Clare Coveney

Background: Osteoarthritis is a degenerative joint disease characterized by progressive loss of articular cartilage and limited capacity for intrinsic repair. A major barrier to developing effective regenerative strategies is the incomplete understanding of the molecular mechanisms regulating chondrogenesis and cartilage maintenance. Cell surface proteins are key mediators of extracellular communication, adhesion, and signaling, yet the chondrogenic surfaceome remains incompletely mapped, with prior studies focusing primarily on mature or cytokine-activated chondrocytes. The aim of this study was to provide a temporal profile of the surfaceome during in vitro chondrogenic differentiation and to identify novel membrane proteins with potential roles in cartilage biology. Methods: We applied a sialoglycoprotein-targeted glycocapture strategy to selectively enrich plasma membrane proteins from chick embryonic limb bud-derived micromass cultures undergoing chondrogenesis. Enriched samples underwent high-resolution shotgun proteomic analysis, and differentially expressed candidates were validated by western blotting, immunocytochemistry, and transient gene silencing. Functional effects on extracellular matrix gene regulation were assessed by quantitative RT-PCR and matrix histochemistry. Results: This approach generated the temporal surfaceome map of chondrogenic progenitors. Among identified candidates, two proteins not previously linked to chondrogenesis, podocalyxin (PODXL) and ciliary neurotrophic factor receptor (CNTFR), were detected at the plasma membrane and confirmed at the protein and transcript levels. Both proteins exhibited time-dependent downregulation during differentiation. Targeted knockdown revealed differential regulation of the fibrocartilage marker COL1A1 expression, indicating non-redundant roles in cell-matrix signaling and survival pathways. Single-cell transcriptomic meta-analysis confirmed expression of both proteins in discrete human articular chondrocyte subpopulations. Conclusions: This study expands the molecular framework of chondrogenesis, identifying PODXL and CNTFR as novel, temporally regulated surfaceome components with distinct roles in extracellular matrix signaling. These findings complement prior proteomic analyses of cytokine-activated mature articular chondrocytes and suggest new candidates for developmental cartilage biomarkers and therapeutic targets for osteoarthritis. Our results provide a resource for future cross-species surfaceome studies and highlight key pathways for further investigation into cartilage lineage specification and matrix adaptation.

Background: Osteoarthritis is a degenerative joint disease characterized by progressive loss of articular cartilage and limited capacity for intrinsic repair. A major barrier to developing effective regenerative strategies is the incomplete understanding of the molecular mechanisms regulating chondrogenesis and cartilage maintenance. Cell surface proteins are key mediators of extracellular communication, adhesion, and signaling, yet the chondrogenic surfaceome remains incompletely mapped, with prior studies focusing primarily on mature or cytokine-activated chondrocytes. The aim of this study was to provide a temporal profile of the surfaceome during in vitro chondrogenic differentiation and to identify novel membrane proteins with potential roles in cartilage biology. Methods: We applied a sialoglycoprotein-targeted glycocapture strategy to selectively enrich plasma membrane proteins from chick embryonic limb bud-derived micromass cultures undergoing chondrogenesis. Enriched samples underwent high-resolution shotgun proteomic analysis, and differentially expressed candidates were validated by western blotting, immunocytochemistry, and transient gene silencing. Functional effects on extracellular matrix gene regulation were assessed by quantitative RT-PCR and matrix histochemistry. Results: This approach generated the first temporal surfaceome map of chondrogenic progenitors. Among identified candidates, two proteins not previously linked to chondrogenesis, podocalyxin (PODXL) and ciliary neurotrophic factor receptor (CNTFR), were detected at the plasma membrane and confirmed at the protein and transcript levels. Both proteins exhibited time-dependent downregulation during differentiation. Targeted knockdown revealed differential regulation of the fibrocartilage marker COL1A1 expression, indicating non-redundant roles in cell-matrix signaling and survival pathways. Single-cell transcriptomic meta-analysis confirmed expression of both proteins in discrete human articular chondrocyte subpopulations. Conclusions: This study expands the molecular framework of chondrogenesis, identifying PODXL and CNTFR as novel, temporally regulated surfaceome components with distinct roles in extracellular matrix signaling. These findings complement prior proteomic analyses of cytokine-activated mature articular chondrocytes and suggest new candidates for developmental cartilage biomarkers and therapeutic targets for osteoarthritis. Our results provide a resource for future cross-species surfaceome studies and highlight key pathways for further investigation into cartilage lineage specification and matrix adaptation. Plain English Summary: Osteoarthritis is the most common joint disease worldwide, causing pain, stiffness, and reduced mobility as the smooth cartilage that covers the ends of bones gradually breaks down. Cartilage has a very limited ability to heal itself, and there are currently no treatments that can stop or reverse the disease. Scientists are working on new ways to repair or replace damaged cartilage, but to do this effectively, they need to understand the molecular “language” that cartilage-forming cells use to develop and maintain healthy tissue. In this study, we examined the set of proteins that are present on the surface of cartilage-forming cells, known as the “surfaceome.” These surface proteins play important roles in how cells connect to their surroundings, receive signals, and work together to build the cartilage matrix. Using a method to selectively capture and analyze these proteins, we discovered two that had never been linked to cartilage development: podocalyxin and ciliary neurotrophic factor receptor. We found that these two proteins may have opposite effects on cartilage quality. Because they can be detected on the outside of cells, they could be developed into markers for early osteoarthritis detection or as targets for new therapies. Our findings open the door to new strategies for diagnosing and treating joint disease by focusing on cell-surface communication.

