High-throughput identification of transient extracellular protein interactions

Abstract

Protein interactions are highly diverse in their biochemical nature, varying in affinity and are often dependent on the surrounding biochemical environment. Given this heterogeneity, it seems unlikely that any one method, and particularly those capable of screening for many protein interactions in parallel, will be able to detect all functionally relevant interactions that occur within a living cell. One major class of interactions that are not detected by current popular high-throughput methods are those that occur in the extracellular environment, especially those made by membrane-embedded receptor proteins. In the present article, we discuss some of our recent research in the development of a scalable assay to identify this class of protein interaction and some of the findings from its application in the construction of extracellular protein interaction networks.

An Extracellular Interactome of Immunoglobulin and LRR Proteins Reveals Receptor-Ligand Networks

“…Consequently, cell surface and secreted proteins comprise a substantial fraction of the human proteome (Almén et al, 2009; da Cunha et al, 2009; Diehn et al, 2006). Although vast amounts of protein interactome data have been generated in the last decade, extracellular and transmembrane proteins are greatly underrepresented in these data sets, due to the technical challenges that extracellular proteins present for systems biology and proteomics approaches (Wright et al, 2010). Producing extracellular molecules requires special conditions enabled by secretion, such as an oxidizing environment (for disulfide bonds) and specific post-translational modifications (predominantly glycosylation) for folding and function.…”

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

“…The physical interactions between the membrane-embedded receptor proteins which mediate many of these contacts have evolved to be extremely weak so as to permit this highly motile behaviour. It has been estimated that monomeric interaction strengths as weak as 50 μM could be sufficient to drive spontaneous interactions at physiological receptor densities 9 which makes detecting novel interactions extremely challenging 2,10 . The AVEXIS method described here provides a sensitive method for detecting transient extracellular protein:protein interactions that can be implemented in a scalable manner with a low false positive rate.…”

Protein microarrays, biosensors, and cell-based methods for secretome-wide extracellular protein–protein interaction mapping

“…Also, techniques like yeast-2-hybrid (Y2H) and affinity-purification mass spectrometry (AP-MS) do not require laborious library constructions and have emerged as powerful alternatives for intercellular PPI mapping. However, because Y2H and AP-MS data sets under represent ePPIs [1,2], there is renewed interest in developing microarrays as a platform for ePPI studies.…”

“…From high-affinity cytokine-receptor interactions to lowaffinity cell-cell adhesion receptor interactions, the diversity of extracellular protein-protein interactions (ePPI) has been well documented [1][2][3][4][5]. Standard methods for intracellular proteinprotein interaction mapping have proven challenging for ePPIs [1,2].…”

“…Standard methods for intracellular proteinprotein interaction mapping have proven challenging for ePPIs [1,2]. The yeast-2-hybrid (Y2H) system functions through an intracellular mechanism and therefore the proper folding and posttranslational processing of secreted or membrane proteins are less likely to occur.…”