATs by JAKs via canonical receptor-driven JAK-STAT signaling is well understood, little is known about JAK-independent mechanisms of STAT activation. Here, we identify the understudied nonreceptor tyrosine kinase TNK1 as a therapeutically targetable, JAK-independent activator of STAT signaling. Using a multiomics approach, we mapped a network of TNK1 substrates associated with protein condensates and proinflammatory signaling, including STAT1. We found that TNK1, but not its sister kinase ACK1, directly phosphorylates STATs at well described STAT-activating JAK sites. In cells, TNK1-mediated STAT1 phosphorylation and activation occurs independently of JAKs. Imaging and interactomics data suggest that TNK1 interacts with STAT1 in cytosolic condensates, which likely compartmentalize TNK1-substrate interactions. We show that an intrinsically disordered proline-rich region in TNK1, which includes a 14-3-3 docking phosphorylation site, is required for the formation of kinase-active TNK1 condensates and STAT1 phosphorylation. Mutations within the proline-rich region that eliminate 14-3-3 binding increase formation of TNK1 condensates, suggesting a model in which 14-3-3 acts as a clamp that constrains the flexible PRR to inhibit condensate formation and STAT1 activation. Finally, we show that TNK1 is a targetable driver of STAT1-mediated inflammation in the gut as inhibition of TNK1 reduces active STAT1 in the colon and ameliorates colitis symptoms in mice.

Parkinson's disease (PD) is a disease of adults involving the loss of dopaminergic neurons after a long, asymptomatic, prodromal period. α-synuclein, LRRK2, and VPS35 are linked to familial PD, however, how these mutations predispose dopamine neurons to death during the early prodromal phases remains unclear. Here, we used in vivo native proximity proteomics (iBioID) and dopaminergic neuron-specific subcellular proteomics across multiple PD models to uncover early alterations preceding neuronal loss. Our analyses identified convergent disruptions in synaptic protein abundance, indicating that presynaptic trafficking defects are early events in PD pathogenesis. Using a targeted CRISPR-based genetic screen in dopamine neurons, we demonstrated that mimicking this misregulation of STXBP1 amplifies vulnerability to α-synuclein, implicating it as a previously underappreciated toxicity buffering factor. These findings highlight convergent mechanisms that sensitize dopamine neuronal degeneration and that presynaptic vesicle SNARE-complex proteins could serve as key targets for disease-modifying therapies in PD and related neurodegenerative disorders.

ABSTRACT:The progression of multiple myeloma (MM), an incurable malignancy of plasma cells, is often associated with the suppression of ferroptosis, a type of cell death driven by iron-dependent lipid peroxidation. The mechanisms underlying this suppression remain largely unknown. Here, we identified serine/threonine kinase 17b (STK17B) kinase as a critical suppressor of ferroptosis in MM. Elevated levels of STK17B are associated with poor overall survival in patients with MM, and STK17B expression is significantly higher in relapsed vs newly diagnosed MM cases. We found that inhibiting STK17B in MM cells increased the labile iron pool, enhanced lipid peroxidation, and sensitized cells to conventional anti-MM therapies. Notably, an orally available, in-house-generated STK17B inhibitor induced ferroptosis and significantly reduced tumor growth in MM xenograft mouse models. Mechanistically, proximity labeling assay combined with the phospho-proteomic analysis identified 2 major regulators of iron uptake and transport as direct targets of STK17B: iron-responsive element binding protein 2 (IREB2), and heat shock protein family B member 1 (HSPB1). We demonstrated that STK17B phosphorylates critical regulatory sites on IREB2 (S157) and HSPB1 (S15), thereby modulating the balance between IREB2 and HSPB1 downstream effectors, proferroptotic transferrin receptor, and antiferroptotic ferritin heavy chain proteins. Furthermore, we demonstrated that STK17B indirectly maintains activating phosphorylation of STAT3, a ferroptosis suppressor and a major driver of MM pathobiology. Our findings uncovered a clinically relevant and targetable STK17B-pIREB2S157/pHSPB1S15 signaling axis that suppresses ferroptosis and contributes to drug resistance in MM.

