Next-generation large-scale binary protein interaction network for Drosophila

Abstract

Generating reference maps of the interactome networks underlying most cellular functions can greatly illuminate genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. Here, we applied state-of-the-art experimental and bioinformatics methods to identify high-confidence binary protein-protein interactions (PPIs) for Drosophila melanogaster. We performed four all-by-all yeast two-hybrid (Y2H) screens of >10,000 Drosophila proteins, resulting in the FlyBi dataset of 8,723 PPIs among 2,939 proteins. As part of this effort, we tested subsets of our data and data from previous PPI datasets using an orthogonal assay, which allowed us to normalize data quality across datasets. Next, we integrated our FlyBi data with previous PPI data, resulting in an expanded, high-confidence binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6,511 proteins. These data are accessible through the Molecular Interaction Search Tool (MIST) and other databases. To assess the utility of the PPI resource, we used novel interactions from the FlyBi dataset to generate an autophagy interaction network that we validated in vivo using two different autophagy-related assays. We found that deformed wings (dwg) encodes a protein that is both a regulator and a target of autophagy. Altogether, the resources generated in this project provide a strong foundation for building high-confidence new hypotheses regarding protein networks and function.

FOXO-regulated Deaf1 controls muscle regeneration through autophagy

“…Decreased and accelerated autophagy in MuSCs leads to similar defects caused by Deaf1 alteration, raising the possibility that Deaf1 may be involved in the regulation of autophagy. In our previous genetic screen, we identified that Deaf1 genetically interacts with Atg1 and that depletion of Deaf1 induces autophagy in larval fat body [18]. Therefore, we investigated if Deaf1 may also regulate autophagy in MuSCs.…”

“…Chromatin Immunoprecipitation was performed on thorax muscle tissues of FOXO-V5;FOXO 25 / FOXO 25 Drosophila (BDSC, 80945) or C2C12 cells, using the Simple ChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technologies, #9005), according to manufacturer's instructions and following the procedures described in our previous paper [18]. Briefly, the C2C12 cells and grinded muscles from FOXO-V5; FOXO 25 /FOXO 25 flies were crosslinked with 1% Formaldehyde and quenched with glycine.…”

“…In addition, proteotoxicity caused by reduced levels of autophagy is another key contributing factor [3,13,14]. Autophagy is a cell survival mechanism that enables cells to adapt to stress by means of degradation and recycling of long-lived proteins or damaged organelles and is regulated by multiple autophagic regulators [15][16][17][18][19]. Autophagic inactivation in aging MuSCs causes the accumulation of damaged mitochondria, which induces ROS and DNA damage and ultimately leads to senescence and apoptosis [13], suggesting that autophagy-regulated proteostasis is essential for preservation of stemness.…”

A Novel TP53 Gene Mutation Sustains Non-Small Cell Lung Cancer through Mitophagy

“…During autophagy, double-membrane vesicles, called autophagosomes, engulf organelles or proteins and subsequently fuse with lysosomes to degrade the contents within the autolysosomes [ 14 ]. This process is strictly regulated by multiple autophagic regulators such as mTORC1, TRAF6, AMPK, ZIPK, TFEB, FOXO, Dwg, and P53 [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Autophagy has been reported to play different roles during various stages of tumor development.…”