Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.

Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds ß integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to ß integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin ß1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome.

Altered protein expression profile associated with phenotypic changes in lung fibroblasts co-cultured with gold nanoparticle-treated small airway epithelial cells.

Despite the availability of toxicity studies on cellular exposure to gold nanoparticles (AuNPs), there is scarcity of information with regard to the bystander effects induced by AuNPs on neighboring cells not exposed to the NPs. In this study, we showed that exposure of small airway epithelial cells (SAECs) to AuNPs induced changes in protein expression associated with functional effects in neighboring MRC5 lung fibroblasts in a co-culture system. Uptake of 20 nm size AuNPs by SAECs was first verified by focused ion beam scanning electron microscopy. Subsequently, pretreated SAECs were co-cultured with unexposed MRC5 lung fibroblasts, which then underwent proteome profiling using a quantitative proteomic approach. Stable-isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry identified 109 proteins (which included 47 up-regulated and 62 down-regulated proteins) that were differentially expressed in the lung fibroblasts co-cultured with AuNP pretreated SAECs. There was altered expression of proteins such as Paxillin, breast cancer anti-estrogen resistance 1 and Caveolin-1, which are known to be involved in the cell adhesion process. Morphological studies revealed that there was a concomitant increase in cell adhesion and altered F-actin stress fiber arrangement involving vinculin in the lung fibroblasts. It is likely that phenotypic changes observed in the underlying lung fibroblasts were mediated by AuNP-induced downstream signals in the pretreated SAECs and cell-cell cross talk.

Protein interactions of phosphatase and tensin homologue (PTEN) and its cancer-associated G20E mutant compared by using stable isotope labeling by amino acids in cell culture-based parallel affinity purification.

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the PI3K pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here, we use a cancer-associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using MS-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type-specific interactors, 2) interactors unique to the G20E mutant, and 3) proteins common to wild-type and mutant. These interactors are involved mainly in cell migration and apoptosis pathways. We further demonstrate that the wild-type-specific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.