Quantitative Proteomic Profiling Research Articles

Label-free liquid chromatography mass spectrometry analysis of changes in broiler liver proteins under transport stress.

Transportation duration and distance are significant concerns for animal welfare, particularly in the poultry industry. However, limited proteomic studies have investigated the impact of transport duration on poultry welfare. In this study, mass spectrometry based bottom up proteomics was employed to sensitively and impartially profile the liver tissue proteome of chickens, addressing the issue of animal stress and welfare in response to transportation before slaughter. The liver exudates obtained from Ross 508 chickens exposed to either short or long road transportation underwent quantitative label-free LC-MS proteomic profiling. This method identified a total of 1,368 proteins, among which 35 were found to be significantly different (p < 0.05) and capable of distinguishing between short and long road transportation conditions. Specifically, 23 proteins exhibited up-regulation in the non stressed group, while 12 proteins showed up-regulation in the stressed group. The proteins identified in this pilot study encompassed those linked to homeostasis and cellular energetic balance, including heat shock proteins and the 5'-nucleotidase domain-containing family. These results contribute to a deeper understanding of the proteome in broiler liver tissues, shedding light on poultry adaptability to transport stress. Furthermore, the identified proteins present potential as biomarkers, suggesting promising approaches to enhance poultry care and management within the industry.

TMT-Based Quantitative Proteomic Profiling of Human Esophageal Cancer Cells Reveals the Potential Mechanism and Potential Therapeutic Targets Associated With Radioresistance.

The recurrence of esophageal squamous cell carcinoma (ESCC) in radiation therapy treatment presents a complex challenge due to its resistance to radiation. However, the mechanism underlying the development of radioresistance in ESCC remains unclear. In this study, we aim to uncover the mechanisms underlying radioresistance in ESCC cells and identify potential targets for radiosensitization. We established two radio-resistant cell lines, TE-1R and KYSE-150R, from the parental ESCC cell lines TE-1 and KYSE-150 through fractionated irradiation. A TMT-based quantitative proteomic profiling approach was applied to identify changes in protein expression patterns. Cell Counting Kit-8, colony formation, γH2AX foci immunofluorescence and comet assays were utilized to validate our findings. The downstream effectors of the DNA repair pathway were confirmed using an HR/NHEJ reporter assay and Western blot analysis. Furthermore, we evaluated the expression of potential targets in ESCC tissues through immunohistochemistry combined with mass spectrometry. Over 2,000 proteins were quantitatively identified in the ESCC cell lysates. A comparison with radio-sensitive cells revealed 61 up-regulated and 14 down-regulated proteins in the radio-resistant cells. Additionally, radiation treatment induced 24 up-regulated and 12 down-regulated proteins in the radio-sensitive ESCC cells. Among the differentially expressed proteins, S100 calcium binding protein A6 (S100A6), glutamine gamma-glutamyltransferase 2 (TGM2), glycogen phosphorylase, brain form (PYGB), and Thymosin Beta 10 (TMSB10) were selected for further validation studies as they were found to be over-expressed in the accumulated radio-resistant ESCC cells and radio-resistant cells. Importantly, high S100A6 expression showed a positive correlation with cancer recurrence in ESCC patients. Our results suggest that several key proteins, including S100A6, TGM2, and PYGB, play a role in the development of radioresistance in ESCC. Our results revealed that several proteins including Protein S100-A6 (S100A6), Protein-glutamine gamma-glutamyltransferase 2 (TGM2), Glycogen phosphorylase, brain form (PYGB) were involved in radio-resistance development. These proteins could potentially serve as biomarkers for ESCC radio-resistance and as therapeutic targets to treat radio-resistant ESCC cells.