Microorganisms, including fungi, adapt to profound changes in their local environment during human infections. After exposure to high temperature and other stress conditions, the opportunistic fungal pathogen Cryptococcus neoformans enacts changes in metabolism, cell wall structure, and transmembrane transport that allow it to survive and proliferate in a mammalian host. This stress response program is regulated by the HECT E3-ubiquitin ligase Rsp5, which is required for growth at high salinity, pH, and temperature. However, the complete set of Rsp5 substrates that direct these molecular changes remains incompletely understood. Here, we demonstrate that C. neoformans Rsp5 confers increased tolerance to temperature and salt stress in part through regulation of the trehalose biosynthesis pathway. Two enzymes in the trehalose biosynthesis pathway, Tps1 and Tps2, are differentially ubiquitinated by Rsp5 after exposure to stress conditions. We directly measured trehalose production after exposure to high temperature and found that a C. neoformans strain lacking Rsp5 is unable to induce trehalose production. Quantitative proteomic analysis of the C. neoformans response to high salinity identified Rsp5-dependent and independent adaptations to osmotic stress, and Rsp5-dependent ubiquitination does not alter the abundance of Tps1 or Tps2. These results suggest that regulation of trehalose biosynthesis is one of the cellular mechanisms by which Rsp5-dependent ubiquitination in C. neoformans facilitates survival in response to stressors encountered in the human infection environment.IMPORTANCECryptococcus neoformans is an opportunistic fungal pathogen that kills over 180,000 people every year, with few effective treatment options. As a yeast that normally lives in the environment, C. neoformans has to survive large changes in its physical environment, including elevated body temperature, which causes human infections. Here, we show how C. neoformans uses a protein modification to regulate production of a fungus-specific metabolic pathway important for survival at human body temperature. Unraveling how environmental fungi tolerate and survive temperature and other stressors will help us to understand how they cause disease and identify new and better ways to treat these deadly infections.

Organic anion transporting polypeptide (OATP) 1B3 plays a clinically significant role in hepatic drug disposition. Lysine acetylation, a key post-translational modification, has not been investigated for OATP1B3. This study determined the lysine acetylation status of OATP1B3 by proteomics and assessed the impact of inhibition of lysine deacetylase (KDAC) 6, a major cytosolic KDAC, on OATP1B3 acetylation and transport function. Proteomics revealed 7 acetylation sites, including 5 with additional ubiquitin-like modifications, and 4 phosphorylation sites (T10, S293, S295, S683). In human embryonic kidney 293 (HEK293)-Myc-FLAG-OATP1B3 cells, preincubation with the selective KDAC6 inhibitor tubacin (TBC) (5 μM, 24 hours), markedly reduced OATP1B3-mediated transport of [3H]cholecystokinin-8 (CCK-8), a specific substrate, and [3H]estradiol-17β-D-glucuronide to 0.15 ± 0.03-fold and 0.19 ± 0.01-fold of the control, respectively, without affecting OATP1B3 mRNA, protein levels, or membrane localization determined by real-time reverse transcription polymerase chain reaction, immunoblotting, and confocal microscopy. TBC treatment increased K664 acetylation to 2.12 ± 1.03-fold of the control (P < .05). Consistently, the acetylation-mimetic K664Q variant exhibited reduced transport compared with the acetylation-null K664R variant (P < .05). Treatment with a second KDAC6 selective inhibitor, WT-161 (3 μM, 5 hours), similarly reduced OATP1B3-mediated [3H]CCK-8 transport. In cultured primary human hepatocytes, TBC treatment for 4, 8, and 24 hours decreased [3H]CCK-8 transport to 0.34 ± 0.02-fold, 0.27 ± 0.03-fold, and 0.37 ± 0.03-fold of the control, respectively (all P < .05). The study reveals a novel post-translational modification of OATP1B3 by lysine acetylation and demonstrates impaired transporter function following KDAC6 inhibition, likely involving increased acetylation at K664, thereby providing new insight into OATP1B3-mediated drug-drug interactions driven by KDAC6 activity. SIGNIFICANCE STATEMENT: This study identifies lysine acetylation as a novel post-translational modification of organic anion transporting polypeptide (OATP)1B3 and demonstrates that altered lysine acetylation following inhibition of lysine deacetylase 6 reduces OATP1B3 transport function. These findings provide a mechanistic basis for altered hepatic drug disposition and highlight a new pathway through which drug-drug interactions involving OATP1B3 may occur.

