Добірка наукової літератури з теми "Proximal interactomics"

Towards a three-dimensional mapping of virus-host proximal protein interactions The SARS-CoV-2 proximal interactome project sets out to build a comprehensive, three-dimensional map of protein virus-host protein interactions inside living human cells. It aims to shed light on unknown mechanisms of infection. It is made possible by a combination of advanced interactomics techniques coupled with algorithms for the 3D visualisation of networks.

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.

Abstract Extensive genetic analyses of Pancreatic Ductal Adenocarcinoma (PDAC) tumors have identified the GTPase KRAS as a major driver of tumorigenesis, with over 90% of tumors possessing oncogenic KRAS mutations, primarily at the mutational hotspot G12. KRAS G12 mutants are constitutively active, and promote cancer growth by hyperdriving multiple downstream effector pathways via direct physical interactions with proteins such as PI3K and RAF. Despite KRASG12Mut being a promising drug target, direct targeting of KRASG12Mut or downstream pathways has shown limited success, with the emergence of rapid resistance. This resistance is associated with compensatory rewiring of parallel pathways, and the emergence of drug-resistant mutations. Further, the failure to translate these therapies into the clinic maybe due to the inability of many preclinical models to incorporate the contribution of the tumor microenvironment (TME). Approaches to study KRAS signalling in complex multicellular environments have often relied on techniques such as transcriptomics that utilise inference to determine signalling dynamics rather than the direct contribution of KRAS to these outputs. Further, these approaches are often unable to determine cell-type specific signalling. A promising methodology to capture cell-specific KRAS signalling in a heterocellular setting is proximity labelling. This technique involves the engineering of cell lines to express a KRASG12Mut transgene fused to a promiscuous biotin ligase (TurboID). The KRASG12Mut-TurboID fusion is capable of labelling proximal proteins within a radius of ~10 nm with a biotin tag. Biotinylated proteins can be isolated and identified using mass spectrometry. As the labelling radius is narrow, with a short labelling time, this approach is ideal for cell-type specific signalling network analysis. Utilising a novel three-dimensional hydrogel scaffold mimicking the human tumor microenvironment, we are characterising the interactomes of KRASG12Mut across a library of patient derived organoids (hPDOs) grown in co-cultures with stromal cells using proximity labelling. Initial characterisation of KRASG12Mut interactomes in a cohort of 10 hPDOs grown in monoculture has highlighted a high degree of diversity among interactors and pathway activation, independent of the type of KRAS mutation. Specifically, unique patterns of interactions were observed in PI3K, mTOR and MAPK pathway components. Further, co-culture of hPDOs with stromal cells, such as fibroblasts and myeloid cells, yields differential engagement of KRAS interactors when compared to hPDOs grown in monoculture indicative of a role for the TME in shaping KRAS signalling. Current work is ongoing to determine the effect of KRAS targeting therapies on adaptive pathway rewiring using this approach. Interrogation of these interactomes has the potential to uncover resistance mechanisms that emerge in response to KRAS pathway inhibition, and identify novel targets for combinatorial therapy for future studies and clinical trials for pancreatic cancer patients. Citation Format: Nasir A. Haider, Joanna Kelly, Duncan Smith, Claus Jorgensen. Utilising interactomics to uncover oncogenic KRAS signalling networks in the context of the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A099.

Given the hydrophilic structure of lysophospholipids (LPLs), their dietary inclusion translates into a better emulsifying capacity of the dietary components. The present study aimed to understand the mechanisms underlying the growth-promoting effect of LPL supplementation by undertaking deep analyses of the proximal intestine and liver interactomes. The Atlantic salmon (Salmo salar) was selected as the main aquaculture species model. The animals were divided into two groups: one was fed a control diet (C-diet) and the other a feed (LPL-diet) supplemented with an LPL-based digestive enhancer (0.1% AQUALYSO®, Adisseo). The LPL-diet had a positive effect on the fish by increasing the final weight by 5% and reducing total serum lipids, mainly due to a decrease in the plasma phospholipid (p < 0.05). In the intestine, the upregulated interactome suggests a more robust digestive capacity, improving vesicle-trafficking-related proteins, complex sugar hydrolysis, and lipid metabolism. In the liver, the LPL-diet promotes better nutrients, increasing several metabolic pathways. The downregulation of the responses to stress and stimuli could be related to a reduced proinflammatory state. This study on the benefits and modes of action of dietary LPLs opens a new window into fish nutrition and could be extended to other productive species.