Esrra regulates Rplp1-mediated translation of lysosome proteins suppressed in metabolic dysfunction-associated steatohepatitis and reversed by alternate day fasting

ObjectiveCurrently, little is known about the mechanism(s) regulating global and specific protein translation during metabolic dysfunction-associated steatohepatitis (MASH; previously known as non-alcoholic steatohepatitis, NASH). MethodsUnbiased label-free quantitative proteome, puromycin-labelling and polysome profiling were used to understand protein translation activity in vitro and in vivo. ResultsWe observed a global decrease in protein translation during lipotoxicity in human primary hepatocytes, mouse hepatic AML12 cells, and livers from a dietary mouse model of MASH. Interestingly, proteomic analysis showed that Rplp1, which regulates ribosome and translation pathways, was one of the most downregulated proteins. Moreover, decreased Esrra expression and binding to the Rplp1 promoter, diminished Rplp1 gene expression during lipotoxicity. This, in turn, reduced global protein translation and Esrra/Rplp1-dependent translation of lysosome (Lamp2, Ctsd) and autophagy (sqstm1, Map1lc3b) proteins. Of note, Esrra did not increase its binding to these gene promoters or their gene transcription, confirming its regulation of their translation during lipotoxicity. Notably, hepatic Esrra-Rplp1-dependent translation of lysosomal and autophagy proteins also was impaired in MASH patients and liver-specific Esrra knockout mice. Remarkably, alternate day fasting induced Esrra-Rplp1-dependent expression of lysosomal proteins, restored autophagy, and reduced lipotoxicity, inflammation, and fibrosis in hepatic cell culture and in vivo models of MASH. ConclusionsEsrra regulation of Rplp1-mediated translation of lysosome/autolysosome proteins was downregulated during MASH. Alternate day fasting activated this novel pathway and improved MASH, suggesting that Esrra and Rplp1 may serve as therapeutic targets for MASH. Our findings also provided the first example of a nuclear hormone receptor, Esrra, to not only regulate transcription but also protein translation, via induction of Rplp1.

Exploring the Molecular Therapeutic Mechanisms of Gemcitabine through Quantitative Proteomics.

Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.

ITRAQ-based Quantitative Proteomic Profiling of the Regulatory Mechanisms on Immune Functions and Blood Lipids in Aged Mice Fed with Lentinula edodes-Derived Polysaccharides

iTRAQ-based Quantitative Proteomic Profiling of the Regulatory Mechanisms on Immune Functions and Blood Lipids in Aged Mice Fed with <em>Lentinula edodes</em>-Derived Polysaccharides

Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems.

Intellectual disability is a heterogeneous disorder, diagnosed using intelligence quotient (IQ) score criteria. Currently, no specific clinical test is available to diagnose the disease and its subgroups due to inadequate understanding of the pathophysiology. Therefore, current study was designed to explore the molecular mechanisms involved in disease perturbation, and to identify potential biomarkers for disease diagnosis and prognosis. A total of 250 participants were enrolled in this study, including 200 intellectually disabled (ID) subjects from the subgroups (mild, moderate, and severe) with age and gender matched healthy controls (n = 50). Initially, IQ testing score and biochemical profile of each subject was generated, followed by label-free quantitative proteomics of subgroups of IQ and healthy control group through nano-LC/MS- mass spectrometry. A total of 310 proteins were identified, among them198 proteins were common among all groups. Statistical analysis (ANOVA) of the subgroups of ID showed 142 differentially expressed proteins, in comparison to healthy control group. From these, 120 proteins were found to be common among all subgroups. The remaining 22 proteins were categorized as exclusive proteins found only in disease subgroups. Furthermore, the hierarchical cluster analysis (HCL) of common significant proteins was also performed, followed by PANTHER protein classification and GO functional enrichment analysis. Results provides that the datasets of differentially expressed proteins, belong to the categories of immune / defense proteins, transfer carrier proteins, apolipoproteins, complement proteins, protease inhibitors, hemoglobin proteins etc., they are known to involvein immune system, and complement and coagulation pathway cascade and cholesterol metabolism pathway. Exclusively expressed 22 proteins were found to be disease stage specific and strong PPI network specifically those that have significant role in platelets activation and degranulation, such as Filamin A (FLNA). Furthermore, to validate the mass spectrometric findings, four highly significant proteins (APOA4, SAP, FLNA, and SERPING) were quantified by ELISA in all the study subjects. AUROC analysis showed a significant association of APOA4 (0.830), FLNA (0.958), SAP (0.754) and SERPING (0.600) with the disease. Apolipoprotein A4 (APOA4) has a significant role in cholesterol transport, and in modulation of glucose and lipid metabolism in the CNS. Similarly, FLNA has a crucial role in the nervous system, especially in the functioning of synaptic network. Therefore, both APOA4, and FLNA proteins represent good potential for candidate biomarkers for the diagnosis and prognosis of the intellectual disability. Overall, serum proteome of ID patients provides valuable information of proteins/pathways that are altered during ID progression.