Hepatic transport protein organic anion transporting polypeptide 1B1 (OATP1B1) is a key determinant of drug-drug interactions. We reported OATP1B1 lysine acetylation recently, however, the lysine deacetylase (KDAC), also known as histone deacetylase (HDAC), involved in its deacetylation remains uninvestigated. This study determined the role of KDAC6/HDAC6, a major cytosolic KDAC, on OATP1B1 acetylation and transport function. Loss-of-function of KDAC6 by CRISPR/Cas9-mediated knockout in HEK293T cells or by treatment with the selective KDAC6 inhibitor tubacin (TBC) (5 μM, 24 hours) in transporter-expressing HEK293 cells markedly reduces OATP1B1-mediated transport of [3H]estradiol-17-ß-D-glucuronide to 0.62 ± 0.095- and 0.28 ± 0.007-fold of control, respectively. TBC treatment did not affect OATP1B1 mRNA, protein levels and colocalization with plasma membrane marker Na/K-ATPase, suggesting a regulation at a post-translational level. TBC treatment also reduces [3H]rosuvastatin accumulation in primary human hepatocytes. Quantitative comparison of post-translational modifications (PTMs) between TBC treatment and control showed concurrent increase in lysine acetylation at K675 (to 5.16 ± 2.7-fold of control) and decrease in dual phosphorylation at Ser659-Ser663 (to 0.34 ± 0.13-fold of control). A variant S659A-S663A-K675Q-OATP1B1 that mimics these concurrent PTM changes reduces OATP1B1-mediated transport compared with the wild-type control, supporting a role for altered PTMs in downregulation of OATP1B1 transport function upon KDAC6 loss-of-function. In addition, K49, a residue important in maintaining OATP1B1 transport function, was identified as acetylated for the first time. This study identifies KDAC6 as a key enzyme regulating lysine acetylation of OATP1B1 and maintaining OATP1B1 transport function, thereby implying a novel KDAC6-linked mechanism in OATP1B1-mediated drug-drug interactions. SIGNIFICANCE STATEMENT: This study identifies lysine deacetylase 6 (KDAC6) as a key enzyme involved in post-translational regulation of OATP1B1. Current findings demonstrate that KDAC6-dependent acetylation-phosphorylation axis modulates OATP1B1 transport function and provides a mechanistic basis by which altered KDAC6 activity may influence OATP1B1-mediated drug-drug interactions.

Central nervous system astrocytes have an intricate, highly branched morphology. Proper development of perisynaptic astrocyte processes is necessary for tripartite synapse formation and function. However, cellular pathways orchestrating this development are largely unknown. Neuroligins (NLs) 1-3 regulate astrocyte morphogenesis via transcellular adhesions with neuronal neurexins. Here, we found an astrocytic NL2-based mechanism governing morphogenesis. Through structure and function studies, we identified a WW-binding motif within the NL2 intracellular domain required for astrocyte morphogenesis. Using cell-specific in vivo proximity labeling (iBioID), we found that each NL displays distinct protein-protein interactions within astrocytes, distinct from the neuronal NL2-binding partners. From these data, we identified a role for WW domain-containing E3 ubiquitin ligase Nedd4l in astrocyte morphogenesis. Biochemical assays revealed Nedd4l ubiquitinates and stabilizes NL2, and this ubiquitination is required for astrocyte morphogenesis. This study shows that NLs have nonoverlapping roles in controlling astrocyte growth and uncovers a molecular mechanism of how NL2 mediates astrocyte morphogenesis.

Coat protein complex II (COPII) mediates anterograde trafficking from the endoplasmic reticulum (ER). While the core COPII machinery is well-characterized, how cells regulate COPII to accommodate large cargoes, including collagens, remains incompletely understood. Here, we show that the cargo-selecting COPII subunit Sec24D is modified by site-specific O-linked β-N-acetylglucosamine (O-GlcNAc) in its N-terminal intrinsically disordered region upon induction of collagen transport. These glycosylations are required for collagen trafficking in human cells and developing zebrafish. Crosslinking proteomics demonstrated that each O-GlcNAcylation influences the Sec24D interactome in a distinct way, regulating nearly all steps of COPII-mediated transport through protein-protein interactions. In particular, myoferlin interacts with glycosylated Sec24D and unexpectedly facilitates fusion of ER exit sites (ERES) and the ER-Golgi intermediate compartment (ERGIC) to enable collagen transport. Our results establish Sec24D O-GlcNAcylation as a dynamic regulator of COPII protein-protein interactions and collagen trafficking and identify myoferlin as a mediator of this process.

