Quantitative Proteomic Analysis Research Articles

PD-1 interactome in osteosarcoma: identification of a novel PD-1/AXL interaction conserved between humans and dogs

The PD-1/PDL-1 immune checkpoint inhibitors revolutionized cancer treatment, yet osteosarcoma remains a therapeutic challenge. In some types of cancer, PD-1 receptor is not solely expressed by immune cells but also by cancer cells, acting either as a tumor suppressor or promoter. While well-characterized in immune cells, little is known about the role and interactome of the PD-1 pathway in cancer. We investigated PD-1 expression in human osteosarcoma cells and studied PD-1 protein–protein interactions in cancer. Using U2OS cells as a model, we confirmed PD-1 expression by western blotting and characterized its intracellular as well as surface localization through flow cytometry and immunofluorescence. High-throughput analysis of PD-1 interacting proteins was performed using a pull-down assay and quantitative mass spectrometry proteomic analysis. For validation and molecular modeling, we selected tyrosine kinase receptor AXL—a recently reported cancer therapeutic target. We confirmed the PD-1/AXL interaction by immunoblotting and proximity ligation assay (PLA). Molecular dynamics (MD) simulations uncovered binding affinities and domain-specific interactions between extracellular (ECD) and intracellular (ICD) domains of PD-1 and AXL. ECD complexes exhibited strong binding affinity, further increasing for the ICD complexes, emphasizing the role of ICDs in the interaction. PD-1 phosphorylation mutant variants (Y223F and Y248F) did not disrupt the interaction but displayed varying strengths and binding affinities. Using bemcentinib, a selective AXL inhibitor, we observed reduced binding affinity in the PD-1/AXL interaction, although it was not abrogated. To facilitate the future translation of this finding into clinical application, we sought to validate the interaction in canine osteosarcoma. Osteosarcoma spontaneously occurs at significantly higher frequency in dogs and shares close genetic and pathological similarities with humans. We confirmed endogenous expression of PD-1 and AXL in canine osteosarcoma cells, with PD-1/AXL interaction preserved in the dog cells. Also, the interacting residues remain conserved in both species, indicating an important biological function of the interaction. Our study shed light on the molecular basis of the PD-1/AXL interaction with the implication for its conservation across species, providing a foundation for future research aimed at improving immunotherapy strategies and developing novel therapeutic approaches.

Quantitative analysis of septin Cdc10 & Cdc3-associated proteome during stress response in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. Fatal outcome of cryptococcosis depends on the ability of C. neoformans to sense and adapt to 37°C. A complex of conserved filament forming GTPases, called septins, composed of Cdc3, Cdc10, Cdc11, and Cdc12, assembles at the mother-bud neck in C. neoformans. Septins Cdc3 and Cdc12 are essential for proliferation of C. neoformans at 37°C and for virulence in the Galleria mellonella model of infection, presumably due to their requirement for septin complex formation, and the involvement in cytokinesis. However, how exactly Cdc3, and Cdc12 contribute to C. neoformans growth at 37°C remains unknown. Based on studies investigating roles of septins in Saccharomyces cerevisiae, septin complex at the mother-bud neck of C. neoformans is predicted to interact with proteins involved in cell cycle control, morphogenesis, and cytokinesis, but the septin-associated proteome in C. neoformans has not been investigated. Here, we utilized tandem mass spectrometry to define C. neoformans proteins that associate with either Cdc3 or Cdc10 at ∼25°C or after the shift to 37°C. Our findings unveil a diverse array of septin-associated proteins, highlighting potential roles of septins in cell division, and stress response. Two proteins, identified as associated with both Cdc3 and Cdc10, the actin-binding protein profilin, which was detected at both temperatures, and ATP-binding multi-drug transporter Afr1, which was detected exclusively at 37°C, were further confirmed by co-immunoprecipitation. We also confirmed that association of Cdc3 with Afr1 was enhanced at 37°C. Upon shift to 37°C, septins Cdc3 and Cdc10 exhibited altered localization and Cdc3 partially co-localized with Afr1. In addition, we also investigated changes to levels of individual C. neoformans proteins upon shift from ∼25 to 37°C in exponentially grown culture and when cells entered stationary phase at ∼25°C. Our study reveals changes to C. neoformans proteome associated with heat and nutrient deprivation stresses and provides a landscape of septin-associated C. neoformans proteome, which will facilitate elucidating the biology of septins and mechanisms of stress response in this fungal pathogen.

Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming.

Repeated antigen exposure leads to T-cell exhaustion, a transcriptionally and epigenetically distinct cellular state marked by loss of effector functions (e.g., cytotoxicity, cytokine production/release), up-regulation of inhibitory receptors (e.g., PD-1), and reduced proliferative capacity. Molecular pathways underlying T-cell exhaustion have been defined for CD8+ cytotoxic T cells, but which factors drive exhaustion in CD4+ T cells, that are also required for an effective immune response against a tumor or infection, remains unclear. Here, we utilize quantitative proteomic, phosphoproteomic, and metabolomic analyses to characterize the molecular basis of the dysfunctional cell state induced by chronic stimulation of CD4+ memory T cells. We identified a dynamic response encompassing both known and novel up-regulated cell surface receptors, as well as dozens of unexpected transcriptional regulators. Integrated causal network analysis of our combined data predicts the histone acetyltransferase p300 as a driver of aspects of this phenotype following chronic stimulation, which we confirmed via targeted small molecule inhibition. While our integrative analysis also revealed large-scale metabolic reprogramming, our independent investigation confirmed a global remodeling away from glycolysis to a dysfunctional fatty acid oxidation-based metabolism coincident with oxidative stress. Overall, these data provide both insights into the mechanistic basis of CD4+ T-cell exhaustion and serve as a valuable resource for future interventional studies aimed at modulating T-cell dysfunction.

Matrine promotes colorectal cancer apoptosis by downregulating shank-associated RH domain interactor expression

BACKGROUND The 5-year survival rate of patients with colorectal cancer (CRC) in China is only 56.9%, highlighting the need for new therapeutic drugs. Previous studies have shown that matrine exhibits antitumor effects by inducing apoptosis. However, the mechanism by which matrine regulates antiapoptotic proteins in CRC remains unclear. AIM To identify apoptotic proteins from proteomics and investigate the role of matrine in impeding CRC apoptosis by regulating these proteins. METHODS Tumor and adjacent normal tissues were collected from 52 patients with CRC who underwent surgery between January and December 2021. Data-independent acquisition quantitative proteomic analysis was performed to identify differentially expressed apoptotic proteins. The selected apoptotic proteins were identified through their association with tumor-node-metastasis (TNM) stage and prognosis, then confirmed by immunohistochemical (IHC) staining in validation cohort. In vitro , the role of matrine or apoptotic proteins on cancer cells were analyzed. RESULTS Compared to normal tissues, 88 anti-apoptotic proteins from proteomic results were selected. Among them, Shank-associated RH domain interactor (SHARPIN) was identified because of its relationship with TNM stage and overall survival in TCGA database. In the IHC-confirmed cohort, SHARPIN was highly expressed in CRC tissues and localized in the cytoplasm. Higher SHARPIN expression was associated with TNM stage, carbohydrate antigen 153 levels, and gross type compared to low expression. SHARPIN knockdown promoted apoptosis, significantly upregulated the expression of Bcl-2 associated agonist of cell death, Bcl-2 associated X protein, caspase 3, and caspase 8, and downregulated B-cell lymphoma-2 (P < 0.05). Importantly, matrine treatment promoted apoptosis and reversed the proliferation, invasion, and migration of CRC cells by repressing SHARPIN. CONCLUSION SHARPIN was identified as an upregulated anti-apoptotic protein in CRC, and matrine exhibited anticancer effects by downregulating its expression. Thus, matrine appears to be a promising drug for CRC.

Quantitative proteomics analysis reveals the pathogenesis of obstructed defecation syndrome caused by abnormal expression of dystrophin.

