Citrullination by peptidylarginine deiminase (PAD) enzymes is a post-translational protein modification implicated in the etiopathogensis of rheumatoid arthritis. Of the five known PAD isoforms, PAD4 is expressed in the nucleus of innate immune cells including synovial macrophages, and studies have shown citrullination of intracellular proteins such as transcription factors and histones can polarize cell phenotype. Here, we report how systemic and local deletion of PAD4 in synovial macrophages from PAD4-/- or PAD4f/fCreLysM mice results in activation of both shared and distinct gene modules in four populations of synovial macrophages. Furthermore, severity of KBxN serum transfer arthritis was increased in PAD4f/fCreLysM mice compared to controls. Furthermore, macrophages isolated from the hindjoints of arthritic PAD4f/fCreLysM displayed increased expression of inflammatory genes compared to macrophages from arthritic PAD4-/- mice. In addition, we report that local intra-articular administration of PAD inhibitor BB-Cl-amidine increased arthritis severity, while systemic administration had no effect. These findings indicate an anti-inflammatory role for intracellular citrullination in synovial macrophages, and highlight the potential confounding effects of extracellular and systemic PAD4 loss of function.

Missing values (MVs) remain a significant barrier to reliable proteomics analysis, particularly in single-cell proteomics, where small amounts of starting material and limits in detection drive missing-not-at-random (MNAR) sparsity. Existing imputation methods typically target either missing-at-random (MAR) or MNAR mechanisms, resulting in a trade-off between replicate consistency and preservation of biological variation, and are largely designed for bulk data. Here, we introduce SoftHybrid, a data-driven imputation framework that jointly models missingness and protein abundance to estimate the probability of MNAR, enabling continuous weighting between MAR- and MNAR-oriented strategies. SoftHybrid requires no external priors (cell type labels, group annotations, predefined missingness assumptions, etc.), enabling fully unsupervised applications. Across ground truth benchmarks and real single-cell proteomics data sets, SoftHybrid outperforms existing methods at low input and matches or exceeds their performance at the minibulk level. By preserving the proteomic structure and abundance accuracy, it enhances the recovery of biologically meaningful signals. SoftHybrid is implemented as an R package and is freely available at GitHub.

Atrial fibrillation (AF) increases energy demand in atrial myocytes, yet the mitochondrial mechanisms underlying this stress remain poorly defined. Using previously published proteomic data from left atrial tissue of AF and sham-operated goats, we performed organelle-specific bioinformatic analyses of the mitochondrial fraction. Over-representation and consensus pathway analyses consistently highlighted enrichment of oxidative phosphorylation (OXPHOS) subunits. Gene set enrichment and network analyses implicated Heat Shock Protein Family A Member 9 (HSPA9) as a potentially central regulatory hub coordinating the dysregulation of Complex I and III subunits, with 69% of regulatory relationships showing pathway concordance. These results indicate a coordinated, system-wide mitochondrial adaptation in AF, integrating energy production, proteostasis, and respiratory chain regulation.

Understanding how mature megakaryocytes (MKs) release their platelets, and crucially, what are the triggers that facilitate this process is of huge impact on human medicine. Controlling this biological process, as well as being able to utilise in vitro produced platelets will be a major therapeutic advancement. Unfortunately, the exact mechanism and mediators that drive thrombopoiesis remain elusive. Here, we seek to identify such mediators through studying the dynamics of platelet production after an acute loss of platelets. Analysis of plasma taken from 19 plateletpheresis donors at various timepoints pre and post donation, identified peak platelet production timepoints (4-8 hours). Analysis of these timepoints by proteomic and metabolomic techniques allowed for the identification of Triiodothyronine (T3), as well as its analogues, GC-1 (Sobetirome), MGL-3196 (Resmetirom) and KB2115 (Eprotirome), as having a direct effect on in vitro platelet production in human cord blood (T3 3 hours 100nM 1.26±0.24 and GC-1 100μM 5.54±1.58, MGL-3196 300μM 6.92±1.38, KB2115 75μM 17.90±5.25 fold change at 12 hours, mean±SD) and iPSC-derived MKs (viral A1ATD1 KB2115 36.1uM 3.36±0.38, inducible QOLG1.1H KB2115 75uM 1.85±0.46 fold change, mean±SD). Receptor specific antagonists revealed that thyroid hormone induced platelet production primarily signals via the non-genomic signalling pathway, integrin αVβ3 (CD51/61, vitronectin receptor), of which MKs highly express. When combined with silk-based-3-dimensional scaffold bioreactor technology, we observe a significant upscaling of platelets (KB2115 2.8±0.79 fold change±SD) that respond positively to agonist stimulation (P-selectin exposure). This shows the direct impact of thyroid on platelet production through integrin αVβ3, which offers interesting therapeutic potential in the field of transfusion medicine.

