Proteomic Analysis Of Cells Research Articles

#302 Proteomic study reveals indoxyl sulfate induced changes in endothelial cells: role of extracellular vesicles in cardiovascular disease

Abstract Background and Aims Chronic kidney disease (CKD) lead to the accumulation of uremic toxins in the bloodstream, among which indoxyl sulfate (IS) is the most prominent. IS causes damage to endothelial cells lining blood vessels, ultimately leading to cardiovascular diseases in patients with CKD. Under physiological conditions, extracellular vesicles (EVs) are released by cells as intercellular communicators; however, after damage, the cargo of the EVs can be modified. This makes EV potential markers of damage, possibly carrying proteins of interest. This study aimed to investigate the impact of IS on endothelial cells and the release of EVs to identify potential therapeutic targets for preventing endothelial injury. Method Human umbilical vein endothelial cells (HUVEC) were cultured under control conditions and with IS exposure (250 μM IS, 24 h, n = 5). The released EVs were isolated from the supernatants by differential centrifugation and characterized by TEM and flow cytometry to confirm their identity as EVs (following the MISEV2018 guidelines). Proteome profiles of both cells and EVs were analyzed using a mass spectrometer (Orbitrap Exploris™ 240), followed by a Gene Set Enrichment Analysis (GSEA) which revealed altered functions. Results A total of 5871 proteins were identified in the proteomic analysis of cells, and 3614 proteins in EVs (with a False Discovery Rate (FDR) < 1%). In cells, 30 proteins were upregulated and 46 were found downregulated in response to IS, while in EVs, 51 proteins were found upregulated and 47 downregulated (IS vs C). GSEA revealed that IS potentiated adipogenesis (p-value < 0.001), TNF-α signaling via NFкB (p-value = 0.006), and xenobiotic metabolism in cells (p-value = 0.012). In contrast, EVs from cells treated with IS exhibited downregulation of extracellular matrix components (p-value = 0.003), myogenesis (p-value 0.009), genes downregulated in response to ultraviolet radiation (p-value = 0.014) and TNF-α signaling via NFкB (p-value = 0.041). Conclusion IS induces a change in protein expression in cells and their EVs, potentiating inflammation and atherosclerotic plaque formation with extracellular matrix loss. All these modifications contribute to endothelial dysfunction, which is the first step in the development of cardiovascular disease in patients with CKD. This study shed light on novel targets for intervention strategies to mitigate cardiovascular complications in patients with CKD. Funding Grants from the Instituto de Salud Carlos III (ISCIII) and cofounded by Fondos Europeo de Desarrollo Regional (FEDER): “PI19/00240” and “PI20/01321” and “FI20/00018” contract by the Instituto de Salud Carlos III and Ayuda de la Línea de Actuación “Excelencia para el Profesorado Universitario de la UAH” (EPU-INV-UAH/2022/001) from Alcala University.

Abstract CT194: N-myristoylation inhibitor zelenirstat: New mechanistic insights and efficacy signals from a first in human phase I study