Obstructed defecation syndrome (ODS) represents the most prevalent form of chronic constipation, affecting a diverse patient population, leading to numerous complications, and imposing a significant burden on healthcare resources. Most ODS patients have insufficient rectal propulsion, but the exact mechanism underlying the pathogenesis of ODS remains unclear. To explore the molecular mechanism underlying the pathogenesis of ODS. A total of 30 pairs of rectal samples were collected from patients with ODS (ODS group) or grade IV prolapsed hemorrhoids without constipation (control group) for quantitative proteomic and bioinformatic analysis. Subsequently, 50 pairs of paraffin-embedded rectal specimens were selected for immunohistochemistry and immunofluorescence studies to validate the analysis results. Human intestinal smooth cell contractile function experiments and electrophysiological experiments were conducted to verify the physiological functions of target proteins. Cellular ultrastructure was detected using transmission electron microscopy. In comparison to the control group, the expression level of dystrophin (DMD) in rectal specimens from ODS patients was markedly reduced. This finding was corroborated using immunohistochemistry and immunofluorescence techniques. The diminished expression of DMD compromised the contractile function of intestinal smooth muscle cells. At the molecular level, nucleoporin protein 153 and L-type voltage-gated calcium channel were found to be overexpressed in intestinal smooth muscle cells exhibiting downregulated DMD expression. Electrophysiological experiments confirmed an excessive influx of calcium ions into these cells. Moreover, vacuolar-like structures which may be associated with excessive calcium influx were observed in the cells by transmission electron microscopy. Decreased DMD expression in intestinal smooth muscle may upregulate L-type voltage-gated calcium channel expression, leading to excessive calcium influx which may cause a decrease in rectal propulsion, thereby contributing to the pathogenesis of ODS.

Quantitative Proteomic Analysis of APP/PS1 Transgenic Mice.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder affecting the central nervous system (CNS), with its etiology still shrouded in uncertainty. The interplay of extracellular amyloid-β (Aβ) deposition, intracellular neurofibrillary tangles (NFTs) composed of tau protein, cholinergic neuronal impairment, and other pathogenic factors is implicated in the progression of AD. The current study endeavors to delineate the proteomic landscape alterations in the hippocampus of an AD murine model, utilizing proteomic analysis to identify key physiological and pathological shifts induced by the disease. This endeavor aims to shed light on the underlying pathogenic mechanisms, which could facilitate early diagnosis and pave the way for novel therapeutic interventions for AD. To dissect the proteomic perturbations induced by Aβ and Presenilin-1 (PS1) in the AD pathogenesis, we undertook a label-free quantitative (LFQ) proteomic analysis focusing on the hippocampal proteome of the APP/PS1 transgenic mouse model. Employing a multi-faceted approach that included differential protein functional enrichment, cluster analysis, and protein-protein interaction (PPI) network analysis, we conducted a comprehensive comparative proteomic study between APP/PS1 transgenic mice and their wild-type C57BL/6 counterparts. Mass spectrometry identified a total of 4817 proteins in the samples, with 2762 proteins being quantifiable. Comparative analysis revealed 396 proteins with differential expression between the APP/PS1 and control groups. Notably, 35 proteins exhibited consistent temporal regulation trends in the hippocampus, with concomitant alterations in biological pathways and PPI networks. This study presents a comparative proteomic profile of transgenic (APP/PS1) and wild-type mice, highlighting the proteomic divergences. Furthermore, it charts the trajectory of proteomic changes in the AD mouse model across the developmental stages from 2 to 12 months, providing insights into the physiological and pathological implications of the disease-associated genetic mutations.

Elucidating the molecular mechanism of angiogenic activity of sulfate glycosaminoglycan derived from fish swim bladder in human umbilical vein endothelial cells

Fish swim bladders are considered a traditional marine aquatic food that has medicinal and nutritional properties. Sulfate glycosaminoglycans (SBSG) obtained from fish swim bladders may have anti-angiogenic activity. However, the anti-angiogenic activity of SBSG and its mechanism has not been reported. Therefore, the present study investigated the effect of SBSG on angiogenesis and examined molecular pathways in human umbilical vein endothelial cells (HUVECs) using cell viability, chicken chorioallantoic membrane (CAM) assay, tube formation assay, and proteomic analyses. The results demonstrated that the SBSG significantly (P < 0.05) suppressed tube formation in the HUVECs while decreasing vascular density in CAM. Quantitative proteome analysis identified 1474 differentially expressed proteins (DEPs) involved in different molecular pathways. In the bladder cancer pathway, 10 proteins were significantly downregulated [matrix metalloproteinase-9 (MMP-9), epidermal growth factor receptor (EGFR), cyclin-dependent kinase 4 (CDK4), fibroblast growth factor receptor 3 (FGFR3), harvey rat sarcoma (H-Ras), rat sarcoma (Ras), mitogen-activated extracellular signal-regulated kinase (MEK), extracellular regulated protein kinases (ERK), receptor tyrosine-protein kinase erbB-2 (ERBB2), and E-Cadherin (E-cad)], whereas 2 proteins were significantly upregulated [thrombospondin-1 (TSP-1) and E-cad]. Notably, SBSG was effectively bound to the active sites of EGFR, MMP-9, and TSP-1, which led to reduced EGFR and MMP-9 protein expression (p < 0.05), and increased TSP-1 (P < 0.05), thereby inhibiting angiogenesis. These findings suggest that SBSG is a potential candidate for the nutraceuticals industry for angiogenesis management.