DNA double-strand breaks (DSBs) are highly toxic DNA lesions that can lead to genomic instability. DSBs can also interfere with other DNA-based processes, including transcription, and thereby jeopardizing cellular function. In situations of persistent DSBs, RNA polymerase II (RNAPII) needs to be removed to facilitate DNA repair. DSB-induced RNAPII removal involves multifaceted ubiquitylation, but the mechanisms involved remain elusive. Our data show that in response to DSBs, the E3 ubiquitin ligase NEDD4, and to a lesser extent CRL3 complexes, catalyse the ubiquitylation of elongating RNAPII, facilitating efficient DSB repair. Specifically, NEDD4 is identified as the specific writer of K63-linked ubiquitin chains on Serine2 phosphorylated (S2P)-RNAPII under stress, while the total pool of RNAPII is found to be modified mainly with K48-linked ubiquitin chains. We find that the ubiquitin ligases NEDD4, WWP2, and CUL3-based complexes exhibit a DNAPK inter-dependency, driving NHEJ repair and proper resolution of transcription defects caused by DSBs.

F-box proteins are the substrate recognition modules of the SCF (SKP1–Cullin–F-box) E3 ubiquitin ligase complex. FBXO42, an understudied member of this family, has recently emerged as a modulator of key cellular processes, including cell cycle progression, the DNA damage response, and glioma stem cell survival. In this study, we define the function of FBXO42 as a major regulator of the protein phosphatase PP4. Phosphoprotein phosphatases (PPPs) have a broad array of substrates, hence necessitating tight regulation. We observe that FBXO42 ubiquitinates the PP4 complex to govern the assembly of regulatory and catalytic subunits, with the net effect of restraining the latter’s phosphatase activity. FBXO42 depletion unleashes PP4 activity, with broad cellular effects, highlighting FBXO42 as a novel regulatory node in ubiquitin-mediated signalling for future therapeutic exploitation. The F-box protein ubiquitin ligase adaptor FBXO42 has been implicated in various key cellular processes. This work shows that FBXO42 limits PP4 phosphatase activity to ensure proper DNA damage signalling, with its loss disrupting phosphorylation dynamics and compromising genome stability. The ubiquitin ligase adaptor FBXO42 limits PP4 phosphatase activity to ensure proper DNA damage signalling.

Nitric oxide (NO) has fundamental roles in numerous physiological and pathophysiological processes. In macrophages, NO produced by inducible nitric oxide synthase (iNOS) modulates metabolic changes that are essential to macrophage activation and plasticity, driving the characteristic metabolic switch from oxidative phosphorylation to glycolysis1,2. Itaconate, derived from the TCA cycle by decarboxylation of cis-aconitate by IRG1 (also referred to as CAD, ACOD1), is one of the most upregulated metabolites during the inflammatory response3. Itaconate regulates macrophage polarization by electrophilically modifying cysteines of key enzymes that control inflammatory states (such as ATF3, Jak1, IFNβ), participate in glycolysis (for example, GAPDH, LDHA) and limit oxidative stress through structural competitive inhibition of succinate dehydrogenase4-9. We recently reported that macrophages that are deficient in iNOS, and subsequent NO generation, produce strikingly higher levels of intracellular itaconate (up to ~15-fold) compared to wild-type cells when stimulated with inflammatory cytokines1,2,10. Here we show that iNOS inhibits IRG1 activity and itaconate levels through a conformation-dependent protein-protein interaction rather than through the production of NO. Using a variety of biochemical and computational approaches, we show that a direct interaction between iNOS and IRG1 occurs within mitochondria, in mouse and human cells, and that it depends on binding of the cofactor BH4 to iNOS but does not require its capability to produce NO. Our findings reveal a non-canonical cellular function for iNOS that places it at the centre of a signalling hub, linking redox signalling and metabolism to modulation of the inflammatory response in macrophages.

