Proteome Of Epithelial Cells Research Articles

Proteomic Differences in Colonic Epithelial Cells in Ulcerative Colitis Have an Epigenetic Basis

The colonic epithelium serves as both a barrier to lumenal contents and a gatekeeper of inflammatory responses. In ulcerative colitis (UC), epithelial dysfunction is a core feature, but little is known about the cellular changes that may underlie disease pathology. We therefore evaluated how the chromatin epigenetics and proteome of epithelial cells differs between health and UC. We sorted live CD326+ epithelial cells from colon biopsies of healthy control (HC) screening colonoscopy recipients and from inflamed or uninflamed colon segments of UC patients on no biologic nor immunomodulator therapy (n= 5-7 subjects per group). Cell lysates were analyzed by proteomic evaluation and nuclei were analyzed for open chromatin with assay for transposase-accessible chromatin using sequencing. Proteins most highly elevated in inflamed UC biopsies relative to HC were those encoded by the HLA-DRA (P= 3.1× 10-33) and CD74 (P= 1.6× 10-27), genes associated with antigen presentation, and the antimicrobial dual oxidase 2 (DUOX2) (P= 3.2× 10-28) and lipocalin-2 (P= 2.2× 10-26) genes. Conversely, the water channel aquaporin 8 was strikingly less common with inflammation (P= 1.9× 10-18). Assay for transposase-accessible chromatin using sequencing revealed more open chromatin around the aquaporin 8 gene in HCs (P= 2.0× 10-2) and more around the DUOX2/DUOXA2 locus in inflamed UC colon (P= 5.7× 10-4), suggesting an epigenetic basis for differential protein expression by epithelial cells in health and disease. Numerous differences exist between the proteome and chromatin of colonic epithelial cells in UC patients and HCs, some of which correlate to suggest specific epigenetic mechanisms regulating the epithelial proteome.

Abstract 3963: Mass spectrometry reveals a downregulation of calreticulin after neoadjuvant chemotherapy in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinomas are one of the deadliest cancer types with limited response to current therapies. Thus, this study examined changes in the proteome of epithelial cells in pancreatic cancer after neoadjuvant chemotherapy with gemcitabine, to identify new therapeutic targets. Methods: Upon receiving patient consent, fresh pancreatic cancer tissue was received from surgical tumor resections at the University Hospital of Bonn. We mechanically and enzymatically digested and purified the tissue to create single-cell suspensions. After staining the cells against CD45 und CD326, we purified epithelial cells, blood cells, and stroma through FACS. To analyze their proteome, the sorted cells were lysed in TEAB buffer and the protein lysates were measured by the mass spectrometry core facility at the University of Bonn. We then analyzed the data using DAVID and Shiny GO. Results: Gene enrichment analyses showed a significantly reduced expression of calreticulin after neoadjuvant chemotherapy in epithelial cells. A direct comparison between untreated and treated epithelium cancer cells using a heat map showed a decreased expression of actin and enzymes of glycolysis in treated cancer cells. Moreover, untreated epithelial cells showed an increase of proteins related to MHC class Ib protein complex assembly and antigen processing and presentation of endogenous peptide antigen via MHC class. In contrast, treated cells showed a higher presence of proteins for nutrient supply from the blood. Furthermore, untreated stroma cells exhibited an increased number of proteins responsible for mesenchymal migration and for actin filament fragmentation. Conclusion: This study shows that neoadjuvant chemotherapy with gemcitabine leads to distinct changes in the proteome of tumor cells in pancreatic cancers. In particular, a decreased expression of the “eat-me-signal” calreticulin impairs macrophage-mediated cancer cell phagocytosis, thereby indicating a mechanism by which cancer cells protect themselves after chemotherapy. Citation Format: Michael C. Stoffel, Elisabeth BH Dingendorf, Dahlia L. Kittel, Glen Kristiansen, Tristan Lerbs. Mass spectrometry reveals a downregulation of calreticulin after neoadjuvant chemotherapy in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3963.