Nicotinamide mononucleotide protects STAT1 from oxidative stress-induced degradation to prevent colorectal tumorigenesis.

Colitis, accompanied by the accumulation of reactive oxygen species (ROS) in the intestinal tract, is a risk factor for colorectal cancer (CRC). Our previous studies indicate that nicotinamide mononucleotide (NMN) replenishment reduces chronic inflammation. In this study, we confirm that NMN supplementation reduces inflammatory cytokine levels and oxidative tissue damage in an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-associated cancer (CAC) model. Mice treated with NMN developed fewer colon tumors than untreated animals under the same AOM/DSS treatment conditions. Quantitative proteomic analysis revealed a decrease in signal transducer and activator of transcription 1 (STAT1) expression in the CAC model. We demonstrate that STAT1 overexpression induces G1 arrest by downregulating CDK6 expression and suppressing tumor cell proliferation and migration. Of note, H2O2 induced trioxidation of the STAT1 protein and promoted its degradation, which was partially reversed by NMN supplementation. Upon H2O2 treatment, Cys155 in STAT1 was oxidized to sulfonic acid, whereas the mutation of Cys155 to alanine abolished ROS-mediated STAT1 degradation. These results indicate that oxidative stress induces STAT1 degradation in tumor cells and possibly in CAC tissues, whereas supplementation with NMN protects STAT1 from oxidation-induced degradation and prevents tumorigenesis. This study provides experimental evidence for the development of NMN-mediated chemoprevention strategies for CRC.

The protein composition of human adenovirus replication compartments.

Human adenoviruses are double-stranded DNA viruses that replicate in the cell nucleus and induce the formation of replication compartments (RCs) that are critical in viral replication and control of virus-host interactions. RCs are specialized virus-induced subnuclear microenvironments where not only viral genome replication and expression are orchestrated but also host proteins that restrict viral replication are co-opted and subverted. The protein composition of these RCs remains largely unexplored. In this study, we isolated adenovirus RC-enriched fractions from infected cells at different times post-infection and employed a tandem mass tag-based quantitative mass spectrometry approach to identify proteins associated with RCs (data available via ProteomeXchange identifier PXD051745). These findings reveal an elaborate network of host and viral proteins potentially relevant for RC formation and function. To validate the RC-protein components identified by mass spectrometry, we employed immunofluorescence and immunoblotting techniques. Proteins previously described to colocalize in RCs in infected cells were identified in the isolated subnuclear fractions. In addition, we validated newly identified proteins associated with RCs, including the high mobility group box 1 (HMGB1), the SET nuclear proto-oncogene, the structure-specific recognition protein 1 (SSRP1), the CCCTC-binding protein (CTCF), and sirtuin 6 (SIRT6). We identified HMGB1 as a protein that binds to the viral DNA binding protein (DBP). Using shRNA knockdowns and inhibitors, we demonstrated that HMGB1 acts as a proviral factor, promoting efficient viral DNA synthesis and progeny production. Our data further suggest potential candidate targets for therapeutic intervention and provide mechanistic insights into the molecular basis of virus-host interactions.IMPORTANCEHuman adenoviruses serve as models for studying respiratory viruses and have provided critical insights into viral genome replication and gene expression, as well as the control of virus-host interactions. These processes are coordinated within virus-induced subnuclear microenvironments known as RCs. We conducted quantitative proteome analyses of RC-enriched subnuclear fractions at different times post-infection with human adenovirus species C type 5, revealing a multifaceted network of proteins that participate in the regulation of gene expression, DNA damage response, RNA metabolism, innate immunity, and other cellular antiviral defense mechanisms. Furthermore, we validated the localization of several host proteins to viral RCs using immunofluorescence microscopy and immunoblotting and identified cellular HMGB1 as a proviral factor late during infection. These findings represent the first analysis of the proteomes of isolated RCs and not only enhance our understanding of nuclear organization during infection but also shed light on the complex interplay between viral and host factors within RCs.