Collagen IV, encoded by genes COL4A1/COL4A2, is a major component of the basement membrane, a specialised extracellular matrix (ECM) structure. Mutations in these genes cause a genetic form of cerebral small vessel disease (cSVD), a leading cause of stroke and dementia. White matter abnormalities are a hallmark of cSVD and are closely linked to cognitive decline and dementia. While white matter defects occur in patients with COL4A1/2 mutations, they remain understudied and their mechanisms are unclear. To address these knowledge gaps, we combined magnetic resonance diffusion tensor imaging, pathology, ultrastructural investigations, behaviour and proteomic analysis of white matter in an established mouse model of cSVD due to a Col4a1 mutation (Col4a1+/Svc). The studies revealed that Col4a1+/Svc mice have reduced myelinating oligodendrocyte pools, axonal myelination defects, and altered white matter structural integrity as well as cognitive impairments. Proteomic analysis of isolated white matter from Col4a1+/Svc mice identified extensive changes to ECM and basement membrane composition. Furthermore, this provided evidence for altered endoplasmic reticulum (ER) biology including ER stress. To determine if white matter defects can be attenuated by targeting protein folding in the ER by promoting collagen IV secretion, we treated mice with the FDA-approved chemical chaperone 4-phenylbutyric acid. This revealed increased myelinating oligodendrocytes and improved axon-glial integrity in Col4a1+/Svc mice. These data provide novel insight into the pathomolecular mechanisms of collagen IV mutations in white matter abnormalities in cSVD and identify a modifiable pathway as a putative therapeutic target.

INTRODUCTION:Microglia have been implicated in the templated spread of tau aggregates in tauopathies through mouse studies. However, it is unclear whether these findings translate to human disease. METHODS:We challenged human induced pluripotent stem cell (iPSC)-derived microglia-like-cells (iMGL) with monomeric and fibrillar recombinant tau and tau purified from Alzheimer's patient brains, examining in detail the uptake, processing, release, and seeding of tau by microglia. RESULTS:iMGL take up tau via lipoprotein receptor-related protein 1 (LRP)1 and heparan sulfate proteoglycans, with leucine-rich repeat kinase 2 affecting LRP1 trafficking. Monomeric tau is digested effectively with minimal effects on iMGL, but recombinant or brain-derived tau fibrils induce chemokine/interferon response subtypes, alongside downregulation of homeostatic genes. Fibrillar tau is degradation-resistant, can escape into the cytoplasm, and becomes phosphorylated on two specific residues. iMGL release partially digested fibrillar tau, including in extracellular vesicles, visualized by cryo-electron microscopy, that seed aggregation in neurons. DISCUSSION:Our study reveals new insights into human microglial responses to tau, highlighting opportunities to limit pathogenic tau spread.

Tendinopathy is a painful overuse disorder marked by a progressive functional decline. Although chronic disease mechanisms have been described, early molecular changes remain poorly defined. Here, we performed temporal proteomic profiling of human patellar tendon biopsies across one, two, and three months of symptom duration and evaluated proteome differences between symptomatic and nonsymptomatic contralateral tendons. Biopsies were collected from participants with unilateral patellar tendinopathy and symptoms lasting three months or less for analysis by data-independent acquisition liquid chromatography-mass spectrometry. Peptide- and protein-level abundances were quantified as part of a bottom-up proteomics workflow and assessed using two-way ANOVA with factors of symptom status and symptom duration. Symptom duration had a significant main effect: 12 proteins and 14 peptides changed significantly over time, with most peptides corresponding to the altered proteins. However, there was no interaction between the symptom status and symptom duration, no sex differences, and no detectable proteomic difference between symptomatic and contralateral tendons. These findings indicate that protein-level changes are detectable within the first three months of tendinopathy, and similar proteomic changes were also observed in contralateral tendons. Together, these observations suggest that early phase patellar tendinopathy is associated with time-dependent proteomic changes that are not confined to the symptomatic tendon.