Quantitative proteomic profiling in brain subregions of mice exposed to open-field low-intensity blast reveals position-dependent blast effects

Quantitative proteomic profiling in brain subregions of mice exposed to open-field low-intensity blast reveals position-dependent blast effects

Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SignificanceCiliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Rolapitant treats lung cancer by targeting deubiquitinase OTUD3

BackgroundLung cancer is cancer with the highest morbidity and mortality in the world and poses a serious threat to human health. Therefore, discovering new treatments is urgently needed to improve lung cancer prognosis. Small molecule inhibitors targeting the ubiquitin-proteasome system have achieved great success, in which deubiquitinase inhibitors have broad clinical applications. The deubiquitylase OTUD3 was reported to promote lung tumorigenesis by stabilizing oncoprotein GRP78, implying that inhibition of OTUD3 may be a therapeutic strategy for lung cancer.ResultsIn this study, we identified a small molecule inhibitor of OTUD3, Rolapitant, by computer-aided virtual screening and biological experimental verification from FDA-approved drugs library. Rolapitant inhibited the proliferation of lung cancer cells by inhibiting deubiquitinating activity of OTUD3. Quantitative proteomic profiling indicated that Rolapitant significantly upregulated the expression of death receptor 5 (DR5). Rolapitant also promoted lung cancer cell apoptosis through upregulating cell surface expression of DR5 and enhanced TRAIL-induced apoptosis. Mechanistically, Rolapitant directly targeted the OTUD3-GRP78 axis to trigger endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP)-DR5 signaling, sensitizing lung cancer cells to TRAIL-induced apoptosis. In the vivo assays, Rolapitant suppressed the growth of lung cancer xenografts in immunocompromised mice at suitable dosages without apparent toxicity.ConclusionIn summary, the present study identifies Rolapitant as a novel inhibitor of deubiquitinase OTUD3 and establishes that the OTUD3-GRP78 axis is a potential therapeutic target for lung cancer.

Abstract 2325: NUWA-ms: A network-based method to infer quantification of missing proteins using multi-cohort proteomics profiles

Abstract Although mass spectrometry is powerful for proteomic quantification, the inherent issue of missing values remains a significant challenge. We found a prevalent lack of quantification of valuable proteins (e.g., immune cell markers and drug targets) in published datasets, limiting the functional utilities of quantitative proteomic profiles. This was exemplified by the substantial lack of quantification of immune cell marker, which impede the dissection of tissue-infiltrating immune cells by deconvolution analysis of cancer proteomic profiles. Thus, we introduce a network-based method NUWA-ms for robust abundance inference of missing proteins using multi-cohort proteomic profiles. By evaluating 561 tumors with paired proteomic and transcriptomic profiles, a significant improvement of deconvolution performance was shown with the aid of NUWA-ms. This was further validated by the comparison between scRNA-seq and proteomic analyses of a same gastric cancer tumor tissue. NUWA-ms applications to cancer proteomic profiles facilitated the inference of CD8+ T cell markers and effector proteins, enabling the associations of CD8+ T cell infiltration with MSI status in colorectal cancer and anti-PD-1 therapy response in melanoma. These findings demonstrated the utilities of NUWA-ms in depicting infiltrating lymphocytes in the tumor microenvironment using proteomic profiles. Furthermore, based on the proteomic profiles of six patient-derived tumor organoids (PDOs), NUWA-ms application helped identifying PDOs sensitive to a targeted therapeutic agent including the ones neglected by the raw MS quantification, and aided in revealing the resistant mechanism. Together, NUWA-ms could enable a robust inference of protein abundance, to help identifying protein biomarkers and therapeutic targets for cancer and other complex diseases. Citation Format: Lihua Cao, Yuhao Xie, Jiale Chen, Man Wang, Tingting Zhao, Heli Yang, Yang Du, Yang Yang, Zhaode Bu, Jiafu Ji, Jianmin Wu. NUWA-ms: A network-based method to infer quantification of missing proteins using multi-cohort proteomics profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2325.