Abstract Background: Prostate cancer (PCa) is the second most diagnosed cancer in men and the second cause of cancer-related death amongst men worldwide. PCa is a heterogeneous disease, and the outcome is worse for patients when the disease progresses from localized PCa to a castrate-resistant disease. Androgen receptor (AR) signaling is crucial for PCa development and has been a major therapeutic target for decades. Despite the success made with hormone therapy to block AR signaling, castration resistant disease can arise through mutations and amplifications of the androgen receptor (AR) and AR splice variants (AR-Vs). Amongst them, AR-V7 and AR-V12 can promote AR signaling independent of androgen. Furthermore, prostate tumors are highly hypoxic due to abnormal tumor vasculature. Hypoxia influences the efficacy of local and systemic treatment strategies, such as radiotherapy, hormone therapy, and chemotherapy, resulting in poorer clinical outcomes for PCa patients. Therefore, a complete understanding of AR and AR variant function, as well as how they are modulated by hypoxia and therapy is warranted for developing new strategies to treat PCa. Methods: To identify the transcriptional apparatus associated with the AR and its variants under normal and hypoxic conditions, we carried out Bio-ID proximity labeling mass spectrometry (BioID-MS). We generated stable androgen-independent human prostate cancer cell lines, PC-3 and DU145, expressing AR and the AR-V7 and AR-V12 variants fused to a MiniTurboID (MTID) enzyme. To delineate the AR proximal interaction network, we treated cells with androgen or vehicle for 3 hours and affinity-purified biotinylated proteins after the addition of biotin for the final 2 hours. Cells were grown in normoxia (20% O2) or treated with hypoxia (95% N2,5% CO2 and 0.5% O2) for 24 h. Nonspecific interactions were filtered using controls and statistical methods to identify high-confidence interactomes. Results: BioID proximity labeling in PC3 cells identified several AR-associated proteins, including many knowninteractors, validating the approach. Hypoxic conditions led to a striking alteration in proximal interactions and many metabolic proteins were highly enriched with AR upon exposure of cells to hypoxia for 24h. Among these, phosphoglycerate kinase 1 (PGK1) was a top hit. PGK1 has been reported to interact with AR to mediate the expression of genes that regulates cell proliferation and apoptosis. Additionally, PGK1 has an essential role in metabolic reprogramming induced by c-MYC and HIF-1α, leading to enhanced tumorigenesis. Conclusion: Bio-ID mass spectrometry was used to map the proximal interactome of AR and its variants, identifying novel interactions partners for further analysis. We found that hypoxia strongly alters interactors with the AR, and we identified PGK1 as a hypoxia induced AR interaction in PCA. Current work to functionally validate this interaction will be presented. Citation Format: Oloruntoba I. Osagie, Jordann Smakk, Deborah E. Citrin, Travis H. Stracker. Identification of critical hypoxia induced factors in castrate resistant prostate 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 4802.

Abstract Chromosomal translocations involving chromosome 9q34 and 22q11 generate the BCR-ABL1 fusion gene. The location of the translocation within the BCR gene dictates which exons are excluded or included in the resulting fusion with the ABL1 gene. The most common translocations produce three main protein products with molecular weights of 190, 210, and 230 kD. Interestingly, the p190 and p210 BCR-ABL1 forms are associated with different clinical characteristics. Specifically, BCR-ABL1+ acute lymphoblastic leukemia (ALL) cases typically harbor the p190 form, whereas chronic myelogenous leukemia (CML) cases typically harbor the p210 form. Despite a great deal of study over many decades devoted to the BCR-ABL1 molecule, neither phosphorylation of putative target proteins at site-specific resolution nor protein-protein interaction differences between the p190 and the p210 forms is well understood, preventing thorough understanding of the signaling programs employed by these variants. Therefore, to examine signaling differences between p190 and p210 we chose a homogeneous system to investigate global phosphorylation of tyrosine residues. Ba/F3 cell lines, which are normally dependent on IL-3 for growth, were transduced with either the p190 or p210 form of BCR-ABL1. Subsequently, these cell lines were selected for successful growth in cytokine free media, indicating that the BCR-ABL1 fusions were signaling and inducing aberrant growth. These two BCR-ABL1 cell lines, as well as the parental Ba/F3 line, were grown in SILAC (stable isotope labeling by amino acids in cell culture) media and lysates were enriched for phosphotyrosine (pTyr) peptides and submitted for mass spectrometric analysis. The resulting 3-state SILAC dataset includes quantitation on 355 pTyr sites in three pairwise comparisons; p190 vs. p210 as well as each BCR-ABL1 cell line vs. its parental control. Many of the known BCR-ABL1 associated phosphorylation sites are recapitulated in our dataset. To our knowledge we report the first data on pTyr site-specific differences between p190 and p210 forms within in the same cell type. Surprisingly, 78 pTyr sites are differentially regulated between p190 and p210, with notable examples including pTyr sites on TEC, SHC1, PEAK1, PTPN6 and LYN. Additionally, sites in the kinase domain of JAK2 and the DNA binding domain of STAT6 showed a reciprocal relationship in p190 vs. p210, suggesting the differential involvement of JAK-STAT pathway. Notably, 28 pTyr sites were differentially regulated exclusively in either p190 or p210 BCR-ABL1 Ba/F3 cells, suggesting that there may be signaling pathways unique to p190 and p210, respectively. To attempt to identify direct substrates of BCR-ABL1 and detect differential binding of p190 and p210, we used the BioID system (Roux et al., JBC 2012). In this system, a specialized biotin ligase (BirA) is used to label adjacent or interacting proteins with a biotin moiety on lysine residues. Specifically, Ba/F3 cells transduced with BirA-tagged p190 or untagged p190 were grown in SILAC media and whole cell lysates were subjected streptavidin immunoprecipitation followed by mass spectrometry. Forty-four proteins were enriched over the control and known interactors such as SHC1, DOK2, and GAB1 were detected, along with several putative novel interactors. Combining both the phosphorylation and interactome datasets revealed all these proteins are hyper-phosphorylated compared to parental cell lines, suggesting these are direct substrates or in proximal complexes with p190 BCR-ABL1. A direct comparison of p190 and p210 interactomes using BioID is pending, and will further elucidate potential differences between these important fusion protein isoforms. In sum, our data suggests that p190 and p210 have at least partially independent signaling cascades that are mediated by differential protein-protein interactions, which may help explain the observed association of p190 with BCR-ABL1+ ALL and p210 with CML. Disclosures No relevant conflicts of interest to declare.