Targeted delivery systems offer a promising approach for selectively modulating cellular processes; yet the intracellular consequences of targeted nutrient delivery to trophoblast cells remain poorly defined. Here, we investigated a previously validated placenta-targeting peptide conjugated to liposomes encapsulating stable isotope-labelled L-arginine and L-lysine to examine cellular uptake and downstream molecular responses in a trophoblast-like cell model. Peptide-dependent uptake of fluorescently labelled liposomes was confirmed in BeWo cells, demonstrating selective internalisation compared with non-targeted controls. Encapsulation of isotope-labelled amino acids enabled direct quantification of intracellular delivery and incorporation into the cellular proteome using stable isotope labelling by amino acids in cell culture (SILAC). Quantitative proteomic analysis revealed coordinated changes in proteins associated with translation, metabolism, and nitric oxide synthase regulation following targeted liposomal uptake. Notably, V-type proton ATPase subunit G1 (ATP6V1G1) and large neutral amino acid transporter small subunit 1 (SLC7A5) showed increased incorporation of labelled amino acids and were independently validated by Western blotting. Together, these findings establish a proof-of-concept platform for targeted intracellular amino acid delivery to trophoblast-like cells and define the resulting proteomic responses. This work provides mechanistic insight into intracellular amino acid utilisation and a framework for future studies in placental cell biology.

Abstract Connective tissues such as skin and cartilage respond to injury in different ways. In cartilage, injury triggers the release of growth factors that activate local repair processes. One such factor, hepatoma-derived growth factor (HDGF), was recently identified by our group and remains poorly characterized. We aimed to study the role of HDGF in connective tissue injury and repair. Release of HDGF from injured skin and cartilage of wildtype (WT) and HDGF-knockout (Hdgf−/−) mice was examined by Western blot. Bulk RNA sequencing was performed in WT and Hdgf−/− tissues, and protein turnover was measured in SILAC-labelled skin biopsies from WT and Hdgf−/− animals via mass spectrometry. In vivo skin wounding was done in WT and Hdgf−/− male mice aged 10, 15 and 26 weeks. Spatial transcriptomics using the CosMx 1k panel was performed on unwounded skin and at days 3, 5, 7 and 10 postwounding in 15-week-old WT and Hdgf−/− male mice. HDGF was released from injured WT skin and cartilage, but not from Hdgf−/− tissues. Comparison of WT and Hdgf−/− skin and cartilage identified five consistently differentially expressed genes, three of which encoded ribosomal components. Proteomic analysis revealed decreased protein synthesis in injured Hdgf−/− skin. Wound healing was delayed in Hdgf−/− male mice compared with age-matched WT controls, characterized by increased granulation tissue and delayed re-epithelialization. This was observed in 15- and 26-week-old, but not 10-week-old, Hdgf−/− male mice. Spatial transcriptomics analysis of unwounded skin identified gene clusters related to innate immunity, inflammation, basal epithelium and stress response as being less prominent in Hdgf−/− mice. In wounded skin, temporal clustering patterns > 10 days after wounding were consistent with a delayed inflammatory response in Hdgf−/−. Our study demonstrates, for the first time, that release of HDGF following injury from multiple connective tissues has a role in repair, possibly by controlling protein synthesis and modulating infiltrating immune cells.

Ca2+/calmodulin-dependent kinase 1 delta (CaMK1δ) plays a central role in regulatory pathways associated with ATP, reduction potential, and Ca2+/calmodulin (CaM). Mass spectrometry (MS)-based structural proteomics incorporating FragPipe and pLink cross-link analysis was used to reveal conformation selection induced by dialysis with ATP, reducing agents, and CaM. The structural changes were mediated via cysteine and phosphate cross-linking and loop-linking of the activation loop within the C-terminal. Phosphate loop-linking was validated by β-elimination and Michael addition (BEMAD) reactions, aligning these findings with phosphoproteomics analyses of phosphorylation events. Oxidizing conditions inhibited the functionality of CaMK1δ wild-type. A novel mechanism of autoinhibition via cysteine cross-linking between the activation loop (αT) and C-terminal (αI) helices was identified. The microenvironment associated with CaMK1δ, including ATP availability, CaM concentration, and reduction potential, modulates the structural rearrangements underlying autophosphorylation. Phosphoproteomics, cysteine and phosphate cross-linking MS, and structural molecular modeling were used to describe kinase activation, allowing the activation of regulatory kinases to be reevaluated. We propose that regulatory kinases respond to an array of kinase family-specific distinct second messengers which can be studied using this multiomics framework, giving significant new insights into PTMs as well as the associated protein structure rearrangements.

Osteoarthritis (OA), a degenerative joint disease, is associated with increased systemic inflammation, chronic pain, and cardiovascular dysfunction. Epidemiological evidence establishes that OA increases the risk of cardiovascular disease (CVD) threefold, yet the causal role of OA's contributions remains underexamined. We assessed cardiac function longitudinally following destabilization of the medial meniscus (DMM) surgery to induce osteoarthritis in mice. DMM-mice exhibited significant, sexually dimorphic alterations in echocardiographic parameters. Female DMM mice developed impaired relaxation with altered E/A ratios, increased E/e' ratios, and prolonged intraventricular relaxation time with no change in ejection fraction, while male DMM mice showed progressive systolic dysfunction with decreasing ejection fraction, increased E/e' ratio, and prolonged intraventricular contraction time. Transcriptomic profiles and biochemical analyses demonstrated divergent cellular responses involving fibrosis and oxidative stress in female mice, whereas autophagic and apoptotic responses were observed in male mice. Using a tumor necrosis factor 2 (TNFR2) agonist shown to reduce systemic inflammation, we investigated its potential therapeutic role in the context of OA-induced cardiovascular dysfunction. TNFR2 agonism proved to be effective both prophylactically and therapeutically for female diastolic dysfunction. While prophylactic and therapeutic administration delayed male systolic dysfunction, the efficacy declined over time. Our findings demonstrate evidence of a novel sexually dimorphic model of OA-induced CVD that recapitulates the sexually dimorphic pattern of patient phenotypes and a promising new therapeutic approach to CVD.

Lithium is the gold standard mood stabiliser used to treat cycling mania and depression in bipolar disorder. Despite seven decades of clinical use, the mechanisms of its mood stabilisation are incompletely understood, fundamentally limiting development of improved alternatives. Two established lithium targets, glycogen synthase kinase 3β (GSK3β) and inositol monophosphatase, both modulate phosphorylation, suggesting lithium may exert broad effects on neuronal phosphorylation networks. We performed a discovery-phase in vitro screen of 140 kinases at 10mM LiCl and demonstrated that lithium inhibits 17 kinases beyond GSK3β. We therefore used untargeted quantitative phosphoproteomics to create a comprehensive map of lithium's phosphorylation signature in mouse synaptoneurosomes collected at dawn and dusk, matching peaks in phosphorylation driven by the sleep/wake cycle. Genes encoding lithium-sensitive phosphoproteins were significantly enriched in bipolar disorder genome-wide association studies, providing independent genomic evidence that these phosphorylation networks are relevant to bipolar pathophysiology. We further refined existing models of lithium’s action by showing that GSK3β inhibition is temporally restricted to dawn, indicating cross talk with sleep/wake cycles of phosphorylation. Overall, our data demonstrate that lithium’s pleiotropic effects may result from coordinated multi-kinase network reorganisation rather than single-target inhibition — a principle with direct implications for rational polypharmacology in mood stabiliser development.

Influenza A viruses (IAV) are clinically important pathogens that cause seasonal epidemics and pandemics in humans. IAV produce pleomorphic, enveloped virions, which can range from a spherical or bacilliform morphology, the predominant form in the most commonly studied laboratory strains, to long filamentous virions which are characteristic of clinical and veterinary isolates. Understanding the structure and function of filamentous virions is crucial for clarifying their role in viral persistence and immune evasion, and for informing the development of therapeutics that target their entry and/or egress pathways. Structural characterisation of influenza virions is challenging however owing to their fragility, heterogeneity and compared to most virus particles, unusually large size. Here, we combined structural and compositional approaches with integrative modelling to define the complete molecular architecture of influenza virions. In doing so we provide the first description of distinctive structural features of IAV filaments, including the selective incorporation of lipids, specific enrichment of viral and host proteins, and a viral cytoskeleton including a secondary helical layer within the viral capsid and extended fibrils of cofilactin. Collectively our findings suggest an important regulatory role for cofilactin in driving filament morphogenesis and provide important insights into the organisation and composition of IAV filamentous virions.

