Proteins are the building blocks of all cells in our bodies and in all living creatures of all kingdoms. They play a role in keeping our cells and bodies functioning. Recent studies have demonstrated that proteins do not only serve their individual purpose, but they interact with other proteins to carry out more function through these protein-protein interactions (PPI).

All the PPIs in a cell form a PPI network. However, identifying a PPI network within human cells has required a tremendous amount of effort and time. Now, bioengineers at the University of California, San Diego (UCSD), have developed a technology capable of revealing the PPIs among thousands of proteins, in a single experiment.

Their findings are published in the journal Molecular Cell in a paper titled, “Revealing protein-protein interactions at the transcriptome scale by sequencing.”

“We describe PROPER-seq (protein-protein interaction sequencing) to map PPIs en masse,” wrote the researchers. “PROPER-seq first converts transcriptomes of input cells into RNA-barcoded protein libraries, in which all interacting protein pairs are captured through nucleotide barcode ligation, recorded as chimeric DNA sequences, and decoded at once by sequencing and mapping.”

A graphical abstract of the three tools that together are known as PROPER-seq. [Zhong Lab]
The researchers claim their tool called PROPER-seq allows researchers to map the PPI network from their cells of interest within several weeks, without any specialized resources such as antibodies or premade gene libraries.

The researchers used the tool PROPER-seq on 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 10,000×10,000 protein pairs in one experiment,” said Kara Johnson, a recent UCSD bioengineering PhD alumna and the first author of this paper.

The researchers also developed a method called “Incubation, ligation and sequencing” (INLISE) to sequence the pair of DNA barcodes that are attached to two interacting proteins. The third component of PROPER-seq is a software package called PROPERseqTools, which incorporates statistical tools to identify the PPIs from the DNA sequencing data. This trio of tools—SMART-display, INLISE, and PROPERseq tools—together is known as PROPER-seq.

The team validated the PROPER-seq-identified PPIs (called PROPER v1.0) with previously characterized PPIs documented in PPI databases. The team found more than 1,300 and 2,400 PPIs in PROPER v1.0 are supported by previous co-immunoprecipitation experiments and affinity purification-mass spectrometry experiments, respectively.

The team experimentally validated four PROPER-seq identified PPIs that have not been reported in the literature. These four PPIs involve PARP1, a critical protein for DNA repair and a drug target of several human cancers, and four other proteins involved in the trafficking of molecules and transcription regulation. These validations suggest mechanistic links between PARP1 and import/export of molecules to/from the nucleus as well as gene transcription.

“PROPER-seq presents a time-effective technology to map PPIs at the transcriptome scale, and PROPER v.1.0 provides a rich resource for studying PPIs,” wrote the researchers.

The researchers hope PROPER-seq can assist researchers in screening many protein pairs and identify PPIs of interest. In addition, the PROPER-seq identified PPIs from different labs can expand the PPI networks’ reference maps and illuminate cell-type-specific PPIs.