BACKGROUND:Bacterial pneumonia remains a leading cause of morbidity and mortality worldwide despite the widespread availability of antibiotics. Novel pneumonia therapies and biomarkers are urgently needed to improve outcomes and advance personalized therapy. Using an established baboon model of S. pneumoniae pneumonia, we sought to characterize the temporal dynamics of pneumonia host responses to identify novel potential diagnostic and therapeutic molecular targets. METHODS:We performed whole blood transcriptomics, unbiased proteomics, and peripheral cytokine measurements serially in baboons inoculated with S. pneumoniae (n = 23) or saline (n = 10) and modeled the peripheral blood host response using principal components analysis and complex sparse logistic regression. Differentially expressed genes were analyzed for pathway analysis. RESULTS:Inoculated animals developed characteristic signs and symptoms of pneumonia. A 39-gene signature was derived that classified S. pneumoniae infection with high accuracy (auROC 0.9 and 0.99 at 24 and 48 h post-inoculation, respectively). Similar performance was observed for 48-h biomarker signatures derived from peripheral blood plasma proteomic and cytokine measurements (both auROC >0.9). The gene signature retained strong diagnostic performance (auROC = 0.88) when transformed to human orthologs and applied to patients with acute respiratory illness (n = 34) or healthy controls (n = 20). Pathway analysis at 48 h identified down-regulation of mitophagy and glucocorticoid signaling in peripheral blood. CONCLUSIONS:We report novel peripheral blood gene and protein expression signatures of S. pneumoniae pneumonia that could improve pneumonia diagnosis and found distinct pathways that may be amenable to modulation. Our findings illustrate how non-human primate models of bacterial pneumonia can successfully translate biomarker discoveries to patients.

Enteroendocrine cells (EECs) are rare sensory cells in the intestinal epithelium that coordinate digestive physiology by secreting a diverse repertoire of peptide hormones. These hormones are the main effectors of EEC function, and their characterization requires direct observation by mass spectrometry due to the specialized protein cleavage and posttranslational modifications that yield their mature forms. Based on the distinct subset of hormones they predominantly secrete, EECs can be categorized into subtypes. How each EEC subtype is specified, however, remains poorly understood. Here we describe EEC subtype differentiation and hormone production in the zebrafish. Using single-cell RNA sequencing data, we identified EEC progenitors and six EEC subtypes in zebrafish and revealed that their expression profiles are consistent across larval and adult stages. Mass spectrometry analysis of isolated zebrafish EECs identified highly processed peptides derived from 18 of 21 hormone coding genes expressed by EECs, yielding a catalog of >400 unique EEC hormone peptides. We assembled reporters for zebrafish EEC subtypes to test the lineage relationships between EEC subtypes and the EEC progenitor population, which expresses neurogenin3. Despite its essential role in mammalian EEC differentiation, we found that selective cytotoxic ablation of neurogenin3+ cells in zebrafish only reduced a subset of EEC subtypes. Finally, we discovered that selective ablation of ghrelin+ EECs reduced a different subset of EEC subtypes, together suggesting that neurogenin3+ and ghrelin+ cells serve as distinct precursors for separate EEC subtypes. We anticipate these observations and resources will facilitate future studies in the zebrafish to discern the developmental biology, physiology, and endocrinology of EEC subtypes. ### Competing Interest Statement The authors have declared no competing interest.

Enteroendocrine cells (EECs) are rare sensory cells in the intestinal epithelium that coordinate digestive physiology by secreting a diverse repertoire of peptide hormones. These hormones are the main effectors of EEC function, and their characterization requires direct observation by mass spectrometry due to the specialized protein cleavage and posttranslational modifications that yield their mature forms. Based on the distinct subset of hormones they predominantly secrete, EECs can be categorized into subtypes. How each EEC subtype is specified, however, remains poorly understood. Here, we describe EEC subtype differentiation and hormone production in the zebrafish. Using single-cell RNA sequencing data, we identified EEC progenitors and six EEC subtypes in zebrafish and revealed that their expression profiles are consistent across larval and adult stages. Mass spectrometry analysis of isolated zebrafish EECs identified highly processed peptides derived from 19 of 23 hormone-coding genes expressed by EECs, including a previously undescribed zebrafish secretin ortholog. We assembled reporters for zebrafish EEC subtypes to test the lineage relationships between EEC subtypes and the EEC progenitor population, which expresses neurogenin 3 (neurog3). Despite its essential role in mammalian EEC differentiation, we found that selective cytotoxic ablation of neurog3+ cells in zebrafish only reduced a subset of EEC subtypes and loss of the neurog3 gene had no impact on EEC numbers. Finally, we discovered that selective ablation of ghrelin+ EECs reduced a different subset of EEC subtypes, together suggesting that neurog3+ and ghrelin+ cells serve as distinct precursors for separate EEC subtypes. We anticipate these observations and resources will facilitate future studies in the zebrafish to discern the developmental biology, physiology, and endocrinology of EEC subtypes.

