No protein is an island. Proteins interact to keep the machinery of the whole organism running. Without protein-protein interactions (PPIs), there would be no biochemical reactions, transmission of messages, immune defenses—in short, no possibility of life. All PPIs in a cell form a complex network critical in health and disease, and require considerable time and resources to decipher with precision.

Researchers led by Sheng Zhong, PhD, professor of bioengineering, University of California, San Diego (USCD), have developed a new technology capable of detecting PPIs among thousands of proteins, in a single experiment.

This tool, called PROPER-seq (protein-protein

This work was done in the lab of Sheng Zhong, PhD, professor of bioengineering, University of California, San Diego.

interaction sequencing), allows researchers to map the protein interaction network in cells in a few weeks, without any special resources such as antibodies or gene libraries.

The development of this breakthrough technology is reported in the Molecular Cell article titled, “Revealing protein-protein interactions at the transcriptome scale by sequencing.” Financial support for the study came from the National Institutes of Health and the Ella Fitzgerald Charitable Foundation.

The authors applied PROPER-seq in human embryonic kidney cells, T lymphocytes, and endothelial cells, and identified 210,518 PPIs involving 8,635 proteins.

“PROPER-seq is capable of scanning the order of 10,000 times 10,000 protein-pairs in one experiment,” said Kara Johnson, PhD, a recent UCSD bioengineering PhD alumna and the first author of this paper, and who is now a scientist at Omniome.

Madhavi K. Ganapathiraju, PhD, associate professor of biomedical informatics and intelligent systems at the University of Pittsburgh, who is unrelated to this study said, “This work is of tremendous value to biomedical science. Proteins and PPIs are central to the functions in cells of all living organisms, and it is through proteins that many genetic aberrations manifest as diseases. Seventy-five percent or more of nearly one million expected PPIs are considered unknown. This work of Dr. Sheng Zhong and collaborators is a big leap ahead towards filling that gap and is an innovative way of using high-throughput sequencing to determine PPIs.”

PROPER-seq labels every interaction with a unique RNA sequence like a barcode. It then reads these barcodes through next-generation sequencing. Zhong’s team developed a technique called SMART-display, which attaches this unique RNA barcode to every protein. They then devised a method called “Incubation, ligation and sequencing” (INLISE) to ligate the pair of DNA barcodes that are attached to two interacting proteins into a chimeric sequence.

The other critical component of PROPER-seq is a software package called PROPERseqTools, which incorporates statistical tools to identify protein-protein interactions from the next-gen DNA sequencing data. This trio of tools—SMART-display, INLISE, and PROPERseqTools—together is known as PROPER-seq.

The team has created a public database with a web interface to download, search, and visualize these PPIs. Collected in the database PROPER v.1.0, PROPER-seq has added more than 200,000 previously uncharacterized protein-protein interactions to the reference human protein interactome.

Matching PPIs detected through PROPER v1.0 with interactions reported in earlier studies, the researchers found 1,300 interactions detected using co-immunoprecipitation and 2,400 interactions detected using affinity purification-mass spectrometry. This wide corroboration validates the new method.

The team also validated four new PPIs in vivo, that they identified using PROPER-seq and that have not been reported in the literature earlier. These involve PARP1, a critical protein for DNA repair and drug target in cancers, and four other proteins—XPO1, MATR3, IPO5, and LEO1—involved in the transport of molecules and transcriptional regulation.

Overall, the scientists detected 17,638 interactions that are computationally predicted but not experimentally validated yet. The complementary evidence now offered by PROPER-seq in support of these interactions buttresses the strong predictive ability of protein structure-based computational models for the detection of protein interactions.

The hubs of the protein interactomes—proteins that interact with numerous other proteins—tend to be synthetic lethal genes. A synthetic lethal gene pair can cause cell death when both genes of the pair are lost. The team found their protein interaction dataset overlaps with 100 synthetic lethal gene pairs. This finding suggests a tantalizing connection between protein and genetic interactions.

One of the limitations of conventional methods of detecting protein interactions is the inability to detect weak, transient, or cell-stage specific interactions. PROPER-seq may be the better option to detect such interactions “because the PPIs are stabilized by crosslinking before they are converted into chimeric DNA sequences.”

The team hopes PROPER-seq will help researchers screen cellular proteins to identify interacting pairs and the growing database of identified interactions will expand the interactome reference maps to illuminate cell-type-specific interactions. They also intend to expand the applicability of PROPER-seq to detect protein-nucleic acid interactions.

PROPER-Seq offers a new experimental approach to detect protein interactions, adding a much-needed method to cross-check PPIs detected using other methods. “Different methods detect different aspects of PPI, some measure direct binding of two proteins, some measure co-complex proteins, and some measure spatial proximity of the proteins. It would be useful to have orthogonal methods to validate the identified PPIs,” said Zhong.

Ganapathiraju added, “Various methods such as this novel bioengineering method developed by Dr. Zhong, or yeast 2-hybrid or mass spectrometry used by others, and computational methods such as what my group uses, all seem to discover a different part of the interactome. When put together, we get to know more about this ‘social network’ of proteins. Of significance, this work of Kara L. Johnson et al., is able to reveal interactomes that are cell-specific, which can be of incredible value in understanding disease-specific and tissue-specific protein interactions.”

Commenting on the prospect of commercializing the new technology, Zhong said, “UCSD has filed a provisional patent on PROPER-seq. Interested parties to license this technology are welcome to contact the UCSD Technology Transfer office.”