Combination of IL-17A/F and TNF-α uniquely alters the bronchial epithelial cell proteome to enhance proteins that augment neutrophil migration

BackgroundThe heterodimer interleukin (IL)-17A/F is elevated in the lungs in chronic respiratory disease such as severe asthma, along with the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Although IL-17A/F and TNF-α are known to functionally cooperate to exacerbate airway inflammation, proteins altered by their interaction in the lungs are not fully elucidated.ResultsWe used Slow Off-rate Modified Aptamer-based proteomic array to identify proteins that are uniquely and/or synergistically enhanced by concurrent stimulation with IL-17A/F and TNF-α in human bronchial epithelial cells (HBEC). The abundance of 38 proteins was significantly enhanced by the combination of IL-17A/F and TNF-α, compared to either cytokine alone. Four out of seven proteins that were increased > 2-fold were those that promote neutrophil migration; host defence peptides (HDP; Lipocalin-2 (LCN-2) and Elafin) and chemokines (IL-8, GROα). We independently confirmed the synergistic increase of these four proteins by western blots and ELISA. We also functionally confirmed that factors secreted by HBEC stimulated with the combination of IL-17A/F and TNF-α uniquely enhances neutrophil migration. We further showed that PI3K and PKC pathways selectively control IL-17A/F + TNF-α-mediated synergistic production of HDPs LCN-2 and Elafin, but not chemokines IL-8 and GROα. Using a murine model of airway inflammation, we demonstrated enhancement of IL-17A/F, TNF-α, LCN-2 and neutrophil chemokine KC in the lungs, thus corroborating our findings in-vivo.ConclusionThis study identifies proteins and signaling mediated by concurrent IL-17A/F and TNF-α exposure in the lungs, relevant to respiratory diseases characterized by chronic inflammation, especially neutrophilic airway inflammation such as severe asthma.

Cell‐Cell and Cell‐Extracellular Matrix Communication Pathways Identified in the Polarized Surface Proteome of Retinal Pigment Epithelial Cells

BackgroundPolarized epithelial cells express distinct sets of surface proteins at the apical and basolateral membranes. This surface proteome is crucial for epithelial physiology as it includes channels and transporters, surface receptors and adhesion molecules among other proteins. We recently developed a quantitative approach to obtain polarized surface proteomes of epithelial cells in culture. This information could benefit efforts to understand epithelial cell/extracellular medium interactions as mechanisms to maintain higher order multi‐tissue structures. Such interactions could represent organ‐specific pathways of homeostasis and disease. In order to exploit the potential of our polarized epithelial surface proteomics approach, we pursued a detailed analysis of members of the Plexin/Semaphorin/Integrin (PSI) superfamily we found in the proteome of retinal pigment epithelial cells. In other cells, plexins are known receptors for semaphorins and mediate cell‐cell communication, while integrins bind components of the extracellular matrix. In epithelial cells, some PSI isoforms have well known roles in cell adhesion, cytoskeletal remodeling and homotypic cell‐cell junctions. However, the wide range of isoforms identified by surface proteomics provides a unique opportunity to analyze less‐known isoforms as well as cell‐type specific pathways.ObjectiveTo identify semaphorins and integrin isoforms in retinal pigment epithelium that mediate interactions with the extracellular matrix and other cell types.MethodsWe identified possible targets of interests based on their surface expression in retinal pigment epithelium and absence in the generic epithelial cell line MDCK. Next, we utilized gene knockdown via CRISPR or shRNA to analyze their role in cell‐adhesion, migration and communication with endothelial cells. To study interaction with endothelial cells in vivo, we measured angiogenesis in a mouse model of matrigel‐induced neovascularization.ResultsWe determined that retinal pigment epithelial cells expressed semaphorin‐4A (Sema4A) and Beta‐8 integrin (ItgB8) at the surface, but they were absent in MDCK epithelial cells. Knockdown of ItgB8 significantly decreased retinal pigment epithelial cell migration in wound healing assays and cell adhesion to uncoated and collagen IV‐coated substrate. Knockdown of Sema4A in retinal pigment epithelial cells enhanced angiogenesis in co‐cultured choroidal explants (contains native endothelial cells that supply the retinal pigment epithelium in vivo). This enhanced angiogenesis suggests a baseline inhibitory role of secreted Sema4A. To test this possibility in vivo, we injected recombinant Sema4A intraocularly in mice, and we observed that this reduced matrigel‐induced neovascularization.ConclusionAnalysis of the surface proteome of epithelial cells led to identification of ItgB8 as mediator of retinal pigment epithelial cell adhesion and migration, and Sema4A as an anti‐angiogenic cell‐cell communication signal.Support or Funding InformationNIH EY008538 and EY008538‐28S1