Candida albicans cells exhibit media specific proteomic profiles during induction of filamentation

Candida albicans is an opportunist pathogen responsible for a broad spectrum of infections, from superficial mycosis to the systemic disease candidiasis. C. albicans has various morphological forms, including unicellular budding yeasts, filamentous pseudohyphae and true hyphae, and the ability to switch from yeast to hyphal forms is a key survival mechanism underlying the adaptation of the pathogen to the microenvironments encountered within the host. Filamentation is regulated by multiple signalling pathways and its induction in different growth media in vitro has often led to conflicting results. In this study, we performed quantitative proteomic analyses to compare the response of C. albicans yeast cells grown in YNB minimal medium to those of cells grown in four media widely used in the literature to induce the yeast-to-hyphae transition: YNB-Serum, YNB-N-acetylglucosamine (YNB-NAG), Lee medium and rich Spider medium. We show that each growth medium induces a unique pattern of response in C. albicans cells, and that some conditions trigger an original and specific adaptive metabolic response, showing significant differences in the intracellular content of the various filamentous forms. Moreover, this comparison of proteomic profiles indicates that the medium used can modify the thiol-dependent redox status of the cells, particularly in YNB-Serum and Lee medium and, to a lesser extent, in Spider medium, confirming the role of oxidative stress in the filamentation process. Overall, our data indicate that some of the media routinely used to induce hyphae cause significant changes in proteomic signature that should be taken account more carefully when exploring the hyphal transition in this pathogen.

4D-DIA-Based Quantitative Proteomic Analysis Reveals the Involvement of TRPV2 Protein in Duck Tembusu Virus Replication

Duck Tembusu virus (DTMUV), a novel positive-sense RNA virus, has caused significant economic losses in the poultry industry of Eastern and Southeast Asia since its outbreak in 2010. Furthermore, the rapid transmission and potential zoonotic nature of DTMUV pose a threat to public health safety. In this study, a 4D-DIA quantitative proteomics approach was employed to identify differentially expressed cellular proteins in DTMUV-infected DF-1 cells, which are routinely used for virus isolation and identification for DTMUV, as well as the development of vaccines against other poultry viruses. One hundred fifty-seven differentially expressed cellular proteins were identified, including 84 upregulated and 73 downregulated proteins at 48 h post-infection, among which CXCL8, DDX3X, and TRPV2 may play crucial roles in viral propagation. Notably, for the upregulated protein TRPV2, the DTMUV replication was inhibited in TRPV2-low-expressing DF-1 cells. In summary, our research represents the application of 4D-DIA quantitative proteomics to analyze the proteomic landscape of DTMUV-infected poultry cells. These findings may provide valuable insights into understanding the interaction mechanism between DTMUV and poultry cells, as well as the identification of disease-resistant host factors in poultry breeding research.

3D in vitro modelling of post-partum cardiovascular health reveals unique characteristics and signatures following hypertensive disorders in pregnancy

BackgroundHypertensive disorders of pregnancy (HDP) affect 2–8% of pregnancies and are associated postpartum with increased cardiovascular disease (CVD) risk, although mechanisms are poorly understood.MethodsHuman induced pluripotent stem cells (iPSC)-derived cardiomyocytes, cardiac fibroblasts and coronary artery endothelial cells were cocultured to form cardiac spheroids (CSs) in collagen type-1 hydrogels containing 10% patient plasma collected five years postpartum [n = 5 per group: normotensive control, gestational hypertension (GH) and preeclampsia (PE)]. Plasma-treated CSs were assessed for cell viability and contractile function and subjected to immunofluorescence staining and imaging. A quantitative proteomic analysis of plasma samples was conducted (controls n = 21; GH n = 5; PE n = 12).ResultsContraction frequency (CF) was increased in PE-treated CSs (CF: 45.5 ± 3.4 contractions/minute, p < 0.001) and GH-treated CSs (CF: 45.7 ± 4.0 contractions/minute, p < 0.001), compared to controls (CF = 21.8 ± 2.6 contractions/min). Only PE-treated CSs presented significantly increased fractional shortening (FS) % (9.95 ± 1.8%, p < 0.05) compared to controls (3.7 ± 1.1%). GH-treated CSs showed a reduction in cell viability (p < 0.05) and an increase in α-SMA expression (p < 0.05). Proteomics analyses identified twenty differentially abundant proteins, with hemoglobin A2 being the only protein perturbed in both GH and PE versus control plasma (p < 0.05).ConclusionsThe innovative patient-relevant CS platforms led to the discovery of biomarkers/targets linked to cell death signaling and cardiac remodeling in GH-induced CVD and vascular/endothelial cell dysfunction in PE-induced CVD.

