Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells.

• Klíčová slova
• AGR2, Colorectal cancer, NPM3, PD-L1,
• MeSH
• antigeny CD274 * metabolismus   genetika   MeSH
• jaderné proteiny metabolismus   genetika   MeSH
• kolorektální nádory * genetika   metabolismus   patologie   MeSH
• lidé MeSH
• mukoproteiny * metabolismus   genetika   MeSH
• nádorové buněčné linie MeSH
• nukleofosmin * MeSH
• onkogenní proteiny * metabolismus   genetika   MeSH
• proteiny * metabolismus   genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• antigeny CD274 * MeSH
• CD274 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• mukoproteiny * MeSH
• NPM3 protein, human MeSH Prohlížeč
• nukleofosmin * MeSH
• onkogenní proteiny * MeSH
• proteiny * MeSH

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and deeper proteome coverage is needed for its molecular characterization. We present comprehensive library of targeted mass spectrometry assays specific for TNBC and demonstrate its applicability. Proteins were extracted from 105 TNBC tissues and digested. Aliquots were pooled, fractionated using hydrophilic chromatography and analyzed by LC-MS/MS in data-dependent acquisition (DDA) parallel accumulation-serial fragmentation (PASEF) mode on timsTOF Pro LC-MS system. 16 individual lysates were analyzed in data-independent acquisition (DIA)-PASEF mode. Hybrid library was generated in Spectronaut software and covers 244,464 precursors, 168,006 peptides and 11,564 protein groups (FDR = 1%). Application of our library for pilot quantitative analysis of 16 tissues increased identification numbers in Spectronaut 18.5 and DIA-NN 1.8.1 software compared to library-free setting, with Spectronaut achieving the best results represented by 190,310 precursors, 140,566 peptides, and 10,463 protein groups. In conclusion, we introduce assay library that offers the deepest coverage of TNBC proteome to date. The TNBC library is available via PRIDE repository (PXD047793).

• MeSH
• chromatografie kapalinová MeSH
• lidé MeSH
• proteom MeSH
• proteomika metody   MeSH
• software MeSH
• tandemová hmotnostní spektrometrie * MeSH
• triple-negativní karcinom prsu * genetika   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• dataset MeSH
• Názvy látek
• proteom MeSH

NF-κB pathway is involved in inflammation; however, recent data shows its role also in cancer development and progression, including metastasis. To understand the role of NF-κB interactome dynamics in cancer, we study the complexity of breast cancer interactome in luminal A breast cancer model and its rearrangement associated with NF-κB modulation. Liquid chromatography-mass spectrometry measurement of 160 size-exclusion chromatography fractions identifies 5460 protein groups. Seven thousand five hundred sixty eight interactions among these proteins have been reconstructed by PrInCE algorithm, of which 2564 have been validated in independent datasets. NF-κB modulation leads to rearrangement of protein complexes involved in NF-κB signaling and immune response, cell cycle regulation, and DNA replication. Central NF-κB transcription regulator RELA co-elutes with interactors of NF-κB activator PRMT5, and these complexes are confirmed by AlphaPulldown prediction. A complementary immunoprecipitation experiment recapitulates RELA interactions with other NF-κB factors, associating NF-κB inhibition with lower binding of NF-κB activators to RELA. This study describes a network of pro-tumorigenic protein interactions and their rearrangement upon NF-κB inhibition with potential therapeutic implications in tumors with high NF-κB activity.

• Klíčová slova
• AlphaPullDown, NF-κB, RELA, breast cancer, interaction, protein complexes, protein correlation profiling, proteomics,
• MeSH
• karcinogeneze metabolismus   MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• mapy interakcí proteinů * MeSH
• nádorové buněčné linie MeSH
• nádory prsu * metabolismus   patologie   MeSH
• NF-kappa B * metabolismus   MeSH
• proteinarginin-N-methyltransferasy metabolismus   MeSH
• signální transdukce MeSH
• transkripční faktor RelA * metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• NF-kappa B * MeSH
• proteinarginin-N-methyltransferasy MeSH
• RELA protein, human MeSH Prohlížeč
• transkripční faktor RelA * MeSH

