Differential Proteomic Analysis Research Articles

Abstract 4449: Hepatic steatosis induced by bioactive lipids

Abstract Introduction: HCC is the most common form of liver cancer and is the sixth most common cancer globally. Despite the clinical advancement of immune checkpoint inhibitors, median survival continues to be less than twenty months and most patients develop resistance making it one of the deadliest malignancies. The rate of liver cancer continues to rise, which is coupled to the high prevalence of steatosis and steatohepatitis that accounts for about 25% in the general population. Pathologically, 80% of liver cancer occurs in patients with an underlying liver disease that displays liver steatosis. Thus, it is paramount to better characterize the transition from steatosis to HCC. Methods: Utilizing a Pten deleted mouse model (PtenloxP/loxP; Alb-Cre+) that recapitulates HCC progression, protein and lipid fractions were isolated to explore differentially expressed proteins and their correlations with bioactive lipid metabolism. Global untargeted differential proteomics was done with fractionation using a qExactive. Lipid fractions were analyzed using a Sciex 6500 for targeted lipidomic analysis. In addition, genetic knockout hepatocyte cell lines of each AKT isoform were used to explore the involvement of the PI3K/AKT signal. Further, a phosphoproteomic enrichment was performed to discover downstream signaling targets. Results: The PTEN regulated PI3K/AKT signal is induced in 54% of all liver cancers and represents the dominant signaling pathway regulating liver cancer progression. Untargeted differential proteomic analysis of Pten deleted mice livers revealed significant dysregulation in oxidative stress and eicosanoid metabolism among the top enriched disease and biological functions when the phenotype progressed from steatosis to HCC. Analysis of publicly available liver cancer patient samples from the NCI Proteomic Data Commons further shows strong correlation between PTEN protein abundance and the expression of enzymes involved in eicosanoid metabolism. Our analysis using a bio-active lipid multi reaction monitoring panel of Pten deleted mouse livers further validates significant decreases in resolving eicosanoid levels along with increases in proinflammatory eicosanoid precursors as steatosis progress to HCC. These data suggest a potential hepatic AKT-dependency in regulating the shift towards proinflammatory eicosanoids. Primary isolated hepatocytes from mouse livers lacking either AKT1 or AKT2 were analyzed to elucidate AKT’s regulatory role in hepatic eicosanoid metabolism. Proteomic and lipidomic analysis of these hepatocytes supported a unique AKT isoform specific role in the regulation of eicosanoids via potential isoform specific signaling interactions with MAPK. Conclusion: Eicosanoid metabolism dysregulation plays a key role in the progression from steatosis to HCC and appears to be regulated in an AKT isoform specific manner via interactions with MAPK signaling. Citation Format: Mario Alba, Ielyzaveta Slarve, Brandon Ebright, Yiren Zhou, Whitaker Cohn, Yunyi Jia, Elizabeth Elton, Jared Khan, Aditi Datta, Lina He, Qi Tang, Pranav Pammidimukkala, Taojian Tu, Phillip Nguyen, Jonathan Katz, Julian Whitelegge, Stan Louie, Bangyan Stiles. Hepatic steatosis induced by bioactive lipids [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 4449.

Abstract 7305: Chemoprevention of colorectal cancer: Role of trace minerals in conjunction with calcium

Abstract Background: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. CRC usually develops from adenomas (CRC precursors) after a long latency period. Prevention is crucial in reducing CRC incidence and progression to cancer. Among several prevention options, calcium has been a widely considered chemo-preventative agent. Aims: Calcium plays a vital role in the regulation of epithelial cell differentiation, which is essential for epithelial growth control. Epidemiological studies have demonstrated that adequate calcium intake is crucial for protection against colonic polyp formation and colorectal cancer. However, recent interventional trials with calcium have had only modest and mixed results, and the question is how to improve efficacy. One potential strategy is to use calcium in combination with additional minerals and trace elements. Results: Our preclinical studies employing monolayer cultures with CRC cell lines and colon organoids cultures from colon polyps have shown that a calcium-rich mineral combination derived from fossilized red algae (Aquamin) is more effective than calcium alone in inducing differentiation, suppressing growth and upregulation of calcium-sensing receptor. When colonic adenomas in culture were subjected to differential proteomic analysis, Aquamin upregulated several proteins involved in proliferation suppression, DNA mismatch repair and inducing differentiation, and apoptosis. Two long-term mouse studies have demonstrated that Aquamin is more effective in suppressing polyp & CRC incidence compared to calcium alone. Based on these findings, we conducted a 90-day FDA-approved trial (a pilot study with ten subjects per arm) comparing the two interventions in human subjects at risk for CRC. Results showed that Aquamin treatment reduced proliferation and increased cell differentiation observed by immunohistochemistry. Proteomic analysis also revealed that the minerals upregulated several proteins related to growth suppression, inducing differentiation and those contributing to cell-cell and cell-matrix adhesion functions and downregulated proteins involved in proliferation and nucleic acid metabolism. Conclusion: These results provide convincing evidence that a multi-mineral combination derived from red algae can provide benefits in preventing CRC that are not seen with calcium alone. Further studies, such as a polyp-prevention trial in human subjects, are warranted to advance these CRC prevention efforts. Citation Format: Isabelle Harber, Shannon McClintock, James Varani, Muhammad N. Aslam. Chemoprevention of colorectal cancer: Role of trace minerals in conjunction with calcium [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 7305.