The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations.

Introduction. The human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular “proteome”). It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI) are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination) or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo.Methods. The biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP) was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry.Results. Using several experimental systems (BirA.A + BAP.B), we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments – 3 paralogues of heterochromatin protein HP1a (CBX5), HP1b (CBX1), HP1g (CBX3), wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA), ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation) and is designed to purify and study the protein composition of chromatin in proximity to the nuclear protein of interest. Using the RAD18 protein as a model, we demonstrated that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2Az has a different pattern of H4 acetylation.Conclusion. Progress in the last decade in cancer drug therapy has led us to the conclusion that the nucleus of eukaryotic cells is an active site for many cellular processes important to the development of cancer. These processes include changes in genetic and epigenetic landscape (e. g. methylation of DNA, modification of histones) and the expression levels of transcription factors, which regulates gene products (e.g. hypoxia-inducible factor 1α (HIF-1α) in chronic anemia, etc.) where protein-protein interactions play important role. Understanding the nature of protein-protein interactions may improve design strategies for small-molecule PPI modulators. PPI assay technologies that closely reflect physiological conditions hold the key to developing specific anti-cancer drugs.

Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry, and topological organization of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level.

Le carcinome à cellules de Merkel (CCM) est un cancer de la peau agressif et de très mauvais pronostic. Ce cancer est lié à la présence du Polyomavirus à cellules de Merkel dans 80% des cas. Ce Polyomavirus exprime deux oncoprotéines virales : sT et LT tronquée. L'expression des deux antigènes T est suffisante à l'émergence du cancer et responsable de la perturbation de checkpoints de signalisation, de la modification du profil épigénétique et de l'évasion immunitaire. L'interactomique et la protéomique nous ont permis d'identifier de nombreux facteurs épigénétiques en relation avec les antigènes T. Nous avons aussi mis en lumières de des mécanismes candidats touchant la régulation de la stabilité protéique, l'expression génique et la stabilité génétique via la voie de réponse aux dommages de l'ADN. Les hypothèses issues de ces analyses ont été testées de manière ciblée. Un lien entre la peptidyl-prolyl cis/trans isomérase et la neddylation a pu être mis en évidence. Nous avons ainsi identifié un mécanisme candidat de régulation de l'activité des complexes d'ubiquitinylation à travers le recrutement de PIN1 et l'isomérisation par un primo de neddylation. L'étude d'EHMT2, régulateur épigénétique d'intérêt, a révélée l'importance de son rôle dans la réponse aux dommages de l'ADN au sein des CCM virus positifs (VP). Nous avons identifié un rôle protecteur de tLT et d'EHTM2 des dommages de l'ADN au sein des CCM-VP. Un rôle de EHMT2 dans la réparation suivant un stress réplicatif est aussi apparu. Nous avons découvert de nouveaux mécanismes importants dans l'oncogénèse des CCM-VP, rapportant pour la première fois un rôle central de tLT dans la gestion des dommages à l'ADN
Merkel cell carcinoma (MCC) is an extremely aggressive skin cancer with a high mortality rate. In 80% of cases, this cancer is linked to the presence of the Merkel cell polyomavirus which expresses two viral oncoproteins, small T and a truncated form of large T. The expression of these proteins is sufficient for carcinogenesis, inducing the disruption of cell cycle checkpoints, changes in the epigenetic profile, and immune evasion. The combination of interactomics and proteomics approaches allowed us to identify numerous epigenetic factors related to the T antigens. These studies have also highlighted potential mechanisms involving the regulation of protein stability, gene expression, and genetic stability via an altered DNA damage response pathway. The hypotheses formulated following these analyses were investigated in targeted assays. A link between the peptidyl-prolyl cis/trans isomerase and neddylation was demonstrated. We have thus identified a new potential mechanism for regulating the activity of ubiquitination complexes involving the recruitment of PIN1 to these complexes, thus their isomerization, by neddylation. The study of the EHMT2 functions, an epigenetic regulator of interest, revealed the importance of its role in the DNA damage response in virus positive MCC (VP-MCC). We were able to identify a protective role of tLT and EHMT2 in DNA damages in VP-MCC cells. More specifically, we also report a role of EHMT2 in solving single strand DNA breaks following replication stress. Our works have enabled the discovery of new important mechanisms in VP-MCC oncogenesis, reporting for the first time a central role of tLT in DNA damages management