BACKGROUND: Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast tumors and was found to increase migration and invasion of MCF7 cells in vitro. Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation. METHODS: Western blotting was used to select potential inhibitor decreasing DSC1 protein level in MCF7 cell line. Using atomic force microscopy we evaluated effect of DSC1 overexpression and modulation on cell morphology. The LC-MS/MS analysis of total proteome on Orbitrap Lumos and RNA-Seq analysis of total transcriptome on Illumina NextSeq 500 were performed to study the molecular mechanisms associated with DSC1. Pull-down analysis with LC-MS/MS detection was carried out to uncover DSC1 protein interactome in MCF7 cells. RESULTS: Analysis of DSC1 protein levels in response to selected inhibitors displays significant DSC1 downregulation (p-value ≤ 0.01) in MCF7 cells treated with NF-κB inhibitor parthenolide. Analysis of mechanic cell properties in response to DSC1 overexpression and parthenolide treatment using atomic force microscopy reveals that DSC1 overexpression reduces height of MCF7 cells and conversely, parthenolide decreases cell stiffness of MCF7 cells overexpressing DSC1. The LC-MS/MS total proteome analysis in data-independent acquisition mode shows a strong connection between DSC1 overexpression and increased levels of proteins LACRT and IGFBP5, increased expression of IGFBP5 is confirmed by RNA-Seq. Pathway analysis of proteomics data uncovers enrichment of proliferative MCM_BIOCARTA pathway including CDK2 and MCM2-7 after DSC1 overexpression. Parthenolide decreases expression of LACRT, IGFBP5 and MCM_BIOCARTA pathway specifically in DSC1 overexpressing cells. Pull-down assay identifies DSC1 interactions with cadherin family proteins including DSG2, CDH1, CDH3 and tyrosine kinase receptors HER2 and HER3; parthenolide modulates DSC1-HER3 interaction. CONCLUSIONS: Our systems biology data indicate that DSC1 is connected to mechanisms of cell cycle regulation in luminal A breast cancer cells, and can be effectively modulated by parthenolide.

• Klíčová slova
• Breast cancer, DIA, DSC1, Metastasis, Proteomics, Pull-down,
• MeSH
• chromatografie kapalinová MeSH
• desmokoliny * metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory * MeSH
• proteom MeSH
• seskviterpeny farmakologie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• desmokoliny * MeSH
• DSC1 protein, human MeSH Prohlížeč
• parthenolide MeSH Prohlížeč
• proteom MeSH
• seskviterpeny MeSH

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein-protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.

• MeSH
• HLA antigeny MeSH
• HLA-A antigeny MeSH
• hmotnostní spektrometrie metody   MeSH
• interferony * MeSH
• lidé MeSH
• proteiny * chemie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• thiolesterasa ubikvitinu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HLA antigeny MeSH
• HLA-A antigeny MeSH
• interferony * MeSH
• proteiny * MeSH
• reagencia zkříženě vázaná MeSH
• thiolesterasa ubikvitinu MeSH
• USP18 protein, human MeSH Prohlížeč

Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture-based LC-MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2-PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein-protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells.

• Klíčová slova
• anterior gradient protein 2, mass spectrometry, protein disulfide isomerase, protein–protein interactions, secretory pathway,
• MeSH
• chromatografie kapalinová MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• mukoproteiny * metabolismus   MeSH
• nádory * MeSH
• onkogenní proteiny * metabolismus   MeSH
• proteindisulfidisomerasy * MeSH
• simulace molekulového dockingu MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• mukoproteiny * MeSH
• onkogenní proteiny * MeSH
• proteindisulfidisomerasy * MeSH

CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular "health". Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP.

• Klíčová slova
• bioinformatics, cell biology, omics, organizational aspects of cell biology, proteomics,
• Publikační typ
• časopisecké články MeSH

Hydrogen can serve as an electron donor for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface and geothermal ecosystems. Nevertheless, the current knowledge of hydrogen utilization by mesophilic acidophiles is minimal. A multi-omics analysis was applied on Acidithiobacillus ferrooxidans growing on hydrogen, and a respiratory model was proposed. In the model, [NiFe] hydrogenases oxidize hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated iron-sulfur proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome aa 3 oxidase via cytochrome bc 1 complex and cytochrome c 4 or the alternate directly to cytochrome bd oxidase, resulting in proton efflux and reduction of oxygen. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome c (ferrireductase) via cytochrome bc 1 complex and a cascade of electron transporters (cytochrome c 4, cytochrome c 552, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux and reduction of ferric iron. The proton gradient generated by hydrogen oxidation maintains the membrane potential and allows the generation of ATP and NADH. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition of the deep terrestrial subsurface.