Abstract Introduction. Zelenirstat (PCLX-001) is a small molecule inhibitor of N-myristoylation, a process that involves addition of the fatty acid myristate to over 200 proteins by two N-myristoyltransferases (NMT1 and 2). These include Src family kinases and c-Abl. Inhibition of N-myristoylation by zelenirstat leads to the degradation of non-myristoylated proteins and cancer cell death. We conducted a phase I trial to evaluate the safety, tolerability, and maximally tolerated dose (MTD) of zelenirstat in patients with refractory cancer. Methods. Differential mass spectrometry was performed in NMT1 and NMT2 CRISPR/Cas9 KOs or zelenirstat-treated HAP1 cells to identify N-myristoylation-regulated proteins. Fully consented patients with advanced solid malignancies or refractory B-cell lymphomas were administered escalating doses of zelenirstat in 28-day cycles until progression or dose-limiting toxicity (DLT). Results. Proteomic analysis of cells with genetic or pharmacological NMT inhibition was performed to gain insights into NMT substrates and function. In addition to the rapid degradation of SFKs, which disrupts the pro-survival signaling of RTKs, we identified multiple respiratory complex I proteins as the most degraded, including NDUFAF4. NMT1 KO and zelenirstat treatment disrupted complex I leading to oxidative phosphorylation (OXPHOS) inhibition, which is essential for both cancer stem cell survival and metastasis. Continuous once-daily zelenirstat at 20 mg to 210 mg was well tolerated, with no dose-limiting toxicities in 24 patients up to and including 210 mg. Gastrointestinal DLTs were observed in the 280 mg cohort, establishing 210 mg as the MTD. Oral absorption was rapid, and pharmacokinetics were suitable for once daily dosing. Seven patients had stable disease as best response, including pancreatic, ovarian and colon cancer patients; one colon cancer patient with 5 prior lines of therapy continues 210 mg beyond 11 cycles with reduction in carcinoembryonic antigen (CEA) and tumor dimensions. Kaplan-Meier analysis revealed longer progression-free survival (log-rank p=0.033) and overall survival (p=0.026) in the patients treated at 210 mg (n=7) when compared with patients treated in lower dose cohorts (n=17). Conclusion. Zelenirstat has a unique dual impact on growth signaling and OXPHOS. Having previously demonstrated pre-clinical efficacy in hematologic cancers in vitro and in vivo, we now demonstrate the therapeutic potential of zelenirstat in patients with refractory advanced solid malignancies, warranting further clinical evaluation in these indications. Citation Format: Luc Gerard Berthiaume, Erwan Beauchamp, Jay Gamma, Rony Pain, Morris A. Kostiuk, Christopher R. Cromwell, Eman W. Moussa, Olivier Julien, Basil P. Hubbard, John Kuruvilla, Laurie H. Sehn, Jennifer Spratlin, Rahima Jamal, Randeep Sangha, John R. Mackey. N-myristoylation inhibitor zelenirstat: New mechanistic insights and efficacy signals from a first in human phase I study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT194.

Ciljanje onkogenih puteva posredstvom laktoferina u hemioprevenciji raka i dopunskoj terapiji - od mehanističkih uvida do kliničkih ispitivanja

Genetic, genomic and proteomic analyses of cells, tissues and body fluids have generated a wealth of precious information about the intricate mechanisms which underlie carcinogenesis and metastasis. Lactoferrin, a multifunctional cationic glycoprotein, has attracted widespread appreciation because of its characteristically novel properties for cancer chemoprevention. Tumor microenvironment is a highly complicated and sophisticated ecosystem, significantly reshaped by a wide variety of treatment regimes. Therefore, lactoferrin-mediated immunostimulatory role reshapes tumor microenvironment and inhibits cancer progression. There is sufficient experimental evidence related to immunostimulatory ability of lactoferrin in tumor microenvironment. Different clinical trials have been conducted for the evaluation of clinical efficacy of lactoferrin in different cancer patients. It is necessary to carefully interpret the clinical evidence and identify the major gaps in our understanding related to the selection of group of cancer patients likely to benefit the most from the combinatorial treatment regime comprised of lactoferrin and chemotherapeutic drugs. Moreover, lack of efficacy should be analyzed by a team of interdisciplinary researchers for a broader and comprehensive understanding of the mechanisms underlying treatment failure.

Genetics meets proteomics: perspectives for large population-based studies.

Proteomic analysis of cells, tissues and body fluids has generated valuable insights into the complex processes influencing human biology. Proteins represent intermediate phenotypes for disease and provide insight into how genetic and non-genetic risk factors are mechanistically linked to clinical outcomes. Associations between protein levels and DNA sequence variants that colocalize with risk alleles for common diseases can expose disease-associated pathways, revealing novel drug targets and translational biomarkers. However, genome-wide, population-scale analyses of proteomic data are only now emerging. Here, we review current findings from studies of the plasma proteome and discuss their potential for advancing biomedical translation through the interpretation of genome-wide association analyses. We highlight the challenges faced by currently available technologies and provide perspectives relevant to their future application in large-scale biobank studies.