Differential Proteomic Analysis of Low-Dose Chronic Paralytic Shellfish Poisoning.

Shellfish poisoning is a common food poisoning. To comprehensively characterize proteome changes in the whole brain due to shellfish poisoning, Tandem mass tag (TMT)-based differential proteomic analysis was performed with a low-dose chronic shellfish poisoning model in mice. A total of 6798 proteins were confidently identified, among which 123 proteins showed significant changes (fold changes of >1.2 or <0.83, p < 0.05). In positive regulation of synaptic transmission, proteins assigned to a presynaptic membrane (e.g., Grik2) and synaptic transmission (e.g., Fmr1) changed. In addition, altered proteins in nervous system development were observed, suggesting that mice suffered nerve damage due to the nervous system being activated. Ion transport in model mice was demonstrated by a decrease in key enzymes (e.g., Kcnj11) in voltage-gated ion channel activity and solute carrier family (e.g., Slc38a3). Meanwhile, alterations in transferase activity proteins were observed. In conclusion, these modifications observed in brain proteins between the model and control mice provide valuable insights into understanding the functional mechanisms underlying shellfish poisoning.

Extracellular vesicles from Staphylococcus aureus promote the pathogenicity of Pseudomonas aeruginosa

Co-infections with Staphylococcus aureus and Pseudomonas aeruginosa are common in patients with chronic wounds, but little is known about their synergistic effect mediated by extracellular vesicles (EVs). In this study, we investigated the effect of EVs derived from S. aureus (SaEVs) on the pathogenicity of P. aeruginosa. By using lipophilic dye, we could confirm the fusion between SaEV and P. aeruginosa membranes. However, SaEVs did not alter the growth and antibiotic susceptible pattern of P. aeruginosa. Differential proteomic analysis between SaEV-treated and non-treated P. aeruginosa was performed, and the results revealed that lipopolysaccharide (LPS) biosynthesis protein in P. aeruginosa significantly increased after SaEV-treatment. Regarding this result, we also found that SaEVs promoted LPS production, biofilm formation, and expression of polysaccharide polymerization-related genes in P. aeruginosa. Furthermore, invasion of epithelial cells by SaEV-pretreated P. aeruginosa was enhanced. On the other hand, uptake of P. aeruginosa by RAW 264.7 macrophages was impaired after pretreatment P. aeruginosa with SaEVs. Proteomic analysis SaEVs revealed that SaEVs contain the proteins involving in host cell colonization, inhibition of host immune response, anti-phagocytosis of the macrophages, and protein translocation and iron uptake of S. aureus. In conclusion, SaEVs serve as a mediator that promote P. aeruginosa pathogenicity by enhancing LPS biosynthesis, biofilm formation, epithelial cell invasion, and macrophage uptake impairment.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

High-Grade Gliomas from Subventricular Zone: Proteomic Drivers of Aggressiveness Using Fluorescence-Guided Multiple Sampling.