• Klíčová slova
• Acidithiobacillus, extremophiles, ferric iron reduction, hydrogen metabolism, multi-omics, oxygen reduction,
• Publikační typ
• časopisecké články MeSH

Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification.

• Klíčová slova
• SWATH-MS, breast cancer, data independent acquisition, proteomics, tissue, transcriptomics, tumor classification,
• MeSH
• fosfatasy genetika   metabolismus   MeSH
• lidé MeSH
• nádory prsu klasifikace   metabolismus   patologie   MeSH
• proteinkinasa CDC2 genetika   metabolismus   MeSH
• proteom analýza   metabolismus   MeSH
• proteomika metody   MeSH
• receptor erbB-2 genetika   metabolismus   MeSH
• receptory pro estrogeny metabolismus   MeSH
• rychlé screeningové testy MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK1 protein, human MeSH Prohlížeč
• ERBB2 protein, human MeSH Prohlížeč
• fosfatasy MeSH
• phosphatidylinositol-3,4-bisphosphate 4-phosphatase MeSH Prohlížeč
• proteinkinasa CDC2 MeSH
• proteom MeSH
• receptor erbB-2 MeSH
• receptory pro estrogeny MeSH

Current prognostic factors are insufficient for precise risk-discrimination in breast cancer patients with low grade breast tumors, which, in disagreement with theoretical prognosis, occasionally form early lymph node metastasis. To identify markers for this group of patients, we employed iTRAQ-2DLC-MS/MS proteomics to 24 lymph node positive and 24 lymph node negative grade 1 luminal A primary breast tumors. Another group of 48 high-grade tumors (luminal B, triple negative, Her-2 subtypes) was also analyzed to investigate marker specificity for grade 1 luminal A tumors. From the total of 4405 proteins identified (FDR < 5%), the top 65 differentially expressed together with 30 previously identified and control markers were analyzed also at transcript level. Increased levels of carboxypeptidase B1 (CPB1), PDZ and LIM domain protein 2 (PDLIM2), and ring finger protein 25 (RNF25) were associated specifically with lymph node positive grade 1 tumors, whereas stathmin 1 (STMN1) and thymosin beta 10 (TMSB10) associated with aggressive tumor phenotype also in high grade tumors at both protein and transcript level. For CPB1, these differences were also observed by immunohistochemical analysis on tissue microarrays. Up-regulation of putative biomarkers in lymph node positive (versus negative) luminal A tumors was validated by gene expression analysis of an independent published data set (n = 343) for CPB1 (p = 0.00155), PDLIM2 (p = 0.02027) and RELA (p = 0.00015). Moreover, statistically significant connections with patient survival were identified in another public data set (n = 1678). Our findings indicate unique pro-metastatic mechanisms in grade 1 tumors that can include up-regulation of CPB1, activation of NF-κB pathway and changes in cell survival and cytoskeleton. These putative biomarkers have potential to identify the specific minor subpopulation of breast cancer patients with low grade tumors who are at higher than expected risk of recurrence and who would benefit from more intensive follow-up and may require more personalized therapy.

• MeSH
• databáze proteinů MeSH
• imunohistochemie MeSH
• izotopové značení MeSH
• Kaplanův-Meierův odhad MeSH
• karboxypeptidasa B metabolismus   MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádory prsu genetika   metabolismus   MeSH
• NF-kappa B metabolismus   MeSH
• proteomika metody   MeSH
• regulace genové exprese u nádorů MeSH
• reprodukovatelnost výsledků MeSH
• stanovení celkové genové exprese metody   MeSH
• stupeň nádoru MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CPB1 protein, human MeSH Prohlížeč
• karboxypeptidasa B MeSH
• nádorové biomarkery MeSH
• NF-kappa B MeSH