Bioengineers at the University of California San Diego have developed a technique that can reveal proton pump inhibitors in thousands of proteins in a single experiment. The tool is called PROPER-seq (protein-protein interaction sequencing) and allows researchers to map PPI networks from their cells of interest within a few weeks without the need for any specialized resources, such as antibodies or prefabricated gene banks.
In general, all protein-protein interactions in the cell form a PPI network. It takes a lot of time and resources to identify PPI networks in human cells. Experiments are needed to identify each individual PPI, and many additional experiments are needed to study the network-level interactions of these protein pairs.
The researchers described the technique in the journal Molecular Cell on August 3. They applied PROPER-seq to human embryonic kidney cells, T lymphocytes and endothelial cells, and identified 210,518 PPIs involving 8,635 proteins.
“PROPER-seq can scan the sequence of 10,000 x 10,000 protein pairs in one experiment,” said Kara Johnson, who recently graduated from the University of California, San Diego with a PhD in bioengineering and the first author of the paper. The research was led by the laboratory of Sheng Zhong, a professor of bioengineering.
The core idea of PROPER-seq is to attach a unique DNA sequence to each PPI, and then read these DNA sequence tags through next-generation sequencing. To realize this idea, Zhong’s team developed a technology called SMART-display, which attaches unique DNA barcodes to each protein. They also designed a method called “Incubation, Ligation and Sequencing” (INLISE) to sequence a pair of DNA barcodes attached to two interacting proteins. The third component of PROPER-seq is a software package called PROPERseqTools, which contains statistical tools for identifying PPIs from DNA sequencing data. These three tools, SMART-display, INLISE, and PROPERseqTools, are collectively called PROPER-seq.
The laboratory will start the PROPER-seq protocol with the cells of interest and obtain the output as a list of identified PPIs. Users can also obtain DNA sequence read counts and other statistical data associated with each identified PPI.
Zhong’s team applied PROPER-seq to human embryonic kidney cells, T lymphocytes, and endothelial cells, and obtained 210,518 PPIs involving 8,635 proteins. The team created a public database with a web interface to download, search, and visualize these PPIs.
The team verified the PPIs identified by PROPER-seq (called PROPER v1.0) and the characteristic PPIs previously recorded in the PPI database. The team found that more than 1,300 and more than 2,400 PPIs in PROPER v1.0 were supported by previous co-immunoprecipitation experiments and affinity purification-mass spectrometry experiments, respectively.
The team also verified four PPIs identified by PROPER-seq through experiments, and these PPIs have not been reported in the literature before. These four PPIs involve PARP1, which is a key protein for DNA repair and a drug target for several human cancers, as well as the other four proteins involved in molecular transport and transcriptional regulation. These verifications indicate that there are mechanistic links between PARP1 and molecules entering and exiting the nucleus and gene transcription.
Their results show that PROPER v1.0 overlaps with more than 17,000 calculated and predicted PPIs that have not been verified by prior experiments. The experimental support provided by PROPER-seq for these previously undescribed PPIs shows that the computational model based on protein structure has reliable predictive power.
The research team found that PROPER v1.0 overlapped with 100 pairs of synthetic lethal (SL) genes. When both genes in the SL gene pair are lost, it will cause cell death. This finding suggests that there is a link between physical interactions (PPIs) and human gene interactions.
Looking to the future, the team hopes that PROPER-seq can help researchers screen many protein pairs and identify PPIs of interest. In addition, the PPIs identified by PROPER-seq from different laboratories can expand the reference map of the PPI network and illuminate the cell type-specific PPIs.