High-grade gliomas (HGGs) are among the most aggressive brain tumors and are characterized by dismally low median survival time. Of the many factors influencing the survival of patients with HGGs, proximity to the subventricular zone (SVZ) is one of the key influencers. In this context, 5-amino levulinic acid fluorescence-guided multiple sampling (FGMS) offers the prospect of understanding patient-to-patient molecular heterogeneity driving the aggressiveness of these tumors. Using high-resolution liquid chromatography-mass spectrometry (MS)/MS proteomics for HGGs from seven patients (four SVZ associated and three SVZ nonassociated), this study aimed to uncover the mechanisms driving the aggressiveness in SVZ-associated (SVZ+) HGGs. Differential proteomics analysis revealed significant dysregulation of 11 proteins, of which 9 proteins were upregulated and 2 were downregulated in SVZ+ HGGs compared to SVZ-non-associated (SVZ-) HGGs. The gene set enrichment analysis (GSEA) of the proteomics dataset revealed enrichment of MYC targets V1 and V2, G2M checkpoints, and E2F targets in SVZ+ HGGs. With GSEA, we also compared the pathways enriched in glioma stem cell subpopulations and observed a similar expression trend for most pathways in our data. In conclusion, this study reveals new and emerging insights on pathways that may potentially contribute to greater aggressiveness in SVZ+ HGGs. Future studies using FGMS in larger cohorts are recommended to help uncover the proteomics and molecular basis of aggressiveness and stemness in HGGs.

Identification of a Novel AML Patient Subset Sensitive to Venetoclax Using Matched Proteomics and Diverse Ex Vivo Models

AML is one of the most common and deadly hematopoietic malignancies. The molecular and biological heterogeneity of AML poses significant challenges to the identification of novel agents to treat it and the optimal triage of patients for the most effective therapeutic regimens. Ex vivo models for drug sensitivity using cell lines and/or primary AML samples have been a fruitful resource for the exploration and discovery of novel compounds in the treatment of AML, and investigators have coupled genomic and transcriptomic profiling with ex vivo models in the hope to further enhance the reliability and accuracy of results. However, primary AML blasts are molecularly complex and fastidious, relying on extracellular matrix (ECM) and signals for optimal survival, making their use for large, high-throughput drug screens impractical. Available AML cell lines are more robust and less dependent upon the ECM but often harbor strong driver alterations (e.g., KMT2A rearrangements, TP53 mutations) that occur in only a small subpopulation of AML cases. Thus, current AML cell lines may not accurately predict agent sensitivity for most patients. Here, we report the results of a novel approach coupling cutting-edge proteomics with ex vivo drug sensitivity models to develop and describe a more diverse and representative set of patient-derived cell models for drug discovery and therapeutic positioning. Initial studies focused on proteomic biomarker signatures and cell lines to predict drug sensitivity for Venetoclax, which has become a stable component for treatment of older and unfit patients with AML. To identify a proteomic biosignature associated with Venetoclax sensitivity, we examined the Beat AML dataset of 210 patient proteomics coupled to ex vivo sensitivity assays. Beat AML patients clustered by unbiased proteomics which resulted in a cluster with a patient population significantly enriched in sensitivity to Venetoclax. A partial least squares discriminant analysis (PLSDA) was used to discover proteins driving the differentiation of Venetoclax sensitive and insensitive clusters. The resulting significant proteins describe a mechanism of altered DNA double stranded break repair and Interleukin-8 (IL-8)-CXCR2 signaling separating patients. These mechanisms were partially comprised of high-quality targets with differential analysis results shown in Fig. 1A. The identities of the targets will be revealed at the time of presentation as they are currently moving forward in preclinical studies. To examine the potential predictive power of the Venetoclax sensitivity biosignature a set of novel ex vivo cell models were derived from a diverse independent population of patients obtained from FHCC/UW Myeloid Disease Repository. This population was not enriched for KMT2A or TP53 mutations. The proteomes of 90 diagnostic AML samples were examined and measured via a tandem mass tag protocol coupled with liquid chromatography with tandem mass spectrometry (TMT-LC-MS/MS). In addition, a proprietary set of novel ex vivo AML cell lines (n=17) representing the diverse genomic phenotype were generated from this same cohort of patients using proprietary media and pyrimidoindole derivative agonists of hematopoietic stem cell self-renewal. As with the primary samples, TMT-LC-MS/MS was utilized to quantify the proteome within the proprietary AML cell lines and 16 commercially available lines. Viability assays with Venetoclax were performed for all ex vivo cell models. Ex vivo cell models were then grouped based on the Venetoclax biosignature. The correlation between the proteomic biosignature for Venetoclax the measured sensitivity to Venetoclax was then examined for the ex vivo cell models (Fig. 1B). The results showed a strong correlation between the biosignature and actual ex vivo sensitivity to Venetoclax in proprietary cell models, but not in the 16 currently available cell models. Genetic drift of cell models from the primary AML patients was examined using differential proteomic analysis and no significant drift was found. The results indicated that the primary AML specimens harbored the Venetoclax sensitivity signature. Our findings reveal that the integration of unbiased patient proteomics with optimized ex vivo cell cultures can be used to identify sensitive patient subsets and exclude non-responders for novel therapeutics, as well as expanding patient access for currently approved therapies.