Abstract P30: Therapeutic activation of pre-B-cell receptor signaling in high-risk pediatric ALL: exploiting TCF3 and MYC-linked vulnerabilities

Abstract P30: Therapeutic activation of pre-B-cell receptor signaling in high-risk pediatric ALL: exploiting TCF3 and MYC-linked vulnerabilities

Quantitative proteomic and metabolomic profiling reveals different osmoregulation mechanisms of tilapia cells coping with different hyperosmotic stress

This study aimed to investigate the different regulatory mechanisms of euryhaline fish under regular hyperosmotic and extreme hyperosmotic stress. The OmB (Oreochromis mossambicus brain) cells were exposed to three treatments: control, regular hyperosmotic stress and extreme hyperosmotic stress. After 12 h exposure, proteomics, metabolomics analyses and integrative analyses were explored.Both kinds of stress lead to lowering cell growth and morphology changes, while under regular hyperosmotic stress, the up-regulated processes related with compatible organic osmolytes synthesis are crucial strategy for the euryhaline fish cell line to survive; On the other hand, under extreme hyperosmotic stress, the processes related with cell apoptosis and cell cycle arrest are dominant. Furthermore, down-regulated pyrimidine metabolism and several ribosomal proteins partially participated in the lowered cell metabolism and increased cell death under both kinds of hyperosmotic stress. The PI3K-Akt and p53 signaling pathways were involved in the stagnant stage of cell cycles and induction of cell apoptosis under both kinds of hyperosmotic stress. However, HIF-1, FoxO, JAK-STAT and Hippo signaling pathways mainly contribute to disrupting the cell cycle, metabolism and induction of cell apoptosis under extreme hyperosmotic stress. SignificanceIn the past, the research on fish osmoregulation mainly focused on the transcription factors and ion transporters of osmoregulation, the processes between osmotic sensing and signal transduction, and the associations between signaling pathways and regulation processes have been poorly understood.Investigating fish cell osmoregulation and potential signal transduction pathways is necessary. With the advancements in omics research, it is now feasible to investigate the relationship between environmental stress and molecular responses.In this study, we aimed to explore the signaling pathways and substance metabolism mode during hyper-osmoregulation in OmB cell line, to reveal the key factors that are critical to cell osmoregulation.

Natural compounds from freshwater mussels disrupt fungal virulence determinants and influence fluconazole susceptibility in the presence of macrophages in Cryptococcus neoformans.

As the prevalence and severity of global fungal infections rise, along with an increasing incidence of antifungal resistance, new strategies to combat fungal pathogens and overcome resistance are urgently needed. Critically, our current methods to overcome fungal infections are limited and drive the evolution of resistance forward; however, an anti-virulence approach to disarm virulence factors of the pathogen and promote host cell clearance is promising. Here, we explore the efficacy of natural compounds derived from freshwater mussels against classical fungal virulence determinants, including thermotolerance, capsule production, stress response, and biofilm formation. We integrate our phenotypic discoveries with state-of-the-art mass spectrometry-based proteomics to identify mechanistic drivers of these antifungal properties and propose innovative avenues to reduce infection and support the treatment of resistant strains.

Temporal Quantitative Proteomic and Phosphoproteomic Profiling of SH-SY5Y and IMR-32 Neuroblastoma Cells during All-Trans-Retinoic Acid-Induced Neuronal Differentiation.