Extracellular polyphosphate signals through Ras and Akt to prime Dictyostelium discoideum cells for development

ABSTRACT Linear chains of five to hundreds of phosphates called polyphosphate are found in organisms ranging from bacteria to humans, but their function is poorly understood. In Dictyostelium discoideum, polyphosphate is used as a secreted signal that inhibits cytokinesis in an autocrine negative feedback loop. To elucidate how cells respond to this unusual signal, we undertook a proteomic analysis of cells treated with physiological levels of polyphosphate and observed that polyphosphate causes cells to decrease levels of actin cytoskeleton proteins, possibly explaining how polyphosphate inhibits cytokinesis. Polyphosphate also causes proteasome protein levels to decrease, and in both Dictyostelium and human leukemia cells, decreases proteasome activity and cell proliferation. Polyphosphate also induces Dictyostelium cells to begin development by increasing expression of the cell–cell adhesion molecule CsA (also known as CsaA) and causing aggregation, and this effect, as well as the inhibition of proteasome activity, is mediated by Ras and Akt proteins. Surprisingly, Ras and Akt do not affect the ability of polyphosphate to inhibit proliferation, suggesting that a branching pathway mediates the effects of polyphosphate, with one branch affecting proliferation, and the other branch affecting development.

Proteomic analysis of proteome and histone post-translational modifications in heat shock protein 90 inhibition-mediated bladder cancer therapeutics.

e16030 Background: Heat shock protein 90 (HSP90) is a chaperone for many proteins integral development of cancer, making inhibition of HSP90 an attractive strategy for cancer treatment. However, little is known about HSP90 inhibition-mediated bladder cancer therapy. Here, we present a quantitative proteomic study that evaluates alterations in protein expression and histone post-translational modifications (PTMs) in bladder cancer in response to HSP90 inhibition. Methods: We assessed the antitumor activity of five HSP90 inhibitors in 5637 bladder cancer cell line using MTS, cell viability, and caspase 3/7 assays. We then performed a proteomic analysis of cells before and after treatment with the HSP90 inhibitors and assessed for changes. Results: We show that the five HSP90 inhibitors (AUY922, ganetespib, SNX2112, AT13387, and CUDC305) potently inhibited the proliferation of bladder cancer 5637 cells. HSP90 inhibitors also had differential effects on cell survival between bladder cancer cells and uroepithelial cells. Our proteomic study quantified 518 twofold up-regulated and 811 twofold downregulated proteins common to both AUY922 and ganetespib treatment (P < 0.05). The subsequent bioinformatic analysis revealed that those differentially expressed proteins were involved in chromatin modifications and cell death-associated pathways. Further, quantitative proteome studies identified 14 different types of PTMs with 93 marks on the core histones, including 34 novel histone marks of butyrylation, citrullination, 2-hydroxyisobutyrylation, propionylation, and succinylation in AUY922- and ganetespib-treated 5637 cells, suggesting that HSP90 inhibitors may exert multiple cytotoxic actions in bladder cancer cells by inhibiting HSP90 activity or altering the structure of chromatin. Conclusions: This study outlines the association between proteomic changes and histone PTMs in response to HSP90 inhibitor treatment in bladder cancer cells, and supports the notion that HSP90 inhibitors both as single agents and in combination might provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration.

Multiplex Proteomic Analysis of Cells in Fine Needle Aspiration Samples: How Soon Can We Expect to Individualize Therapy by Comparing Pre- and Posttreatment Biopsies?