TMT quantitative proteomics reveals key proteins relevant to microRNA-1-mediated regulation in osteoarthritis

Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2, Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.

Quantitative proteomics analysis reveals the key proteins related to semen quality in Niangya yaks

BackgroundProteins related to sperm motility and sperm morphology have an important impact on sperm function such as metabolism, motility and fertilisation etc. An understanding of the key proteins related to semen quality in Niangya yaks would help to provide support for breeding. However, the key proteins that affect semen quality in Niangya yaks remain unclear.MethodsHerein, we applied tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC‒MS/MS) to analyze the expression levels of sperm proteins in groups of high- and low-quality semen from Niangya yaks. And fifteen differentially expressed proteins (DEPs) were randomly selected for expression level validation by parallel reaction monitoring (PRM).ResultsOf the 2,092 quantified proteins, 280 were identified as DEPs in the high-quality group versus the low-quality group. Gene Ontology (GO) analysis revealed that in terms of biological pathways, the DEPs were mainly involved in metabolic processes, cell transformation processes, and single organism metabolic processes. In terms of cell composition, the DEPs were mainly located in the cell membrane, organelle, molecular complex. In terms of molecular functions, the most abundant functions of the DEPs were catalytic activity, binding activity, transport activity, and enzyme regulation activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs were mainly involved in the cytokine and cytokine receptor interaction, notch signaling pathway, lysine biosynthesis, renal function-related protein and proteasome pathway. From protein-protein interaction (PPI) analysis of DEPs involved in important pathways, 6 related proteins affecting the semen quality of Niangya yaks were identified. And the results of the PRM and TMT analysis were consistent.ConclusionsThe differential sperm proteomic analysis of high- and low-quality semen from Niangya yaks, revealed 6 proteins (PSMC5, PSMD8, PSMB3, HSP90AA1, UGP2 and HSPB1), were mainly concentrated in energy production and metabolism, might play important roles in semen quality, which could serve as candidates for the selection and breeding of Niangya yaks.

Identification of Potentially Novel Molecular Targets of Endometrial Cancer Using a Non-Biased Proteomic Approach.

The present study was aimed at identifying novel proteins in endometrial cancer (EC), employing proteomic analysis of tissues obtained after surgery. A differential MS-based proteomic analysis was conducted from whole tissues dissected from biopsies from post-menopausal women, histologically confirmed as endometrial cancer (two endometrioid and two serous; n = 4) or normal atrophic endometrium (n = 4), providing 888 differentially expressed proteins with 246 of these previously documented elsewhere as expressed in EC and 372 proteins not previously demonstrated to be expressed in EC but associated with other types of cancer. Additionally, 33 proteins not recorded previously in PubMed as being expressed in any forms of cancer were also identified, with only 26 of these proteins having a publication associated with their expression patterns or putative functions. The putative functions of the 26 proteins (GRN, APP, HEXA, CST3, CAD, QARS, SIAE, WARS, MYH8, CLTB, GOLIM4, SCARB2, BOD1L1, C14orf142, C9orf142, CCDC13, CNPY4, FAM169A, HN1L, PIGT, PLCL1, PMFBP1, SARS2, SCPEP1, SLC25A24 and ZC3H4) in other tissues point towards and provide a basis for further investigation of these previously unrecognised novel EC proteins. The developmental biology, disease, extracellular matrix, homeostatic, immune, metabolic (both RNA and protein), programmed cell death, signal transduction, molecular transport, transcriptional networks and as yet uncharacterised pathways indicate that these proteins are potentially involved in endometrial carcinogenesis and thus may be important in EC diagnosis, prognostication and treatment and thus are worthy of further investigation.