The primary mechanism and subcellular localisation of α-synuclein toxicity in Parkinson's disease pathogenesis remain unknown. We spatially and temporally resolved proteomic and transcriptomic changes in human iPSC-derived dopaminergic neurons with increasing burden of pathological α-synuclein. We found that misfolded α-synuclein proteoforms, signified by the formation of nanoscale intraneuronal puncta, are associated with impaired translocon function at the endoplasmic reticulum (ER). We show that α-synuclein interacts with Sec61A in iPSC-derived dopaminergic neurons and in post-mortem brain tissue from patients with Parkinson's disease. This interaction interferes with the co-translational translocation of ER-processed proteins including the vacuolar-type ATPase V0a1 subunit, glucocerebrosidase, and Cathepsin B, causing defective organelle function such as reduced lysosomal acidification, leading to increased extracellular vesicle release of α-synuclein. Defective ER-translocation was associated with increased ribosomal UFMylation and proteasomal recruitment but not activation of the unfolded protein response. Reduction of pathological α-synuclein by either CRISPRi to decrease α-synuclein expression or pharmacological activation of proteasomal degradation with repurposed drugs mitigates the ER defect. Our study offers a unifying mechanistic link between α-synuclein pathology and dysregulation of diverse organelle-associated proteins that are both Sec61A translocon substrates and genetic modifiers of Parkinson's disease risk. Our data also provide a therapeutic rationale for proteasomal activation in early Parkinson's disease.

SUMMARY The ubiquitin specific protease 28 (USP28) is implicated in tumorigenesis by controlling the turnover of substrates including the oncogene c-MYC and the ubiquitin ligase FBW7. Here, we describe small molecule inhibitors of USP25 and USP28, leading to cancer cell cycle arrest and death. However, genetic deletion of USP25/28 does not replicate this effect. An integrated –omics approach revealed off-target effects for thienopyridine and thienopyrazine carboxamide compounds in protein translation. Chemoproteomics and biochemical analyses suggested binding of such compounds to a region near the ribosome complex polypeptide exit tunnel. Structural analysis of a USP28-inhibitor complex enabled the design of modified USP25/28 inhibitor molecules which minimized translation-related off-target effects. In distinction to earlier compounds, the optimized inhibitors were non-toxic to breast cancer cells yet retained potent anti-proliferative activity in squamous lung carcinoma cells, where USP28 is associated with disease progression. Together, our results demonstrate that refined USP25/28 inhibitors can selectively suppress tumor growth by targeting c-MYC driven pathways, offering a more precise therapeutic strategy for treating squamous lung cancers whilst minimizing undesired cytotoxicity.

BACKGROUND:Lung cancer is the leading cause of cancer mortality worldwide despite the availability of low-dose computed tomography (LDCT) for screening in high-risk populations. METHODS:To develop an approach and identify blood-based protein signatures for lung cancer that can be deployed across platforms, we combined data-independent acquisition mass-spectrometry (DIA-MS) and proximity extension assay (PEA) with explainable artificial intelligence (XAI)-led machine learning (ML) for plasma-based biomarker discovery. Using a cohort of 490 lung cancer patients and 124 matched controls, ML models were trained to predict lung cancer and XAI was used to characterise networks of model-consistent features. We then introduced a DNA-aptamer based proteomic approach to assess cross-platform concordance and define a cross-platform signature. This signature was subsequently evaluated using an external cohort. RESULTS:Here we show that ML models achieve an AUROC of 0.91 [95% CI: 0.88-0.93] and 0.97 [95% CI: 0.92-0.98] in DIA-MS and PEA, respectively, using a 80/20% train/holdout split. XAI further characterises networks of model-consistent features related to chemotaxis, cell adhesion, wound healing and immune response. Introduction of the DNA-aptamer proteomic approach identifies a cross-platform signature, with performances of 0.88 [95% CI: 0.80-0.90] and 0.88 [95% CI: 0.81-0.95] in DIA-MS and PEA, respectively. Assessment of this signature in an external cohort separates lung cancer from control cases. CONCLUSIONS:This study develops an approach combining multi-dimensional proteomics with XAI-ML and demonstrates the characterisation of cross-platform biomarker signatures for lung cancer.