PRISMA: Protein Interaction Screen on Peptide Matrix Reveals Interaction Footprints and Modifications- Dependent Interactome of Intrinsically Disordered C/EBPβ

Abstract

SummaryCCAAT enhancer-binding protein beta (C/EBPβ) is a pioneer transcription factor that specifies cell differentiation. C/EBPβ is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPβ differs depending on alternative translation initiation and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPβ alters protein interactions and cell differentiation, suggesting that a C/EBPβ PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPβ using an array technique based on spot-synthesized C/EBPβ-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of ∼1,300 proteins in nuclear extracts, many with chromatin modifying, chromatin remodeling, and RNA processing functions. The results suggest that C/EBPβ acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function.

Peptide array–based interactomics

“…A screening technique targeting transient SLiM-based interactions along the primary structure was recently developed. The technique, PrISMa (Protein Interaction Screen on a Peptide Matrix), is based on a membrane-bound array of overlapping peptides, spanning the entire sequence of the protein of interest, creating a sliding window for the detection of SLiM-mediated interactions [ 11 ] (Fig. 1d, f ).…”

“…1g ). The technique was used to map the interactome of the transcription factor C/EBPβ, identifying a large number of new interaction partners [ 11 ].…”

Peptide-based Interaction Proteomics

“…High Throughput Peptide-based Interaction Proteomics-Recently, the advantageous parts of both above-mentioned peptide pull-down approaches have been combined (Fig. 2): Instead of probing a peptide array with a single prey protein, peptide arrays were probed with whole cell lysates and interacting proteins were identified using quantitative mass spectrometry (28,29,40). In all three cases, 14 -18 mer peptides were synthesized on cellulose membranes via the SPOT technology that were then incubated with cell extracts to identify binding partners via mass spectrometry.…”

“…The two other studies therefore employed quantification to distinguish specific from nonspecific binders. Dittmar and co-workers used a sliding window approach (overlapping "tiled" peptides) to map binding partners along the sequence of C/EBPbeta (28). They also assessed the impact of PTMs because SPOT synthesis permits inclusion of phosphorylation, methylation (mono, di, tri), citrullination, acetylation, crotonylation, sumoylation and many more.…”

“…For the actual pull-down experiment, it is advantageous to use a cell extract that contains the putative interaction partners. For example, for interactions in the neuronal context it makes sense to use extract from neuronal cell lines (29), and for interaction partners of nuclear proteins nuclear extracts are a good choice (28). Whatever extract is used, it should be rather concentrated (5 mg protein/ml) to increase the sensitivity of the screen (28,29).…”