ITRAQ-based differential proteomic analysis of high- and low-virulence strains of Spiroplasma eriocheiris

Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

How iron-bearing minerals affect the biological reduction of Sb(V): A newly discovered function of nitrate reductase

As a toxic element of global concern, the elevated concentration of antimony (Sb) in the environment has attracted increasing attention. Microorganisms have been reported as important driving forces for Sb transformation. Iron (Fe) is the most important metal associated element of Sb, however, how Fe-bearing minerals affect the biological transformation of Sb is still unclear. In this study, the effects of Fe-bearing minerals on biological Sb(V) reduction were investigated by employing a marine Shewanella sp. CNZ-1 (CNZ-1). Our results showed that the presence of hematite, magnetite and ferrihydrite (1 g/L) resulted in a decrease in Sb(III) concentration of ~19–31 % compared to the Fe(III)-minerals free system. The calculated Sb(V) reduction rates are 0.0256 (R2 0.71), 0.0389 (R2 0.87), 0.0299 (R2 0.96) and 0.0428 (R2 0.95) h−1 in the hematite-, magnetite-, ferrihydrite-supplemented and Fe(III)-minerals free systems, respectively. The cube-shaped Sb2O3 was characterized as a reductive product by using XRD, XPS, FTIR, TG and SEM approaches. Differential proteomic analysis showed that flagellar protein, cytochrome c, electron transfer flavoprotein, nitrate reductase and polysulfide reductase (up-regulation >1.5-fold, p value <0.05) were supposed to be included in the electron transport pathway of Sb(V) reduction by strain CNZ-1, and the key role of nitrate reductases was further highlighted during this reaction process based on the RT–qPCR and confirmatory experiments. Overall, these findings are beneficial to understand the environmental fate of Sb in the presence of Fe-bearing minerals and provide guidance in developing the bacteria/enzyme-mediated control strategy for Sb pollution.

Proteomic Study of Middle Ear Effusion and Its Clinical Application for Otitis Media with Effusion.

Proteins found in middle ear effusion play crucial roles in the physiological and pathological processes of otitis media with effusion (OME), influencing the etiology and clinical characteristics of this disease. The qualitative and quantitative composition of these proteins depending on the underlying pathogenesis of middle ear effusion. Understanding their physiological and pathological functions is of great importance. We collected samples from 19 volunteers diagnosed with OME. After offline separation using high-pH reversed-phase liquid chromatography (RPLC), the pooled sample was subjected to LC-MS/MS analysis to obtain a comprehensive profile of the OME proteome. Functional analysis was performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity Pathway Analysis (IPA) annotations. Data-independent acquisition (DIA) technology was utilized to analyze samples and fix whether the OME proteome could replicate the pathophysiological features associated with this disease. We conducted a differential proteomic analysis between patients with simple OME and patients who had received radiotherapy. The radiotherapy-reduced group was further divided into two subgroups: nasopharyngeal carcinoma (NPC) and other types of carcinoma. Parallel reaction monitoring (PRM) technology was used for validation of 36 differentially expressed proteins (DEPs). A number of 732 proteins were identified in the OME proteome database. Among them, 527 proteins were quantified using peak intensity-based semi-quantification (iBAQ), covering a wide dynamic range of approximately 8 orders of magnitude. Based on the functional analysis, we proposed a hypothetical mechanism of OME. This study managed to put up an inclusive analysis of the OME proteome, establishing the first human OME proteome database. We focused on differential proteomic analysis among different groups to gain a more comprehensive concept of the OME proteome and search for meaningful biomarkers.

Sanguinarine induces apoptosis in osteosarcoma by attenuating the binding of STAT3 to the single-stranded DNA-binding protein 1 (SSBP1) promoter region.

Osteosarcoma, a primary malignant bone tumour prevalent among adolescents and young adults, remains a considerable challenge despite protracted progress made in enhancing patient survival rates over the last 40 years. Consequently, the development of novel therapeutic approaches for osteosarcoma is imperative. Sanguinarine (SNG), a compound with demonstrated potent anticancer properties against various malignancies, presents a promising avenue for exploration. Nevertheless, the intricate molecular mechanisms underpinning SNG's actions in osteosarcoma remain elusive, necessitating further elucidation. Single-stranded DNA-binding protein 1 (SSBP1) was screened out by differential proteomic analysis. Apoptosis, cell cycle, reactive oxygen species (ROS) and mitochondrial changes were assessed via flow cytometry. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) were used to determine protein and gene levels. The antitumour mechanism of SNG was explored at a molecular level using chromatin immunoprecipitation (ChIP) and dual luciferase reporter plasmids. Our investigation revealed that SNG exerted an up-regulated effect on SSBP1, disrupting mitochondrial function and inducing apoptosis. In-depth analysis uncovered a mechanism whereby SNG hindered the JAK/signal transducer and activator of transcription 3 (STAT3) signalling pathway, relieved the inhibitory effect of STAT3 on SSBP1 transcription, and inhibited the downstream PI3K/Akt/mTOR signalling axis, ultimately activating apoptosis. The study delved further into elucidating the anticancer mechanism of SNG in osteosarcoma. Notably, we unravelled the previously undisclosed apoptotic potential of SSBP1 in osteosarcoma cells. This finding holds substantial promise in advancing the development of novel anticancer drugs and identification of therapeutic targets.