Individualized therapy of the future will be guided by panels of biomarkers that include proteins, nucleic acids, and metabolites. Among these analyte classes, proteins can provide direct functional information central to the disease. The added bonus is that the protein, or phosphoprotein, biomarker is the drug target itself and/or the direct readout of the signaling activity being inhibited. An example is the selection of kinase inhibitor therapy based on phosphorylated receptor tyrosine kinases, such as human epidermal growth factor receptor 2, protein kinase B, epidermal growth factor receptor, MET receptor tyrosine kinase, or cytoplasmic signaling proteins, such as mammalian target of rapamycin. Yet it has not been easy to quantitatively measure a panel of protein/phosphoprotein biomarkers in tissue biopsy samples with high precision, sensitivity, and accuracy, which are essential attributes if the data are used to select patient therapies. Addressing this challenge, Ullal et al. (1) described a new technology that uses DNA barcode labeling of antibodies for multiplex proteomic/phosphoprotein analysis of cells in a tumor fine needle aspiration (FNA).2 FNAs are challenging clinical specimens that contain relatively few cells compared to a tissue biopsy. Why is this method important, how does it compare to existing multiplexed clinical proteomic technologies, and when can we hope to see this platform routinely used in the clinical laboratory? The author's platform (1) is divided into serial stages. In the first stage, the cells are passed through a microfluidic chamber where tumor cells are separated from host cells. Here, the cell mixture is exposed to antibodies tagged with magnetic particles. This permits the removal of contaminating immune cells or positive selection of tumor cells. In the second stage, the cancer cells are incubated with a cocktail of antibodies that are labeled with DNA “barcodes.” Each barcode is a different 70-nucleotide sequence. The DNA …

Amplification Target ADRM1: Role as an Oncogene and Therapeutic Target for Ovarian Cancer

Approximately 25,000 ovarian cancers are diagnosed in the U.S. annually, and 75% are in the advanced stage and largely incurable. There is critical need for early detection tools and novel treatments. Proteasomal ubiquitin receptor ADRM1 is a protein that is encoded by the ADRM1 gene. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. Its overexpression correlated significantly with shorter time to recurrence and overall survival. Array-CGH and microarray expression of ovarian cancer cell lines provided evidence consistent with primary tumor data that ADRM1 is a 20q13 amplification target. Herein, we confirm the ADRM1 amplicon in a second ovarian cancer cohort and define a minimally amplified region of 262 KB encompassing seven genes. Additionally, using RNAi knock-down of ADRM1 in naturally amplified cell line OAW42 and overexpression of ADRM1 via transfection in ES2, we show that (1) ADRM1 overexpression increases proliferation, migration, and growth in soft agar, and (2) knock-down of ADRM1 results in apoptosis. Proteomic analysis of cells with ADRM1 knock-down reveals dysregulation of proteins including CDK-activating kinase assembly factor MAT1. Taken together, the results indicate that amplified ADRM1 is involved in cell proliferation, migration and survival in ovarian cancer cells, supporting a role as an oncogene and novel therapeutic target for ovarian cancer.

Proteomic analysis of cells in the early stages of herpes simplex virus type‐1 infection reveals widespread changes in the host cell proteome

During infection by herpes simplex virus type-1 (HSV-1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV-1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV-1 replication, 2-DE and LC-MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock- and HSV-1-infected HEp-2 cells revealed a total of 103 protein spot changes. Of these, 63 were up-regulated and 40 down-regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV-1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV-1 infection.

Amiodarone induces stress responses and calcium flux mediated by the cell wall inSaccharomyces cerevisiae

We used a proteomic approach to study effects of amiodarone on cells of the yeast Saccharomyces cerevisiae. Amiodarone has been shown to have antifungal activity in vitro and causes a massive increase in cytoplasmic calcium levels ([Ca2+]cyt). Proteomic analysis of cells exposed to amiodarone show that this drug elicits stress responses and points to involvement of proteins associated with the cell wall. We tested several of those proteins for involvement in the Ca2+ flux. In particular, the amiodarone-induced Ca2+ flux was decreased in bgl2Delta cells, which have altered levels of beta-glucan and chitin. The involvement of the cell wall in the Ca2+ flux induced by amiodarone treatment was tested by addition of yeast cell-wall components. While mannan inhibited the rise in [Ca2+]cyt, beta-glucan potentiated the Ca2+ flux by 4.5-fold, providing evidence that the cell wall is directly involved in controlling this Ca2+ flux. This conclusion is corroborated by the inhibition of the Ca2+ flux by calcofluor, which is known to bind to cell-wall chitin and inhibit cell growth. Zymolyase treatment altered the kinetics of amiodarone-induced calcium flux and uncoupled the inhibitory effect of calcofluor. These effects demonstrate that the cell-wall beta-glucan regulates calcium flux elicited by amiodarone.