LAP1 Interactome Profiling Provides New Insights into LAP1’s Physiological Functions

Figures

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  Figure 1. Schematic representation of the experimental design of this study [32]. IP, immunoprecipitation; LAP1, lamina-associated polypeptide 1; LC–MS/MS, liquid chromatography coupled to tandem mass spectrometry; MS, mass spectrometry; PPI, protein–protein interaction.

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  Figure 2. Identification of LAP1-interacting proteins. (a) Summarized results of the in silico analysis leading to the identification of previously known interacting proteins of human LAP1 and mouse Lap1, retrieved from online databases. (b) Summarized results of the LC–MS/MS analysis leading to the identification of proteins that were significantly co-immunoprecipitated with LAP1 in control and LAP1 E482A fibroblast cell lines. The number of novel LAP1-interacting proteins uncovered in control fibroblasts is also indicated. (c) LAP1 co-immunoprecipitation assay coupled to immunoblotting analysis validating several LC–MS/MS-identified proteins as LAP1 interactors in control fibroblasts. Whole cell lysates obtained from control and LAP1 E482A fibroblast cell lines were immunoprecipitated using a LAP1-specific antibody bound to Dynabeads Protein G. The respective negative controls were performed by incubating fibroblasts’ total protein extracts with Dynabeads Protein G-conjugated rabbit IgG. The presence of LAP1 and/or its binding partners Ku70, emerin and H2AX in the immunoprecipitates (right), as well as in the initial whole cell lysates (left), of control and LAP1 E482A fibroblasts was…

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  Figure 3. Functional characterization of LAP1-interacting proteins. (a) Functional classification based on PANTHER protein class categories. From a total of 375 proteins under analysis, 288 protein class hits distributed across 20 main categories were retrieved using the PANTHER online resource [33]. (b,c) Functional enrichment analysis of (b) GO biological process and (c) GO cellular component categories. Some enriched GO terms (summarizing the broad spectrum of over-represented annotations) retrieved from the STRING database [34] are shown. DNA, deoxyribonucleic acid; GO, Gene Ontology; LAP1, lamina-associated polypeptide 1; PANTHER, Protein ANalysis THrough Evolutionary Relationships; RNA, ribonucleic acid.

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  Figure 4. LAP1 (TOR1AIP1) protein interactome. The network, which comprises LAP1-interacting proteins (identified by gene name) retrieved from online databases and/or detected by LC–MS/MS in control fibroblasts, was constructed using the Cytoscape software [36]. The list of 375 LAP1-interacting proteins was organized into 13 functional groups (as indicated in the figure). Nodes with a circle shape (○) or diamond shape (◊) represent LAP1-interacting proteins found exclusively in online databases or control fibroblasts, respectively, while nodes with a square shape (□) symbolize LAP1-interacting proteins identified in both online databases and control fibroblasts. Nodes with a red contour line indicate LAP1-interacting proteins detected by LC–MS/MS in control fibroblasts but not in LAP1 E482A fibroblasts. Edges denote interactions between LAP1 and its binding partners. DNA, deoxyribonucleic acid; ER, endoplasmic reticulum; LAP1, lamina-associated polypeptide 1; LC–MS/MS, liquid chromatography coupled to tandem mass spectrometry; RNA, ribonucleic acid.

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  Figure 5. PPI and gene–disease association network of ‘Nuclear organization/Transcription regulation/DNA replication and repair’ functional protein group. The network comprises differentially expressed proteins found in LAP1 E482A fibroblasts [32] as well as LAP1-interacting proteins (identified by gene name). The PPI data were retrieved from the STRING database [34] and the PPI network was constructed using the Cytoscape software [36]. Nodes with a black label represent LAP1-interacting proteins, while nodes with a green or red label symbolize, respectively, upregulated or downregulated proteins (the black asterisk following the name indicates deregulated proteins that are LAP1 interactors). Blue edges denote physical/functional associations between LAP1-interacting proteins and LAP1 E482A fibroblasts’ differentially expressed proteins, whereas grey edges correspond to associations between proteins of the same proteomic dataset. The gene–disease association data were retrieved from the DisGeNET online platform [37]. The different colors filling the nodes’ interior represent specific diseases/pathological phenotypes (as indicated in the figure) associated to the proteins. The total number…

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  Figure 6. PPI and gene–disease association network of ‘Protein processing in the ER/Protein (re)folding, stability and proteasomal degradation’ functional protein group. The network comprises differentially expressed proteins found in LAP1 E482A fibroblasts [32] as well as LAP1-interacting proteins (identified by gene name). The PPI data were retrieved from the STRING database [34] and the PPI network was constructed using the Cytoscape software [36]. Nodes with a black label represent LAP1-interacting proteins, while nodes with a green or red label symbolize, respectively, upregulated or downregulated proteins (the black asterisk following the name indicates deregulated proteins that are LAP1 interactors). Blue edges denote physical/functional associations between LAP1-interacting proteins and LAP1 E482A fibroblasts’ differentially expressed proteins, whereas grey edges correspond to associations between proteins of the same proteomic dataset. The gene–disease association data were retrieved from the DisGeNET online platform [37]. The different colors filling the nodes’ interior represent specific diseases/pathological phenotypes (as indicated in the figure) associated to the proteins.…