Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform
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Evaluation Summary:
This manuscript reports the development of a proteo-genomic screening methodology to identify protein-protein interactions between secreted proteins and their cell surface receptors. The authors use a CRISPRa-based approach to overexpress membrane proteins in cells and then use magnetic cell sorting to identify receptors that bind candidate ligands. This approach leads to the identification of several novel interaction pairs that are validated biochemically, including receptor tyrosine phosphatase ligands and other interactions with implications for immune system function. The work is of interest to a wide variety of fields including biochemistry and signaling.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Secreted proteins, which include cytokines, hormones, and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors (‘orphans’) or are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening are not optimized for extracellular interactions. Cell-based screening is a promising technology for the deorphanization of ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high-value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass transmembrane (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding assays. The newly deorphanized ligands include three receptor protein tyrosine phosphatase (RPTP) ligands and a chemokine-like protein that binds to killer immunoglobulin-like receptors (KIRs). These new interactions provide a resource for future investigations of interactions between the human-secreted and membrane proteomes.
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Evaluation Summary:
This manuscript reports the development of a proteo-genomic screening methodology to identify protein-protein interactions between secreted proteins and their cell surface receptors. The authors use a CRISPRa-based approach to overexpress membrane proteins in cells and then use magnetic cell sorting to identify receptors that bind candidate ligands. This approach leads to the identification of several novel interaction pairs that are validated biochemically, including receptor tyrosine phosphatase ligands and other interactions with implications for immune system function. The work is of interest to a wide variety of fields including biochemistry and signaling.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
This manuscript reports the development of a proteo-genomic screening methodology to identify protein-protein interactions between secreted proteins and their cell surface receptors. The authors use a CRISPRa-based approach to overexpress membrane proteins in cells and then use magnetic cell sorting to identify receptors that bind candidate ligands. This approach leads to the identification of several novel interaction pairs that are validated biochemically, including receptor tyrosine phosphatase ligands and other interactions with implications for immune system function. The work is of interest to a wide variety of fields including biochemistry and signaling.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Siepe et al. developed a high-throughput screen designed to identify novel protein-protein interactions in the extracellular human proteome. Their CRISPRa-based method induced the expression of transmembrane receptors such that they could be screened for binding to proteins of interest. Major strengths of this approach include the ability to screen multiple ligands in parallel, the ability to identify low-affinity interactions, and the availability of custom single- and multi-pass transmembrane protein libraries for selective target screening. A potential weakness is that low-affinity binders and non-specific interactions can be difficult to distinguish in certain cases, and these scenarios require more complex statistical analysis. The authors also note that the CRISPRa strategy cannot …
Reviewer #1 (Public Review):
In this manuscript, Siepe et al. developed a high-throughput screen designed to identify novel protein-protein interactions in the extracellular human proteome. Their CRISPRa-based method induced the expression of transmembrane receptors such that they could be screened for binding to proteins of interest. Major strengths of this approach include the ability to screen multiple ligands in parallel, the ability to identify low-affinity interactions, and the availability of custom single- and multi-pass transmembrane protein libraries for selective target screening. A potential weakness is that low-affinity binders and non-specific interactions can be difficult to distinguish in certain cases, and these scenarios require more complex statistical analysis. The authors also note that the CRISPRa strategy cannot induce the expression of multi-subunit receptors that may be required for some ligands. The screen was tested against a curated set of ligand candidates and identified more than twenty novel interactions with intriguing biological implications. Both the method and newly discovered interactions will be of immediate scientific interest given the growing need to identify receptors for orphan ligands. Overall, this technology should function as a powerful new tool for ligand deorphanization in the extracellular space.
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Reviewer #2 (Public Review):
The authors implemented a novel interaction screening strategy where binders to soluble bait are identified by selecting/sorting cells expressing a library of putative receptors, followed by sequencing of selected cells to identify receptors. The expression library is created by CRISPR activation. The method's shortcomings are explained well in the article (at the end) which need not be repeated here. The new set of interactions discovered/validated are surprising and interesting, which may lead to the opening of the relevant fields to new avenues of thought and research. While co-expression data hint at vague biological functions, none are tested, which is beyond the scope of the manuscript. I have no major concerns about the methodology, which appears to be executed well.
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Reviewer #3 (Public Review):
The authors use a previously developed technology, CRISPR activation screening, in which pooled sgRNAs are used to guide an RNA-associated regulatory complex (MS2-p65-HSF1 transcriptional activators) to promoter regions resulting in increased expression of a specific target gene. The authors screen two different pooled libraries TM1 (single pass) and TM2+ (multiple pass) with 20 different recombinant biotinylated soluble ligands and identified 22 novel interactions. These interactions were further characterized by SPR and cell-based binding experiments; however, several of the interactions are low affinity and were not characterized for any activity or function beyond the relatively weak biochemical binding. Therefore, while the data provide evidence of potential novel interactions, the biological relevance …
Reviewer #3 (Public Review):
The authors use a previously developed technology, CRISPR activation screening, in which pooled sgRNAs are used to guide an RNA-associated regulatory complex (MS2-p65-HSF1 transcriptional activators) to promoter regions resulting in increased expression of a specific target gene. The authors screen two different pooled libraries TM1 (single pass) and TM2+ (multiple pass) with 20 different recombinant biotinylated soluble ligands and identified 22 novel interactions. These interactions were further characterized by SPR and cell-based binding experiments; however, several of the interactions are low affinity and were not characterized for any activity or function beyond the relatively weak biochemical binding. Therefore, while the data provide evidence of potential novel interactions, the biological relevance remains unclear.
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