About

At CCSB, the Human Interactome Mapping Project has grown in several distinct stages primarily defined by the number of human protein-coding genes amenable to screening for which at least one Gateway-cloned Open Reading Frame (ORF) was available at the time of the project. As of today, three proteome-scale human PPI datasets are available via this web portal.

Furthermore, efforts to optimize our pipeline, build a framework for quality control, and benchmark new Y2H assay versions have led to the production of PPI datasets generated in systematic screens on smaller sets of our human ORF collection.

 

We also make available at this website all PPIs identified in screens using different protein isoforms from the same gene as generated by alternative splicing.

 

Finally, we also make available for search and download a subset of PPIs curated from the literature, referred to as Lit-BM dataset. These PPIs are annotated with multiple experimental evidences (different publications or different methods) as curated by many different curation efforts and collected in the metaserver Mentha. For a PPI to be part of Lit-BM, at least two experimental evidences need to be available of which at least one has to stem from a method that can detect binary PPIs (described in detail in Rolland et al Cell 2014). We have shown that Lit-BM PPIs recover at much higher rates when tested in binary protein interaction detection assays, compared to PPIs with only one experimental evidence or only non-binary experimental evidences, and that Lit-BM PPIs recover at similar rates to PPIs detected in our screens (Rolland et al Cell 2014).

 

 

Proteome-scale efforts

HI-I-05: Our first iteration at mapping the human protein interactome (Rual et al Nature 2005) screened a space (Space I) of ~8,000 ORFs corresponding to ~7,000 protein-coding genes, and identified ~2,700 high-quality binary PPIs. This search space represents ~12% of the complete search space, assuming a total of ~20,000 protein-coding genes.

 

 

HI-II-14: The second phase of the human interactome mapping project (Rolland et al Cell 2014) generated a dataset of ~14,000 binary PPIs following two screens of a matrix of ~13,000 x 13,000 protein-coding genes (Space II). This search space covers ~42% of the complete search space, a more than three-fold increase with respect to our first attempt.

 

HI-III-18: The next phase of the human interactome project is underway. The human ORF collection being screened has been expanded to ~17,500 protein-coding genes (Space III) and covers ~77% of the complete search space. ~52,000 PPIs identified from screening space III nine times with three different variations of the Y2H assay are provided prior to publication for search and download (note: email required for downloading unpublished data).

 

 

Other CCSB PPI datasets

Venkatesan-09: To estimate the coverage and size of the human interactome (Venkatesan et al Nature Methods 2009), four Y2H screens were performed on a set of ~1,800 DB-X fusion proteins (or baits, representing ~1,700 unique genes) against ~1,800 AD-Y proteins (or preys, representing ~1,800 unique genes), corresponding to ~10% of the available protein-coding genes and ~1% of the full search space. This dataset contains ~200 high-quality binary PPIs.

 

Yu-11: To develop a novel Stitch-seq interactome mapping protocol, a Y2H screen was carried out inside Space II (Yu et al Nature Methods 2011). Stitch-seq combines PCR stitching with next-generation sequencing, and increases the efficiency and cost effectiveness of Y2H screening. The resulting dataset contains ~1,200 PPIs among proteins encoded by ~1,100 human protein-coding genes.

 

Yang-16: To assess the extent to which different protein isoforms generated by alternative splicing from the same gene perform different functions within the cell, we have successfully cloned multiple isoforms for 161 genes and screened those for PPIs against all human ORFs from space II (Yang et al Cell 2016). ~700 PPIs have been identified.

Test-Space: To develop, optimize, and benchmark improvements to the mapping pipeline and variations of the Y2H assay used for ongoing screening efforts (HI-III-18), independent, reciprocal screens on a search space of ~1,800 x ~1,800 genes, constituting ~1% of the full search space, were completed using six different variations of the Y2H assay. This unpublished dataset of ~2,900 PPIs is available for search and upon registration for download.

Pilot-Screen: To further benchmark and test our adapted screening pipeline for the HI-III-18 screening phase we conducted one screen of ~10% of space III. This screen resulted in 1,300 PPIs that are available via this web portal.

  

Y2H screen description

Y2H bait and prey libraries: ORFs from the hORFeome collection were transferred by Gateway recombinational cloning (Invitrogen) into Y2H destination vectors pDEST-DB to generate Gal4 DNA binding domain hybrid proteins (DB-ORF) and pDEST-AD-CYH2, pDEST-AD-QZ213, and pDEST-AD-AR68 to generate Gal4 activation domain hybrid proteins (AD-ORF) as described previously (Rolland et al Cell 2014).

 

Yeast strains: Yeast strains Y8930 (Genotype: MATa leu2-3,112 trp1-901 his3Δ200 ura3-52 gal4Δ gal80Δ GAL2::ADE2 GAL1::HIS3@LYS2 GAL7::lacZ@met2 cyh2R) and Y8800 (Genotype: MATa leu2-3,112 trp1-901 his3Δ200 ura3-52 gal4Δ gal80Δ GAL2::ADE2 GAL1::HIS3@LYS2 GAL7::lacZ@met2 cyh2R) were transformed with individual DB-ORF and AD-ORF constructs, respectively.

 

Y2H Vector details:

 

Name

pDEST-DB

pDEST-AD-CHY2

pDEST-QZ213

pDEST-AD-AR68

Fusion

Gal4-DB

(aa 1-147)

Gal4-AD

(aa 768-881)

Gal4-AD

(aa 768-881)

Gal4-AD

(aa 768-881)

Fusion location

N-term

N-term

N-term

C-term

Promoter

Truncated ADH1 promoter (-701 to +1)

Truncated ADH1 promoter (-701 to +1)

Truncated ADH1 promoter (-410 to +1)

Truncated ADH1 promoter (-410 to +1)

Yeast replication ori

CEN

CEN

2micron

2micron

Linker

SRSNQ

GGSNQ

ICMAYPYDVPDYASLGGHMAMEAPS

VDGTA

Terminator

ADH1 Term

ADH1 Term

ADH1 Term

ADH1 Term

Selection marker

AmpR

AmpR

AmpR

AmpR

         

 

Y2H screens: Fresh overnight cultures of individual Y8930:DB-ORF yeast strains were mated against Y8800:AD-ORF mini-libraries containing ~1,000 different Y8800:AD-ORF yeast strains. After overnight growth at 30ºC in liquid rich medium (YEPD), mated yeast cells were transferred into SC-Leu-Trp liquid media to select for diploids. After overnight incubation at 30ºC diploid yeast cells were spotted onto SC-Leu-Trp-His+1mM 3AT solid media to select for activation of the GAL1::HIS3 reporter gene.

 

Y2H assay versions: Combinations of different yeast strains and vectors result in different Y2H assay versions as described in the table below. Assay version 0 was used to generate the HI-I-05 and Venkatesan-09. Assay version 1 was used to generate HI-II-14, Yu-11, Yang-16, Test-Space, Pilot-Screen, and screens 1-3 of HI-III-18. Assay version 2 was used to generate screens 4-6 and assay version 3 for screens 7-9 of HI-III-18.

 

Assay version

DB vector

AD vector

DB yeast strain  

AD yeast strain

0

pDEST-DB

pDEST-AD-CHY2

MaV203

MaV103

1

pDEST-DB

pDEST-AD-CHY2

Y8930

Y8800

2

pDEST-DB

pDEST-QZ213

Y8930

Y8800

6

pDEST-DB

pDEST-AD-AR68

Y8930

Y8800

 

External datasets used to annotate PPI data in this portal

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