Virtual interactomics represents a rapidly developing scientific area on the boundary line of bioinformatics and interactomics. Protein-related virtual interactomics then comprises instrumental tools for prediction, simulation, and networking of the majority of interactions important for structural and individual reproduction, differentiation, recognition, signaling, regulation, and metabolic pathways of cells and organisms. Here, we describe the main areas of virtual protein interactomics, that is, structurally based comparative analysis and prediction of functionally important interacting sites, mimotope-assisted and combined epitope prediction, molecular (protein) docking studies, and investigation of protein interaction networks. Detailed information about some interesting methodological approaches and online accessible programs or databases is displayed in our tables. Considerable part of the text deals with the searches for common conserved or functionally convergent protein regions and subgraphs of conserved interaction networks, new outstanding trends and clinically interesting results. In agreement with the presented data and relationships, virtual interactomic tools improve our scientific knowledge, help us to formulate working hypotheses, and they frequently also mediate variously important in silico simulations.

• Publication type
• Journal Article MeSH

BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.

• MeSH
• Chromatography, Liquid MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Humans MeSH
• Microfilament Proteins genetics   isolation & purification   MeSH
• Neoplasms genetics   pathology   MeSH
• Surface Plasmon Resonance * MeSH
• LIM Domain Proteins genetics   isolation & purification   MeSH
• Streptavidin chemistry   MeSH
• Tandem Mass Spectrometry MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Nucleoside diphosphate kinase 7, non-metastatic cells 7 (NME7) is an acknowledged member of ciliome and is involved in the biogenesis or function of cilia. As obesity and diabetes are common in several ciliopathies, we aimed to analyze changes of gene expression within Nme7 interactome in genetically designed rat models of metabolic syndrome. We assessed the liver transcriptome by Affymetrix microarrays in adult males of 14 PXO recombinant inbred rat strains and their two progenitor strains, SHR-Lx and BXH2. In the strains with the lowest expression of Nme7, we have identified significant enrichment of transcripts belonging to Nme7 interactome. In the subsequent network analysis, we have identified three major upstream regulators - Hnf4a, Ppara and Nr1h4 and liver steatosis (p=0.0001) and liver necrosis/cell death (apoptosis of liver cells, p=0.0003) among the most enriched Tox categories. The mechanistic network reaching the top score showed substantial overlap with Assembly of non-motile cilium and Glucose metabolism disorder gene lists. In summary, we show in a genetic model of metabolic syndrome that rat strains with the lowest expression of Nme7 present gene expression shifts of Nme7 interactome that are perturbing networks relevant for carbohydrate and lipid metabolism as well as ciliogenesis.

• MeSH
• Species Specificity MeSH
• Gene Regulatory Networks genetics   MeSH
• Rats MeSH
• Metabolic Syndrome genetics   metabolism   MeSH
• Lipid Metabolism physiology   MeSH
• Disease Models, Animal * MeSH
• Nucleoside-Diphosphate Kinase biosynthesis   genetics   MeSH
• Rats, Inbred SHR MeSH
• Gene Expression Profiling methods   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The main objective of this study was to propose a novel methodology to approach challenges in molecular biology. Akirin/Subolesin (AKR/SUB) are vaccine protective antigens and are a model for the study of the interactome due to its conserved function in the regulation of different biological processes such as immunity and development throughout the metazoan. Herein, three visual artists and a music professor collaborated with scientists for the functional characterization of the AKR2 interactome in the regulation of the NF-κB pathway in human placenta cells. The results served as a methodological proof-of-concept to advance this research area. The results showed new perspectives on unexplored characteristics of AKR2 with functional implications. These results included protein dimerization, the physical interactions with different proteins simultaneously to regulate various biological processes defined by cell type-specific AKR-protein interactions, and how these interactions positively or negatively regulate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in a biological context-dependent manner. These results suggested that AKR2-interacting proteins might constitute suitable secondary transcription factors for cell- and stimulus-specific regulation of NF-κB. Musical perspective supported AKR/SUB evolutionary conservation in different species and provided new mechanistic insights into the AKR2 interactome. The combined scientific and artistic perspectives resulted in a multidisciplinary approach, advancing our knowledge on AKR/SUB interactome, and provided new insights into the function of AKR2-protein interactions in the regulation of the NF-κB pathway. Additionally, herein we proposed an algorithm for quantum vaccinomics by focusing on the model proteins AKR/SUB.