Label-free quantitative proteomic analysis reveals the characteristics of ovarian development from stage II to III in Chinese sturgeon (Acipenser sinensis)

Chinese sturgeon (Acipenser sinensis) is an endangered anadromous and rare fish. The ovary development from stage II to III is being challenged by captive conditions, which seriously hinders the artificial reproduction of Chinese sturgeon. A comparative proteomic analysis on the ovaries at stage II and stage III of Chinese sturgeon was conducted in this study using the label-free quantitative method to uncover the intricate physiological processes. The findings revealed 200 up-regulated differentially expressed proteins (DEPs) and 150 down-regulated DEPs in the ovary at stage III compared with stage II, and 11 DEPs (including UCHL1, VTG, CTSD, and ZP, etc.) involved in the vitellogenesis and oocyte growth. Moreover, the up-regulated DEPs exhibited significant enrichment in the pathways linked to protein synthesis (ribosome, protein processing in the endoplasmic), metabolism and energy production (valine, leucine and isoleucine degradation, TCA cycle, oxidative phosphorylation, and fatty acid degradation), suggesting more active protein processing and energy metabolism at stage III. Meanwhile, APOB was down-regulated while VTG was up-regulated in stage III ovary, indicating the uptake of yolk protein and lipid through VTG at this stage. The parallel reaction monitoring (PRM) quantification of 16 DEPs solidly validated the outcomes of label-free analysis. This study provided powerful information for understanding the physiological processes of ovarian development in Chinese sturgeon, and potential biomarkers in predicting the vitellogenic stage.

Perinatal iron deficiency alters the cardiac proteome and mitochondrial function in neonatal offspring.

Aims: Iron deficiency (ID) is common during gestation and early infancy and can alter developmental trajectories with lasting consequences on cardiovascular health. Iron plays a critical role in systemic oxygen transport (via hemoglobin) and aerobic respiration (as a component of mitochondrial complexes). Perinatal ID has been shown to cause cardiac dysfunction in neonates, but the mechanisms underlying these changes have not been characterized. Here, we examined the effects of perinatal ID on cardiac mitochondrial function in the early postnatal period in rats. Methods and Results: Female rats were fed an iron-restricted or iron-replete diet before and during pregnancy. Offspring hearts were collected postmortem for quantitative shotgun proteomic analysis (postnatal days [PD]0 and 28) and mitochondrial function was assessed by high-resolution respirometry (at PD0, 14 and 28). Markers of oxidative stress were measured by fluorescence microscopy and assessment of antioxidant gene expression profiles. Both male and female ID pups had reduced body weight and increased relative heart weights at all time points assessed, despite recovering from anemia by PD28. Proteomics analysis revealed dysregulation of mitochondrial proteins by ID, and these differences were most pronounced in males. In male hearts, ID increased mitochondrial content and decreased normalized mitochondrial respiration through the NADH-pathway, succinate-pathway, and FAO-pathway. Conclusions: ID causes changes in cardiac mitochondrial function in neonates, which may reflect an inadequate or maladaptive compensation during the transition from intrauterine to extrauterine life. Further, the results presented herein, which were stratified by offspring sex, underscore the need for follow-up studies to directly assess differences in the way male and female offspring cope with ID as a perinatal stressor.

Proteome Profiling of Experimental Autoimmune Encephalomyelitis Mouse Model and the Effect of a SUMO E1 Inhibitor.