The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein–protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53.

Amyloid-beta (Aβ) deposition in the brain is closely linked with the development of Alzheimer’s disease (AD). Unfortunately, therapies specifically targeting Aβ deposition have failed to reach their primary clinical endpoints, emphasizing the need to broaden the search strategy for alternative targets/mechanisms. Transglutaminase-2 (TG2) catalyzes post-translational modifications, is present in AD lesions and interacts with AD-associated proteins. However, an unbiased overview of TG2 interactors is lacking in both control and AD brain. Here we aimed to identify these interactors using a crossbreed of the AD-mimicking APP23 mouse model with wild type and TG2 knock-out (TG2^−/−) mice. We found that absence of TG2 had no (statistically) significant effect on Aβ pathology, soluble brain levels of Aβ_1–40 and Aβ_1–42, and mRNA levels of TG family members compared to APP23 mice at 18 months of age. Quantitative proteomics and network analysis revealed a large cluster of TG2 interactors involved in synaptic transmission/assembly and cell adhesion in the APP23 brain typical of AD. Comparative proteomics of wild type and TG2^−/− brains revealed a TG2-linked pathological proteome consistent with alterations in both pathways. Our data show that TG2 deletion leads to considerable network alterations consistent with a TG2 role in (dys)regulation of synaptic transmission and cell adhesion in APP23 brains.

Exercise is a non-pharmacological intervention that can enhance bone regeneration and improve the management of bone conditions like osteoporosis or metastatic bone cancer. Therefore, it is gaining increasing importance in an emerging area of regenerative medicine—regenerative rehabilitation (RR). Osteocytes are mechanosensitive and secretory bone cells that orchestrate bone anabolism and hence postulated to be an attractive target of regenerative exercise interventions. However, the human osteocyte signalling pathways and processes evoked upon exercise remain to be fully identified. Making use of a computer-controlled bioreactor that mimics exercise and the latest omics approaches, RNA sequencing (RNA-seq) and tandem liquid chromatography-mass spectrometry (LC-MS), we mapped the transcriptome and secretome of mechanically stretched human osteocytic cells. We discovered that a single bout of cyclic stretch activated network processes and signalling pathways likely to modulate bone regeneration and cancer. Furthermore, a comparison between the transcriptome and secretome of stretched human and mouse osteocytic cells revealed dissimilar results, despite both species sharing evolutionarily conserved signalling pathways. These findings suggest that osteocytes can be targeted by exercise-driven RR protocols aiming to modulate bone regeneration or metastatic bone cancer.

Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and β2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-β2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-β2 inhibition. This study also highlights the first association between ADRβ2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRβ2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.

The expected five-year survival rate from a stage III ovarian cancer diagnosis is a mere 22%; this applies to the 7000 new cases diagnosed yearly in the UK. Stratification of patients with this heterogeneous disease, based on active molecular pathways, would aid a targeted treatment improving the prognosis for many cases. While hundreds of genes have been associated with ovarian cancer, few have yet been verified by peer research for clinical significance. Here, a meta-analysis approach was applied to two carefully selected gene expression microarray datasets. Artificial neural networks, Cox univariate survival analyses and T-tests identified genes whose expression was consistently and significantly associated with patient survival. The rigor of this experimental design increases confidence in the genes found to be of interest. A list of 56 genes were distilled from a potential 37,000 to be significantly related to survival in both datasets with a FDR of 1.39859 × 10⁻¹¹, the identities of which both verify genes already implicated with this disease and provide novel genes and pathways to pursue. Further investigation and validation of these may lead to clinical insights and have potential to predict a patient’s response to treatment or be used as a novel target for therapy.