Proteomic and Transcriptomic Analyses to Decipher the Chitinolytic Response of Jeongeupia spp.

In nature, chitin, the most abundant marine biopolymer, does not accumulate due to the action of chitinolytic organisms, whose saccharification systems provide instructional blueprints for effective chitin conversion. Therefore, discovery and deconstruction of chitinolytic machineries and associated enzyme systems are essential for the advancement of biotechnological chitin valorization. Through combined investigation of the chitin-induced secretome with differential proteomic and transcriptomic analyses, a holistic system biology approach has been applied to unravel the chitin response mechanisms in the Gram-negative Jeongeupia wiesaeckerbachi. Hereby, the majority of the genome-encoded chitinolytic machinery, consisting of various glycoside hydrolases and a lytic polysaccharide monooxygenase, could be detected extracellularly. Intracellular proteomics revealed a distinct translation pattern with significant upregulation of glucosamine transport, metabolism, and chemotaxis-associated proteins. While the differential transcriptomic results suggested the overall recruitment of more genes during chitin metabolism compared to that of glucose, the detected protein-mRNA correlation was low. As one of the first studies of its kind, the involvement of over 350 unique enzymes and 570 unique genes in the catabolic chitin response of a Gram-negative bacterium could be identified through a three-way systems biology approach. Based on the cumulative data, a holistic model for the chitinolytic machinery of Jeongeupia spp. is proposed.

Characterization of biochemical properties of the skin mucus of turbot (Scophthalmus maximu) and differential proteomic analysis of skin mucus in response to Vibrio anguillarum

Fish mucus is the first defensive barrier to fight off pathogens in teleosts. In this study, we reported the antibacterial activity of turbot (Scophthalmus maximus) skin mucus, and the results showed that mucus exhibited antibacterial effects against both Gram-positive and Gram-negative bacteria. Moreover, biochemical parameters assays suggested that turbot skin mucus exhibited strong total antioxidant capacity (TAC), protease activity and lysozyme activity. To explore the roles of skin mucus in defense against pathogens, we processed proteomic profiling of turbot skin mucus before and after Vibrio anguillarum infection by LC-MS/MS approach. Differentially expressed proteins (DEPs) were identified among V. anguillarum treated turbot skin mucus at 0 h, 4 h and 48 h post-infection, respectively. A total of 3,324,612 secondary spectrograms were obtained, containing 38,681 unique peptides which were corresponded to 4987 proteins. The DEPs were markedly enriched in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with immunity, biological adhesion and cell junction, revealing complex changes of turbot skin mucus during pathogens infection. Collectively, this study can help us better understand the characteristics of turbot skin mucus in infection immunity and the biological function of skin mucus in response to bacterial invasion.

Exposure to High Dosage of Gold Nanoparticles Accelerates Growth Rate by Modulating Ribosomal Protein Expression.

Gold nanoparticles (AuNPs) have been utilized in various biomedical applications including diagnostics and drug delivery. However, the cellular and metabolic responses of cells to these particles remain poorly characterized. In this study, we used bacteria (Escherichia coli and Bacillus subtilis) and a fungus (Saccharomyces cerevisiae) as model organisms to investigate the cellular and metabolic effects of exposure to different concentrations of citrate-capped spherical AuNPs with diameters of 5 and 10 nm. In different growth media, the synthesized AuNPs displayed stability and microorganisms exhibited uniform levels of uptake. Exposure to a high concentration of AuNPs (1012 particles) resulted in a reduced cell division time and a 2-fold increase in cell density in both bacteria and fungus. The exposed cells exhibited a decrease in average cell size and an increase in the expression of FtsZ protein (cell division marker), further supporting an accelerated growth rate. Notably, exposure to such a high concentration of AuNPs did not induce DNA damage, envelope stress, or a general stress response in bacteria. Differential whole proteome analysis revealed modulation of ribosomal protein expression upon exposure to AuNPs in both E. coli and S.cerevisiae. Interestingly, the accelerated growth observed upon exposure to AuNPs was sensitive to sub-minimum inhibitory concentration (sub-MIC) concentration of drugs that specifically target ribosome assembly and recycling. Based upon these findings, we hypothesize that exposure to high concentrations of AuNPs induces stress on the translation machinery. This leads to an increase in the protein synthesis rate by modulating ribosome assembly, which results in the rapid proliferation of cells.