We consider the problem of de novo peptide sequencing in tandem mass spectrometry, where the goal is to predict the underlying peptide sequence given a spectrum’s fragment peaks and precursor information. We present PLMNovo, a constrained learning framework that leverages pre-trained protein language models (PLMs) to guide the training process. In particular, we cast peptide-spectrum matching as a constrained optimization problem that enforces alignment between spectrum and peptide embeddings produced by a spectrum encoder and a PLM, respectively. We use a Lagrangian primal-dual algorithm to train the spectrum encoder and the peptide decoder by solving the proposed constrained learning problem, while optionally fine-tuning the pre-trained PLM. Through numerical experiments on established benchmarks, we demonstrate that PLMNovo outperforms several state-of-the-art deep learning-based de novo sequencing algorithms. ### Competing Interest Statement The authors have declared no competing interest.

During mitosis, eukaryotic cells cease anterograde trafficking from the endoplasmic reticulum (ER) toward the Golgi. This cessation corresponds with the dispersal of the COPII transport protein, Sec24C, from juxtanuclear ER exit sites (ERES) into a diffusely cytosolic pool. Redistribution of Sec24 paralogs and other core COPII proteins may underlie the mitotic pause in secretion and may be required for the equal inheritance of endomembrane organelles and machinery by both daughter cells. Therefore, it is important to understand the mechanisms governing the mitotic relocalization of COPII components. Here, we explore the role of post-translational modifications (PTMs) of the model COPII protein Sec24C in this phenotypic switch during mitosis. In interphase, Sec24C is modified by O-linked β-N-acetylglucosamine (O-GlcNAc), and we show that this glycan is rapidly removed upon mitotic entry, influencing the timing of Sec24C dispersal. Additionally, we identify novel, cell cycle phase-enriched phosphorylation events on Sec24C, including phosphosites that regulate the stability and localization of the protein, providing the first systematic characterization of dynamic PTMs on any Sec24 protein. Together, our data support the hypothesis that phosphorylation and glycosylation of Sec24C act in concert to induce rapid dispersal upon mitotic entry and may promote equal partitioning of the endomembrane system to daughter cells after division.

Coat protein complex II (COPII) mediates anterograde trafficking from the endoplasmic reticulum (ER). While the core COPII machinery is well-characterized, how cells regulate COPII to accommodate large cargoes, including collagens, remains incompletely understood. Here, we show that the cargo-selecting COPII subunit Sec24D is modified by site-specific O-linked β-N-acetylglucosamine (O-GlcNAc) in its N-terminal intrinsically disordered region upon induction of collagen transport. These glycosylations are required for collagen trafficking in human cells and developing zebrafish. Crosslinking proteomics demonstrated that each O-GlcNAcylation influences the Sec24D interactome in a distinct way, revealing novel mediators of COPII function. In particular, Sec24D glycosylation is required for its interaction with myoferlin, which unexpectedly facilitates fusion of ER exit sites (ERES) and the ER-Golgi intermediate compartment (ERGIC) to enable collagen transport. Our results establish Sec24D O-GlcNAcylation as a dynamic regulator of COPII protein-protein interactions and collagen trafficking and identify myoferlin as a novel mediator of this process.

The nervous system is primarily composed of neurons and glia, and the communication between them has profound roles in regulating the development and function of the brain. Neuron-glia signal transduction is known to be mediated by secreted signals through ligand-receptor interactions on the cell membrane. Here we show a new mechanism for neuron-glia signal transduction, wherein neurons transmit proteins to glia through extracellular vesicles, activating glial signaling pathways. We find that in the amphid sensory organ of Caenorhabditis elegans, different sensory neurons exhibit varying aging rates. This discrepancy in aging is governed by the cross-talk between neurons and glia. We demonstrate that early aged neurons can transmit heat shock proteins to glia via extracellular vesicles. These neuronal heat shock proteins activate the glial IRE1-XBP1 pathway, leading to the transcriptional regulation of chondroitin synthases to protect glia-embedded neurons from aging-associated functional decline. Therefore, our studies unveil a new mechanism for neuron-glia communication in the nervous system and provide new insights into our understanding of brain aging.

