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

Abstract

Approximately one quarter of all human genes encode proteins that function in the extracellular space or serve to bridge the extracellular and intracellular environments. Physical associations between these secretome proteins serve to regulate a wide range of biological activities and consequently represent important therapeutic targets. Moreover, some extracellular proteins are targeted by pathogens to allow host access or immune evasion. Despite the importance of extracellular protein-protein interactions, our knowledge in this area has remained sparse. Weak affinities and low abundance have often hindered efforts to identify these interactions using traditional methods such as biochemical purification and cDNA library expression cloning. Moreover, current large-scale protein-protein interaction mapping techniques largely under represent extracellular protein-protein interactions. This review highlights emerging biosensor and protein microarray technology, along with more traditional cell-based techniques, that are compatible with secretome-wide screens for extracellular protein-protein interaction discovery. A combination of these approaches will serve to rapidly expand our knowledge of the extracellular protein-protein interactome.

Defining the Ligand Specificity of the Deleted in Colorectal Cancer (DCC) Receptor

“…To help our understanding of axon guidance, the full complement of extracellular signaling molecules must be uncovered. The application of technologies for screening extracellular protein-protein interactions is an emerging approach for identifying unknown axon-guidance molecules [38]. In one example, a screening methodology called AVEXIS was used to interrogate the interactions among 150 zebrafish receptors from the leucine-rich-repeat and/or Ig superfamily expressed in the nervous system.…”

“…Here we have utilized an extracellular protein microarray that allows efficient screening of nearly one-third of the known human secreted or cell-surface expressed, single-transmembrane-containing genes [24], [38]. Prior work has shown that the microarray approach is able to robustly identify the majority of expected interactions within the Ig receptor family, including those having low binding affinities [24], [39].…”

Technologies for Proteome-Wide Discovery of Extracellular Host-Pathogen Interactions

“…Protein microarrays offer the unique advantage of requiring minimal consumption of protein reagents, fast readouts, and relatively more affordable instrumentation. Typically, a fluorescently labeled or tagged protein of interest (the bait) is generated as a recombinant product and screened against all proteins in the array [10,53]. Despite the increased availability of highcoverage protein arrays, very few are focused on extracellular proteins and therefore are not suitable for study of hostpathogen ePPIs.…”

“…The protein microarrays have represented one of the most fruitful approaches for unbiased determination of ePPIs, including host-pathogen interactions. Nevertheless, one of the main limitations of this technology is the need to generate comprehensive libraries, a process that is resource consuming and often not available to many researchers [53,93]. Consequently, although some of the available arrays were designed to cover a significant fraction of the human proteome, any discoveries made using these platforms are limited to the proteins present in each array.…”

“…Robust methodologies that permit unbiased characterization of ePPI in the absence of preexisting hypotheses are thus needed to elucidate the binding partners and molecular functions of most pathogenencoded molecules. Excellent reviews have recently revisited the currently available technologies for proteome-wide ePPI discovery [4,8,[53][54][55]. Here we discuss the application of some of these technologies to the study of host-pathogen interaction and describe some of the major findings that have recently impacted research in the field of extracellular host-pathogen recognition.…”