Rapid Remodeling of the Host Epithelial Cell Proteome by the Listeriolysin O (LLO) Pore-forming Toxin

Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria modulate host gene transcription, thereby modifying the set of proteins synthetized by the infected cell. Bacteria can also target pre-existing host proteins and modulate their post-translational modifications or trigger their degradation. Analysis of protein levels variations in host cells during infection allows to integrate both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the toxin Listeriolysin O (LLO), secreted by the bacterial pathogen Listeria monocytogenes. We showed that a short-term treatment with LLO remodels the host cell proteome by specifically decreasing the abundance of 149 proteins. The same decrease in host protein levels was observed in different epithelial cell lines but not in macrophages. We show in particular that this proteome remodeling affects several ubiquitin and ubiquitin-like ligases and that LLO leads to major changes in the host ubiquitylome. Strikingly, this toxin-induced proteome remodeling involves only post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data reveal that different bacterial pore-forming toxins induce important host proteome remodeling, that may impair epithelial cell functions.

Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis

Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens.

Evaluating the potential of a novel oral lesion exudate collection method coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery

IntroductionEarly diagnosis of Oral Squamous Cell Carcinoma (OSCC) increases the survival rate of oral cancer. For early diagnosis, molecular biomarkers contained in samples collected non-invasively and directly from at-risk oral premalignant lesions (OPMLs) would be ideal.MethodsIn this pilot study we evaluated the potential of a novel method using commercial PerioPaper absorbent strips for non-invasive collection of oral lesion exudate material coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery.ResultsOur evaluation focused on three core issues. First, using an "on-strip" processing method, we found that protein can be isolated from exudate samples in amounts compatible with large-scale mass spectrometry-based proteomic analysis. Second, we found that the OPML exudate proteome was distinct from that of whole saliva, while being similar to the OPML epithelial cell proteome, demonstrating the fidelity of our exudate collection method. Third, in a proof-of-principle study, we identified numerous, inflammation-associated proteins showing an expected increase in abundance in OPML exudates compared to healthy oral tissue exudates. These results demonstrate the feasibility of identifying differentially abundant proteins from exudate samples, which is essential for biomarker discovery studies.ConclusionsCollectively, our findings demonstrate that our exudate collection method coupled with mass spectrometry-based proteomics has great potential for transforming OSCC biomarker discovery and clinical diagnostics assay development.

Intestinal Epithelial Cell Proteome from Wild-Type and TNFΔARE/WT Mice: Effect of Iron on the Development of Chronic Ileitis

Environmental factors substantially contribute to the development of chronic intestinal inflammation in the genetically susceptible host. Nutritional components like iron may act as pro-oxidative mediators affecting inflammatory processes and cell stress mechanisms. To better characterize effects of dietary iron on epithelial cell responses under the pathological conditions of chronic intestinal inflammation, we characterized the protein expression profile (proteome) in primary intestinal epithelial cells (IEC) from iron-adequate and low-iron fed wild-type (WT) and TNFDeltaARE/WT mice. We performed all possible comparisons between the 4 groups according to genotype or diet. Histological analysis of iron-adequate fed TNFDeltaARE/WT mice (approximately 0.54 mg of iron/day) revealed severe ileal inflammation with a histopathology score of 8.3+/-0.91 (score range from 0-12). Interestingly, low-iron fed mice (approximately 0.03 mg of iron/day) were almost completely protected from the development of inflammatory tissue destruction (histopathology score of 2.30+/-0.73). In total, we identified 74 target proteins with significantly altered steady state expression levels in primary IEC using 2D-gel electrophoresis (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS). Interestingly, the overlap between the comparison of iron-adequate fed WT and TNFDeltaARE/WT mice (inflamed conditions) and the comparison between the iron-adequate and iron-low fed TNFDeltaARE/WT mice (absence of inflammation) revealed 4 contrarily regulated proteins including aconitase 2, catalase, intelectin 1 and fumarylacetoacetate hydrolase (FAH). These proteins are associated with energy homeostasis, host defense, oxidative and endoplasmic reticulum (ER) stress responses. In conclusion, the iron-low diet affected the epithelial cell proteome and inhibited the development of chronic intestinal inflammation, suggesting a critical role for nutritional factors in the pathogenesis of IBD.