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases, causing demyelination and inflammation in the central nervous system. The pathology of MS has been extensively studied using the experimental autoimmune encephalomyelitis (EAE) mouse model. However, the molecular mechanisms are still largely unclear and require further investigation. In this study, we carried out quantitative proteomic analysis of the brain and spinal cord tissues in mice induced with EAE using a data-independent acquisition strategy and identified 744 differentially regulated proteins in the brain and 741 in the spinal cord. The changed proteins were highly related with phagocytosis, lysosomal enzymes, inflammasome activation, complements, and synaptic loss processes. Moreover, gene set enrichment analysis revealed the elevation of the SUMOylation process in EAE with the increase of SUMOylation-related enzymes and modification targets. Furthermore, to test the possibility of treating MS by targeting SUMOylation, we explored the application of a selective SUMO E1 inhibitor, TAK-981. Intriguingly, TAK-981 suppressed the global SUMOylation level in the brain and significantly alleviated the symptoms of EAE in mice. Our findings contribute to a better understanding of MS pathology, reveal the important role of SUMOylation in disease progression, and demonstrate the potential of the SUMO E1 inhibitor as a novel treatment for MS.

Targeting TSPEAR-AS2 suppresses tumor growth and interferon signaling in esophageal cancer

Dysregulation of long non-coding RNAs (lncRNAs) and interferon signaling contributes to tumorigenesis and progression; however, their crosstalk in tumor biology remains poorly understood. Here, we showed that TSPEAR-AS2, an lncRNA mediated by METTL1, activates the interferon signaling pathway in esophageal cancer (ESCA). Clinically, we conducted a pan-cancer investigation and identified TSPEAR-AS2 as a novel ESCA-related lncRNA that predicts a worse patient prognosis. The high expression of TSPEAR-AS2 was validated in multiple ESCA cohorts, demonstrating that this lncRNA had good diagnostic performance. METTL1, an RNA N7-methylguanosine (m7G) methyltransferase, was positively correlated with TSPEAR-AS2 expression in ESCA tissues, and METTL1 was found to induce TSPEAR-AS2 expression in ESCA cells. Functionally, the up-regulation of TSPEAR-AS2 promoted proliferation, cell cycle progression, migration, and stemness in ESCA cells, whereas its knockdown attenuated these malignant phenotypes. Furthermore, both TSPEAR-AS2 deficiency and antisense oligonucleotide (ASO)-based intratumoral intervention could significantly suppress tumor growth in vivo. Mechanistically, quantitative proteomic analysis revealed that TSPEAR-AS2 ablation altered the expression of functional proteins related to interferon signaling pathways, such as HLA-E, IRF3, and IFITs. These findings identify a previously unrecognized role of the METTL1/TSPEAR-AS2/interferon signaling axis in ESCA development and suggest that TSPEAR-AS2 is a potential prognostic indicator and therapeutic vulnerability for this malignancy.

TMT-based quantitative proteomics analysis of defense responses induced by the Bph3 gene following brown planthopper infection in rice.

The brown planthopper (BPH) is an economically significant pest of rice. Bph3 is a key BPH resistance gene. However, the proteomic response of rice to BPH infestation, both in the presence and absence of Bph3, remains largely unexplored. In this study, we employed tandem mass tag labeling in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins (DEPs) in rice samples. We detected 265 and 125 DEPs via comparison of samples infected with BPH for 2 and 4 days with untreated samples of the BPH-sensitive line R582. For the Bph3 introgression line R373, we identified 29 and 94 DEPs in the same comparisons. Bioinformatic analysis revealed that Bph3 significantly influences the abundance of proteins associated with metabolic pathways, secondary metabolite biosynthesis, microbial metabolism in diverse environments, and phenylpropanoid biosynthesis. Moreover, Bph3 regulates the activity of proteins involved in the calcium signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction. Our results indicate that Bph3 enhances the resistance of rice to BPH mainly by inhibiting the down-regulation of proteins associated with metabolic pathways; calcium signaling, the MAPK signaling pathway, and plant hormone signal transduction might also be involved in BPH resistance induced by Bph3.