The human neuroblastoma cell lines SH-SY5Y and IMR-32 can be differentiated into neuron-like phenotypes through treatment with all-trans-retinoic acid (ATRA). After differentiation, these cell lines are extensively utilized as in vitro models to study various aspects of neuronal cell biology. However, temporal and quantitative profiling of the proteome and phosphoproteome of SH-SY5Y and IMR-32 cells throughout ATRA-induced differentiation has been limited. Here, we performed relative quantification of the proteomes and phosphoproteomes of SH-SY5Y and IMR-32 cells at multiple time points during ATRA-induced differentiation. Relative quantification of proteins and phosphopeptides with subsequent gene ontology analysis revealed that several biological processes, including cytoskeleton organization, cell division, chaperone function and protein folding, and one-carbon metabolism, were associated with ATRA-induced differentiation in both cell lines. Furthermore, kinase-substrate enrichment analysis predicted altered activities of several kinases during differentiation. Among these, CDK5 exhibited increased activity, while CDK2 displayed reduced activity. The data presented serve as a valuable resource for investigating temporal protein and phosphoprotein abundance changes in SH-SY5Y and IMR-32 cells during ATRA-induced differentiation.

Disruption of CFAP418 interaction with lipids causes widespread abnormal membrane-associated cellular processes in retinal degenerations.

Syndromic ciliopathies and retinal degenerations are large heterogeneous groups of genetic diseases. Pathogenic variants in the CFAP418 gene may cause both disorders, and its protein sequence is evolutionarily conserved. However, the disease mechanism underlying CFAP418 mutations has not been explored. Here, we apply quantitative lipidomic, proteomic, and phosphoproteomic profiling and affinity purification coupled with mass spectrometry to address the molecular function of CFAP418 in the retina. We show that CFAP418 protein binds to the lipid metabolism precursor phosphatidic acid (PA) and mitochondrion-specific lipid cardiolipin but does not form a tight and static complex with proteins. Loss of Cfap418 in mice disturbs membrane lipid homeostasis and membrane-protein associations, which subsequently causes mitochondrial defects and membrane-remodeling abnormalities across multiple vesicular trafficking pathways in photoreceptors, especially the endosomal sorting complexes required for transport (ESCRT) pathway. Ablation of Cfap418 also increases the activity of PA-binding protein kinase Cα in the retina. Overall, our results indicate that membrane lipid imbalance is a pathological mechanism underlying syndromic ciliopathies and retinal degenerations which is associated with other known causative genes of these diseases.

Proteomic analysis of extracellular vesicle cargoes mirror the cardioprotective effects of rivaroxaban in patients with venous thromboembolism.

Venous thromboembolism (VTE) remains a significant cause of morbidity and mortality worldwide. Rivaroxaban, a direct oral factor Xa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties; yet, these remain poorly characterized. Extracellular vesicles (EVs) are considered proinflammatory messengers regulating a myriad of (patho)physiological processes and may be highly relevant to the pathophysiology of VTE. The effects of Rivaroxaban on circulating EVs in VTE patients remain unknown. We have established that differential EV biosignatures are found in patients with non-valvular atrial fibrillation anticoagulated with Rivaroxaban versus warfarin. Here, we investigated whether differential proteomic profiles of circulating EVs could also be found in patients with VTE. We performed comparative label-free quantitative proteomic profiling of enriched plasma EVs from VTE patients anticoagulated with either Rivaroxaban or warfarin using a tandem mass spectrometry approach. Of the 182 quantified proteins, six were found to be either exclusive to, or enriched in, Rivaroxaban-treated patients. Intriguingly, these proteins are involved in negative feedback regulation of inflammatory and coagulation pathways, suggesting that EV proteomic signatures may reflect both Rivaroxaban's anti-coagulatory and anti-inflammatory potential. These differences suggest Rivaroxaban may have pleiotropic effects, supporting the reports of its emerging anti-inflammatory and cardiovascular-protective characteristics relative to warfarin.

Label-free quantitative proteomic profiling reveals differential plasma protein expression in patients with obesity after treatment with liraglutide.

