Multiplex matrix network analysis of protein complexes in the human TCR signalosome

Abstract

Multiprotein complexes transduce cellular signals through extensive interaction networks, but the ability to analyze these networks in cells from small clinical biopsies is limited. To address this, we applied an adaptable multiplex matrix system to physiologically relevant signaling protein complexes isolated from a cell line or from human patient samples. Focusing on the proximal T cell receptor (TCR) signalosome, we assessed 210 pairs of PiSCES (proteins in shared complexes detected by exposed surface epitopes). Upon stimulation of Jurkat cells with superantigen-loaded antigen-presenting cells, this system produced high-dimensional data that enabled visualization of network activity. A comprehensive analysis platform generated PiSCES biosignatures by applying unsupervised hierarchical clustering, principal component analysis, an adaptive nonparametric with empirical cutoff analysis, and weighted correlation network analysis. We generated PiSCES biosignatures from 4-mm skin punch biopsies from control patients or patients with the autoimmune skin disease alopecia areata. This analysis distinguished disease patients from the controls, detected enhanced basal TCR signaling in the autoimmune patients, and identified a potential signaling network signature that may be indicative of disease. Thus, generation of PiSCES biosignatures represents an approach that can provide information about the activity of protein signaling networks in samples including low-abundance primary cells from clinical biopsies.

Homeostatic Plasticity Requires Remodeling of the Homer-Shank Interactome

“…The quantitative multiplex immunoprecipitation (QMI) panel described in this report contains 21 validated synapse-associated proteins-receptors, scaffolds, and downstream signaling molecules (Table 1) and measures 380 binary PiSCES for each experiment (Lautz et al, 2018(Lautz et al, , 2019. QMI takes advantage of fluorophore-embedded microspheres ("beads") to immunoprecipitate multiple protein targets from a single sample simultaneously (Lautz et al, 2018;Smith et al, 2016). Biotinyated probe antibodies detect co-associated proteins, and, following streptavidin-PE secondary labeling, the median fluorescence intensity (MFI) of each IP-probe combination is measured on a flow cytometer and averaged across technical replicates (see methods) ( Fig.…”

“…To better understand the PIN rearrangements that occur during homeostatic scaling, we identified a set of high-confidence PiSCES that changed individually after drug treatment and belonged to a module of interactions that correlated with the drug treatment using a combination of two statistical tests: 1) adaptive nonparametric analysis with an empirical alpha cutoff (ANC) and 2) weighted correlation network analysis (CNA) (Langfelder and Horvath, 2008), as described in (Smith et al, 2016). ANC, which identifies PiSCES with different bead distributions between conditions, identified 27 PiSCES that changed significantly with TTX treatment and 16 that changed with BIC treatment (multiple-comparison-corrected P-value<0.05); 8 PiSCES were significant in both conditions (Table S1).…”

“…So why not use an N-of-12 design for all experiments? ANC analysis becomes more stringent with increasing Ns, identifying fewer significant PiSCES for each N over 4 (Smith et al, 2016), while CNA identifies modules of PiSCES that change together, but may not be true positives. Moreover, samples run in parallel show high interexperimental consistencey (Fig.…”

“…QMI was performed as described previously (Lautz et al, 2018;Smith et al, 2016). Briefly, a master mix containing equal numbers of each antibody-coupled Luminex bead was prepared and distributed to lysates containing equal amounts of protein and incubated overnight on a rotator at 4°C.…”

“…Biotinylated detection (probe) antibodies were added to the appropriate wells and incubated at 4°C with gentle agitation for 1 hour. The resulting bead-probe complexes were washed 3 times with Fly-P buffer, incubated for 30 minutes with streptavidin-PE on ice, washed another 3 times, resuspended in 125µl ice cold Fly-P buffer, and processed for fluorescence using a customized refrigerated Bio-Plex 200 (Smith et al, 2016). Antibody clone names, catalog numbers, and lot numbers are the same as in (Lautz et al, 2018).…”