Intestinal Epithelial Cell Proteome in IL-10 Deficient Mice and IL-10 Receptor Reconstituted Epithelial Cells: Impact on Chronic Inflammation

The interaction of nonpathogenic enteric bacteria with intestinal epithelial cells (IEC) in the absence of host-derived Interleukin 10 (IL-10) may contribute to the development of chronic inflammation. Functional proteome analysis of primary IEC from Enterococcus faecalis-monoassociated WT and IL-10-/- mice as well as IL-10 receptor reconstituted IEC revealed expression changes of proteins that are involved in endoplasmic reticulum stress, energy metabolism, and apoptosis, suggesting a protective role for IL-10 at the epithelial cell level.

Intestinal epithelial cell signalling and chronic inflammation: From the proteome to specific molecular mechanisms

Advancing knowledge regarding the cellular mechanisms of intestinal inflammation has led to a better understanding of the disease pathology in patients with inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis. It has become clear from numerous studies that enteric bacteria are a critical component in the development and prevention/treatment of chronic intestinal inflammation. An emerging new paradigm suggests that changes in the homeostasis of bacteria- and host-derived signal transduction at the intestinal epithelial cell (IEC) level may lead to a break in barrier function and the development of adaptive immune disturbances. The functional loss of anti-inflammatory host-derived signals in the gut including the immunosuppressive cytokines Interleukin 10 (IL-10) and transforming growth factor (TGF)-β are of high relevance to the pathogenesis of IBD. The development of analytical tools including two-dimensional (2D) high-resolution protein separation techniques and peptide mass fingerprinting via high-sensitivity mass-spectrometers (MS) allows the quantitative assessment of protein expression changes in disease-relevant cell types. By using these advanced methods, the characterization of the epithelial cell proteome from murine models of experimental colitis and human IBD patients identified novel disease-related mechanisms with respect to the regulation of the glucose-regulated endoplasmic reticulum stress response protein 78 (grp-78). In conclusion, the identification and functional analysis of differentially expressed proteins in purified intestinal target cell types will help to add important insights to the understanding of the molecular pathogenesis of these immune-mediated chronic intestinal disorders.

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome.

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.

The tumour-associated antigen EpCAM upregulates the fatty acid binding protein E-FABP

The epithelial cell adhesion molecule, EpCAM, is a transmembrane glycoprotein associated with both benign and malignant proliferation. In cancer cells, expression levels of this tumour-associated antigen correlate positively with the grade of dysplasia and are also a negative prognostic factor for breast cancer patients. De novo expression of EpCAM resulted in the rapid upregulation of the proto-oncogene c-Myc along with enhanced cell proliferation and metabolism. Here, we analyzed the effects of EpCAM onto the proteome of epithelial cells. The epidermal fatty acid binding protein, E-FABP, was identified as a new EpCAM-regulated protein. E-FABP is a major target of c-Myc and was rapidly upregulated upon induction of EpCAM. Additionally, E-FABP levels correlated with the amount of EpCAM in permanent squamous cell carcinoma lines and in vivo in primary head and neck carcinomas. Taken together, these results provide further evidence for the direct involvement of EpCAM in signalling processes, gene regulation, and cellular metabolism supporting its important role in tumour biology.