Treatment and management of obesity is clinically challenging. The inclusion of GLP-1 receptor agonists (GLP1RA) in the medical management of obesity has proven to be efficacious. However, mechanisms underlying the molecular changes arising from GLP1RA treatment in patients with obesity remain to be elucidated. A single-center, prospective study was undertaken to evaluate the changes in the plasma proteins after liraglutide 3 mg therapy in twenty patients (M/F: 7/13) with obesity (mean BMI 40.65 ± 3.7 kg/m2). Anthropometric and laboratory parameters were measured, and blood samples were collected at two time points: baseline, before initiating treatment (pretreatment group, PT), and after three months of receiving the full dose liraglutide 3 mg (posttreatment group, PoT). An untargeted label-free LC MSMS mass spectrometric approach combined with bioinformatics and network pathway analysis was used to determine changes in the proteomic profiles. The mean age of the study participants was 36.0 ± 11.1 years. A statistically significant change was observed in weight, BMI and HbA1c levels between the PT and PoT groups (paired t-test, P < 0.001). A significant dysregulation was noted in the abundances of 151 proteins (31 up and 120 downregulated) between the two groups. The potential biomarkers were evaluated using receiver operating characteristic (ROC) curves. The top ten proteins (area under the curve (AUC) of 0.999 (95% CI)) were identified as potential biomarkers between PT and PoT groups and included Cystatin-B, major vault protein, and plastin-3, which were upregulated, whereas multimerin-2, large ribosomal P2, and proline-rich acidic protein 1 were downregulated in the PoT group compared with the PT group. The top network pathway identified using ingenuity pathway analysis (IPA), centered around dysregulation of MAPK, AKT, and PKc signaling pathways and related to cell-to-cell signaling and interaction, cellular assembly and organization, cellular compromise and a score of 46 with 25 focus proteins. Through label-free quantitative proteomic analysis, our study revealed significant dysregulation of plasma proteins after liraglutide 3 mg treatment in patients with obesity. The alterations in the proteomic profile between the PT and PoT groups demonstrated a decrease in levels of proteins involved in inflammation and oxidative stress pathways. On the other hand proteins involved in the glycolytic and lipolytic metabolic pathways as well as those participating in cytoskeletal and endothelial reorganization were observed to be increased. Understanding actions of liraglutide at a molecular and proteomic levels provides a holistic look into how liraglutide impacts metabolism, induces weight loss and improves overall metabolic health.

Myeloid Cell Trim59 Deficiency Worsens Experimental Ischemic Stroke and Alters Cerebral Proteomic Profile.

Tripartite motif containing 59 (TRIM59) is a ubiquitin ligase and is involved in the pathogenesis of various diseases, including cancers, sepsis, and other immune-related diseases. However, it has not been defined whether TRIM59 plays a role in ischemic stroke in mice. This study determined the influence of Trim59 deficiency on experimental stroke outcomes and the cerebral proteomic profile using myeloid cell Trim59 conditional knockout (Trim59-cKO) mice and a label-free quantitative proteomic profiling technique. The possible mechanisms by which TRIM59 affected stroke onset were elucidated by in vivo and in vitro experiments. Immunofluorescence staining results showed that TRIM59 expression was up-regulated after cerebral ischemia and co-localized with macrophages. Myeloid cell Trim59 deficiency exacerbated ischemic injury on day 3 after experimental stroke. In proteomic analysis, 23 differentially expressed proteins were identified in ischemic brain of Trim59-cKO mice as compared to Trim59flox/flox mice. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the differentially expressed proteins were enriched in complement and coagulation cascades. Protein-protein interaction analysis suggested the central role of clusterin in the interaction network. ELISA and Western blot assays confirmed the reduced levels of clusterin protein in the ischemic brains of Trim59-cKO mice. Further experimental results showed that clusterin was expressed in neurons. Conditional co-culture experiments of primary neurons and bone marrow-derived macrophages demonstrated that LPS stimulated macrophages to secrete complement C3. In addition, TRIM59 may affect the changes in clusterin expression in an indirect manner by influencing the secretion of complement C3 in macrophages. In vivo experiments also proved a significant increase in C3 levels in the brains of Trim59-cKO mice after ischemia. Myeloid cell Trim59 deficiency aggravated ischemic stroke outcomes in conjunction with a distinct cerebral proteomic profile, and the underlying mechanism may be related to the regulation of macrophage C3 expression by TRIM59.