Combined Mendelian randomization and quantitative proteomics analysis to study the influence of thyroid dysfunction on acute ischemic stroke

AbstractAcute ischemic stroke (AIS) is characterized by high morbidity and mortality, making it crucial to identify the risk factors that influence its occurrence and prognosis. Although individuals with thyroid dysfunction exhibit altered stroke patterns, evidence from observational studies remains inconsistent. Herein, we investigated the influence of thyroid dysfunction on stroke progression and prognosis. We combined Mendelian randomization (MR) and tandem mass tag (TMT)‐based quantitative proteomics analysis to study the influence of thyroid dysfunction on AIS. Differentially expression proteins (DEPs) were subsequently identified, functional enrichment analysis was performed, and a protein–protein interaction (PPI) network was constructed. Protein alterations were further validated by western blot. MR analysis revealed a causal association between thyroid disorders and ischemic stroke. DEP analysis identified 38 downregulated proteins and five upregulated proteins. Functional enrichment analysis and PPI network construction highlighted the importance of immune response activation and acute phase pathways, along with the suppression of focal adhesion, regulation of the actin cytoskeleton, and platelet activation pathways. Vasodilator‐stimulated phosphoprotein, MYL12B, MYL6, and TPM4 were identified as key DEPs significantly associated with pathological pathways and were verified by western blot. The identification of these key proteins and pathways provides new perspectives for investigating the progression and prognosis of AIS.

Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography

To better understand host cell protein (HCP) retention in adeno-associated virus (AAV) downstream processes, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to quantitatively profile residual HCPs for four AAV serotypes (AAV2, -5, -8, and -9) produced with HEK293 cells and purified using POROS CaptureSelect AAVX affinity chromatography. A broad range of residual HCPs were detected in affinity eluates after purification (N total = 2,746), and HCP profiles showed universally present species (N universal = 1,117) and species unique to one or more AAV serotype. SWATH-MS revealed that HCP persistence was dominated by high-abundance conserved species (HACS), which appeared across all serotype conditions studied. Due to the notable contribution of these species to overall residual HCP levels, physical and functional characteristics of HACS were examined to determine trends that coincide with persistence. Subnetwork interaction mapping and Gene Ontology function enrichment analysis revealed extensive physical interactions between these proteins and significant enrichment for biological processes, molecular functions, and reactome pathways related to protein folding, nucleic acid binding, and cellular stress. The abundant and conserved nature of these HCPs and their functions offers a new perspective for mechanistic evaluations of impurity retention for AAV downstream processes.

Prolonged cultivation enhances the stimulatory activity of hiPSC mesenchymal progenitor-derived conditioned medium

BackgroundHuman induced pluripotent stem cells represent a scalable source of youthful tissue progenitors and secretomes for regenerative therapies. The aim of our study was to investigate the potential of conditioned medium (CM) from hiPSC-mesenchymal progenitors (hiPSC-MPs) to stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (MSCs). We also investigated whether prolonged cultivation or osteogenic pre-differentiation of hiPSC-MPs could enhance the stimulatory activity of CM.MethodsMSCs were isolated from 13 donors (age 20–90 years). CM derived from hiPSC-MPs was added to the MSC cultures and the effects on proliferation and osteogenic differentiation were examined after 14 days and 6 weeks. The stimulatory activity of hiPSC-MP-CM was compared with the activity of MSC-derived CM and with the activity of CM prepared from hiPSC-MPs pre-cultured in growth or osteogenic medium for 14 days. Comparative proteomic analysis of CM was performed to gain insight into the molecular components responsible for the stimulatory activity.ResultsPrimary bone marrow-derived MSC exhibited variability, with a tendency towards lower proliferation and tri-lineage differentiation in older donors. hiPSC-MP-CM increased the proliferation and alkaline phosphatase activity of MSC from several adult/aged donors after 14 days of continuous supplementation under osteogenic conditions. However, CM supplementation failed to improve the mineralization of MSC pellets after 6 weeks under osteogenic conditions. hiPSC-MP-CM showed greater enhancement of proliferation and ALP activity than CM derived from bone marrow-derived MSCs. Moreover, 14-day cultivation but not osteogenic pre-differentiation of hiPSC-MPs strongly enhanced CM stimulatory activity. Quantitative proteomic analysis of d14-CM revealed a distinct profile of components that formed a highly interconnected associations network with two clusters, one functionally associated with binding and organization of actin/cytoskeletal components and the other with structural constituents of the extracellular matrix, collagen, and growth factor binding. Several hub proteins were identified that were reported to have functions in cell-extracellular matrix interaction, osteogenic differentiation and development.ConclusionsOur data show that hiPSC-MP-CM enhances early osteogenic differentiation of human bone marrow-derived MSCs and that prolonged cultivation of hiPSC-MPs enhances CM-stimulatory activity. Proteomic analysis of the upregulated protein components provides the basis for further optimization of hiPSC-MP-CM for bone regenerative therapies.Graphical