A differential proteomic analysis on HuH7 human hepatocarcinoma cell line naturally overexpressing or hypoexpressing LDLR: New proteins/pathways involved in cholesterol and triglycerides metabolism

A differential proteomic analysis on HuH7 human hepatocarcinoma cell line naturally overexpressing or hypoexpressing LDLR: New proteins/pathways involved in cholesterol and triglycerides metabolism

Proteomics of Plasma and Plasma-Treated Podocytes: Application to Focal and Segmental Glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syndrome (INS), a glomerulopathy of presumably immune origin that is attributed to extrarenal pathogenic circulating factors. The recurrence of FSGS (rFSGS) after transplant occurs in 30% to 50% of cases. The direct analysis of patient plasma proteome has scarcely been addressed to date, mainly due to the methodological difficulties associated with plasma complexity and dynamic range. In this study, first, we compared different methods of plasma preparation, second, we compared the plasma proteomes of rFSGS and controls using two preparation methods, and third, we analyzed the early proximal signaling events in podocytes subjected to patient plasma, through a combination of phosphoproteomics and lipid-raft proteomics (raftomics). By combining immunodepletion and high pH fractionation, we performed a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n = 4). In both the soluble- and the EV-protein sets from the rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid-binding proteins, generally associated with lipoproteins, was found to be decreased in the soluble set from the rFSGS patients. In addition, three amino acid transporters involved in mTORC1 activation were found to be significantly increased in the EV from the rFSGS. Next, we incubated human podocytes for 30 min with 10% plasma from both groups of patients. The phosphoproteomics and raftomics of the podocytes revealed profound differences in the proteins involved in the mTOR pathway, in autophagy, and in cytoskeleton organization. We analyzed the correlation between the abundance of plasma and plasma-regulated podocyte proteins. The observed changes highlight some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating-factor activity in podocytes.

Unbiased differential proteomic profiling between cancer-associated fibroblasts and cancer cell lines

Cancer-associated fibroblasts (CAFs) are a key component of tumors. We aimed to profile the proteome of cancer cell lines representing three common cancer types (lung, colorectal and pancreatic) and a representative CAF cell line from each tumor type to gain insight into CAF function and novel CAF biomarkers. We used isobaric labeling, liquid chromatography and mass spectrometry to evaluate the proteome of 9 cancer and 3 CAF cell lines. Of the 9460 proteins evaluated, functional enrichment analysis revealed an upregulation of N-glycan biosynthesis and extracellular matrix proteins in CAFs. 85 proteins had 16-fold higher expression in CAFs compared to cancer cells, including previously known CAF markers like fibroblast activation protein (FAP). Novel overexpressed CAF biomarkers included heat shock protein β-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1). SiRNA knockdown of the genes encoding these proteins did not reduce contractility in lung CAFs, suggesting they were not crucial to this function. Immunohistochemical analysis of 30 tumor samples (10 lung, 10 colorectal and 10 pancreatic) showed restricted HSPB6 and PTGS1 expression in the stroma. Therefore, we describe an unbiased differential proteome analysis of CAFs compared to cancer cells, which revealed higher expression of HSPB6 and PTGS1 in CAFs. Data are available via ProteomeXchange (PXD040360). SignificanceCancer-associated fibroblasts (CAFs) are highly abundant stromal cells present in tumors. CAFs are known to influence tumor progression and drug resistance. Characterizing the proteome of CAFs could give potential insights into new stromal drug targets and biomarkers. Mass spectrometry-based analysis comparing proteomic profiles of CAFs and cancers characterized 9460 proteins of which 85 proteins had 16-fold higher expression in CAFs compared to cancer cells. Further interrogation of this rich resource could provide insight into the function of CAFs and could reveal putative stromal targets. We describe for the first time that heat shock protein β-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1) are overexpressed in CAFs compared to cancer cells.