Mitochondrial dynamics enable cellular adaptation to fluctuations in energy demand, such as those imposed on skeletal muscle by exercise, metabolic disorders, or aging. Here, we report a novel pathway that modulates mitochondria dynamics in skeletal muscle involving the scaffolding protein ankyrin-B. Rare variants in ankyrin-B, encoded by ANK2, increase risk for cardio-metabolic syndrome in humans and mice. We show that mice selectively lacking skeletal muscle ankyrin-B have reduced endurance exercise capacity without alterations in muscle strength or systemic glucose regulation. Muscle fibers in these mice have increased oxidative stress, reduced fatty acid oxidation, and enlarged and hyperconnected mitochondria. We found that ankyrin-B interacts with and is required for efficient mitochondria recruitment of fission modulators and sarcoplasmic reticulum-mitochondria coupling. Thus, we conclude that ankyrin-B enables substrate adaptability and bioenergetic homeostasis under energetic stress, and exercise capacity by promoting efficient mitochondrial fission in skeletal muscle.

The cystine-xCT transporter/glutathione/GPX4 axis is the canonical pathway protecting cells from ferroptosis. Whereas GPX4-targeting ferroptosis-inducing compounds (FINs) act independently of mitochondria, xCT-targeting FINs require mitochondrial lipid peroxidation, though the mechanism remains unclear. Because cysteine is also a precursor for coenzyme A (CoA) biosynthesis, here, we demonstrated that CoA supplementation selectively prevented ferroptosis triggered by xCT inhibition by regulating the mitochondrial thioredoxin system. Our data showed that CoA regulated the in vitro enzymatic activity of mitochondrial thioredoxin reductase-2 (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolished its enzymatic activity and ability to protect cells against ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2. Furthermore, the fibroblasts from patients with disrupted CoA metabolism had increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis led to an oxidized thioredoxin system, increased mitochondrial lipid peroxidation, and loss of cell viability, which were all rescued by ferrostatin-1. These findings identified CoA-mediated posttranslational modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

BACKGROUND:Reactive oxygen species (ROS) augment the activation of vascular smooth muscle cells (SMCs) and promote neointimal hyperplasia evoked by arterial injury or atherogenesis. We have previously shown that small nucleolar RNAs (snoRNAs) from the Rpl13a locus are key regulators of cellular ROS levels. METHODS:Using mice deficient in the Rpl13a snoRNAs, we tested whether these snoRNAs regulate SMC activation in vitro and in vivo. Carotid endothelial denudation was used to provoke neointimal hyperplasia in wild-type (WT) and snoRNA knockout (snoKO) mice, which lack all four intronically-encoded Rpl13a snoRNAs. Primary SMCs from WT and snoKO mice were used for in vitro functional and proteomic analyses. HEK293T cells with specific snoRNA deletions were used to test for snoRNA-guided 2'-O-methylation of mRNA. RESULTS:Arterial ROS levels, inflammation, and carotid artery neointimal hyperplasia were reduced in snoKO compared with WT mice. In vitro, snoKO SMCs demonstrated lower ROS levels and less migration, proliferation, and inflammatory signaling than WT SMCs. Reduced ROS levels in snoKO SMCs and aortas correlated with upregulation of the mitochondrial protein COX4I2, which is associated with reduced mitochondrial ROS under normoxic conditions. Deleting the snoRNA U32A in human HEK293T cells decreased 2'-O-methylation of COX4I2 mRNA and upregulated COX4I2 protein without changing COX4I2 mRNA levels. Silencing Cox4i2 in snoKO SMCs upregulated SMC ROS to WT levels. CONCLUSIONS:Rpl13a snoRNAs are important drivers of SMC activation and neointimal hyperplasia. Rpl13a snoRNAs augment SMC ROS levels, at least in part, by post-transcriptional downregulation of COX4I2 expression.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

We aimed to identify serum biomarkers that predict knee osteoarthritis (OA) before the appearance of radiographic abnormalities in a cohort of 200 women. As few as six serum peptides, corresponding to six proteins, reached AUC 77% probability to distinguish those who developed OA from age-matched individuals who did not develop OA up to 8 years later. Prediction based on these blood biomarkers was superior to traditional prediction based on age and BMI (AUC 51%) or knee pain (AUC 57%). These results identify a prolonged molecular derangement of joint tissue before the onset of radiographic OA abnormalities consistent with an unresolved acute phase response. Among all 24 protein biomarkers predicting incident knee OA, the majority (58%) also predicted knee OA progression, revealing the existence of a pathophysiological “OA continuum” based on considerable similarity in the molecular pathophysiology of the progression to incident OA and the progression of established OA.