THE CELL “SURFACEOME”: A STRATEGY TO TARGET SENESCENT CELLS IN VIVO

Abstract Senescent cell accumulation drives aging and related pathologies including metabolic disorders and neurodegeneration, and their selective elimination is a promising therapeutic approach to treat multiple diseases of aging in humans. However, developing drugs to eliminate senescent cells requires specific biomarkers to selectively identify and target these cells in vivo. In this study, we have performed a comprehensive and quantitative proteomic profiling of surface proteomes (surfaceomes) of senescent cells to identify potential senescence biomarker candidates in human tissues. We induced senescence using ionizing radiation in a variety of cell types including lung fibroblasts, monocytes, vascular smooth muscle cells, aortic endothelial cells and preadipocytes. Senescence was validated using a panel of canonical markers including reduced proliferation, increased senescence-associated β-galactosidase activity, and increased expression of p21, p16, IL6, and others. The cell surface proteome was enriched using an optimized surface biotinylation approach (Glyco-cell surface capture) followed by data-independent acquisition (DIA) LC-MS/MS analysis on a Q-Exactive HF Orbitrap mass spectrometer. Our analysis revealed novel cell surface targets that were further validated by flow cytometry and immunohistochemistry. These results provide clinically useful information for establishing biomarkers and therapeutic targets to aid in the translation of senescence-targeted therapies to treat age-associated diseases.

EXTH-31. EXPLOITING THE GENETIC DEPENDENCY ON PI3K/MTOR SIGNALING FOR THE TREATMENT OF H3-ALTERED DIFFUSE MIDLINE GLIOMA

Abstract Recurring activating mutations, amplifications and allelic loss of the negative repressors of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling genes are overarching contributors to the poor survival of patients with H3K27-altered diffuse midline gliomas (DMG) diagnosed in the pons (diffuse intrinsic pontine glioma – DIPG), highlighting the clinical potential of therapies that target this axis. Employing a loss-of-function CRISPR-Cas9 gene deletion screen, we identified PIK3CA and MTOR as survival dependences in DMG/DIPG patient derived cell line models (n=36), highlighting the clinical potential of targeting PI3K/Akt/mTOR. The brain-penetrant, PI3K/mTOR inhibitor paxalisib is currently under clinical investigation in DIPG (NCT03696355 and NCT05009992), but dosing limiting toxicities including hyperglycaemia, mucositis and skin irritation limit patient compliance. To exploit this therapeutic vulnerability, we optimized the preclinical use of paxalisib, identifying both precision medicine combination strategies and systemic interventions that reduced side effects and elicited profound tumor control in vivo. Mimicking the patient experience, paxalisib elevated blood glucose levels, promoting the phosphorylation of the insulin receptor (p-INSR) in patient derived DIPG xenograft models. Combining paxalisib with the anti-glycemic drug metformin, synergistically extended the survival of two DIPG xenograft models, decreasing p-Akt, p-mTOR and p-INSR in vivo. Phosphoproteomic profiling of DIPG cells identified paxalisib induced intracellular calcium-regulated PKC signaling. The brain penetrant PKC inhibitor enzastaurin extended the survival of DIPG xenograft models alone, and synergistically combined with paxalisib, enhanced using metformin in three DIPG xenograft models, as well as the immunocompetent H3f3aK27M, PdgfraD842Vmut, Trp53-/- murine brainstem glioma model. Mechanisms of adaptation/plasticity were assessed by ATAC-Seq and quantitative proteomic profiling xenograft tumors refractory to treatment. Here we address the intrinsic neoplastic sequela of DIPG, by combined targeting of PI3K/Akt/mTOR using paxalisib, compensatory PKC signaling using enzastaurin, coupled with strategies to manage treatment-related side-effects and reduced efficacy using metformin; providing the preclinical rationale for the addition of metformin to NCT05009992.