• Publication type
• Journal Article MeSH

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Adenosine analogs & derivatives   metabolism   MeSH
• AlkB Homolog 5, RNA Demethylase genetics   metabolism   MeSH
• Molecular Sequence Annotation MeSH
• Alpha-Ketoglutarate-Dependent Dioxygenase FTO genetics   metabolism   MeSH
• Transcription, Genetic MeSH
• Gene Ontology MeSH
• HEK293 Cells MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Protein Interaction Mapping MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Methyltransferases genetics   metabolism   MeSH
• Oxidoreductases, N-Demethylating genetics   metabolism   MeSH
• RNA, Untranslated genetics   metabolism   MeSH
• DNA Repair MeSH
• Protein Isoforms genetics   metabolism   MeSH
• DNA Replication MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) is essential for correct functioning of innate immune responses. The ADAR1p110 isoform is mainly nuclear and ADAR1p150, which is interferon (IFN) inducible, is predominately cytoplasmic. Using three different methods - co-immunoprecipitation (co-IP) of endogenous ADAR1, Strep-tag co-IP and BioID with individual ADAR1 isoforms - a comprehensive interactome was generated during both homeostasis and the IFN response. Both known and novel interactors as well as editing regulators were identified. Nuclear proteins were detected as stable interactors with both ADAR1 isoforms. In contrast, BioID identified distinct protein networks for each ADAR1 isoform, with nuclear components observed with ADAR1p110 and components of cytoplasmic cellular condensates with ADAR1p150. RNase A digestion distinguished between distal and proximal interactors, as did a double-stranded RNA (dsRNA)-binding mutant of ADAR1 which demonstrated the importance of dsRNA binding for ADAR1 interactions. IFN treatment did not affect the core ADAR1 interactomes but resulted in novel interactions, the majority of which are proximal interactions retained after RNase A treatment. Short treatment with high molecular weight poly(I:C) during the IFN response resulted in dsRNA-binding-dependent changes in the proximal protein network of ADAR1p110 and association of the ADAR1p150 proximal protein network with some components of antiviral stress granules.

• MeSH
• Adenosine Deaminase * metabolism   genetics   MeSH
• Cell Nucleus * metabolism   MeSH
• Cytoplasm * metabolism   MeSH
• RNA, Double-Stranded metabolism   genetics   MeSH
• RNA Editing MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Interferons metabolism   genetics   MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Poly I-C pharmacology   MeSH
• Protein Isoforms * metabolism   genetics   MeSH
• RNA-Binding Proteins * metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article 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.

• MeSH
• Carcinogenesis metabolism   MeSH
• Humans MeSH
• Protein Interaction Mapping MeSH
• Protein Interaction Maps * MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms * metabolism   pathology   MeSH
• NF-kappa B * metabolism   MeSH
• Protein-Arginine N-Methyltransferases metabolism   MeSH
• Signal Transduction MeSH
• Transcription Factor RelA * metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

De-etiolation is the first developmental process under light control allowing the heterotrophic seedling to become autotrophic. The phytohormones cytokinins (CKs) largely contribute to this process. Reversible phosphorylation is a key event of cell signaling, allowing proteins to become active or generating a binding site for specific protein interaction. 14-3-3 proteins regulate a variety of plant responses. The expression, hormonal regulation, and proteomic network under the control of 14-3-3s were addressed in tomato (Solanum lycopersicum L.) during blue light-induced photomorphogenesis. Two isoforms were specifically investigated due to their high expression during tomato de-etiolation. The multidisciplinary approach demonstrated that TFT9 expression, but not TFT6, was regulated by CKs and identified cis-regulating elements required for this response. Our study revealed >130 potential TFT6/9 interactors. Their functional annotation predicted that TFTs might regulate the activity of proteins involved notably in cell wall strengthening or primary metabolism. Several potential interactors were also predicted to be CK-responsive. For the first time, the 14-3-3 interactome linked to de-etiolation was investigated and evidenced that 14-3-3s might be involved in CK signaling pathway, cell expansion inhibition and steady-state growth rate establishment, and reprograming from heterotrophy to autotrophy. BIOLOGICAL SIGNIFICANCE: Tomato (Solanum lycopersicum L.) is one of the most important vegetables consumed all around the world and represents probably the most preferred garden crop. Regulation of hypocotyl growth by light plays an important role in the early development of a seedling, and consequently the homogeneity of the culture. The present study focuses on the importance of tomato 14-3-3/TFT proteins in this process. We provide here the first report of 14-3-3 interactome in the regulation of light-induced de-etiolation and subsequent photomorphogenesis. Our data provide new insights into light-induced de-etiolation and open new horizons for dissecting the post-transcriptional regulations.

• MeSH
• Chromatography, Affinity MeSH
• Protein Interaction Maps * MeSH
• 14-3-3 Proteins metabolism   MeSH
• Proteomics * MeSH
• Plant Proteins metabolism   MeSH
• Seedlings growth & development   MeSH
• Solanum lycopersicum growth & development   MeSH
• Light * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• NIH 3T3 Cells MeSH
• Cyclin-Dependent Kinases genetics   metabolism   MeSH
• Peptide Elongation Factor 1 genetics   metabolism   MeSH
• Endosomal Sorting Complexes Required for Transport genetics   metabolism   MeSH
• Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases genetics   metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Humans MeSH
• Protein Interaction Mapping MeSH
• Mice MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Proteomics methods   MeSH
• Receptor, Fibroblast Growth Factor, Type 3 genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Signal Transduction genetics   MeSH
• Gene Expression Profiling MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Amino Acid Motifs MeSH
• Phosphorylation genetics   MeSH
• Histone-Lysine N-Methyltransferase genetics   metabolism   MeSH
• HIV Integrase genetics   metabolism   MeSH
• HIV enzymology   genetics   MeSH
• Humans MeSH
• Mediator Complex Subunit 1 genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Myeloid-Lymphoid Leukemia Protein genetics   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH