Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality

Charles Hillier,Mercedes Pardo,Lu Yu,Ellen Bushell,Theo Sanderson,Tom Metcalf,Colin Herd,Burcu Anar,Julian C Rayner,Oliver Billker,Jyoti S Choudhary

doi:10.1016/j.celrep.2019.07.019

Abstract

SummaryMalaria represents a major global health issue, and the identification of new intervention targets remains an urgent priority. This search is hampered by more than one-third of the genes of malaria-causing Plasmodium parasites being uncharacterized. We report a large-scale protein interaction network in Plasmodium schizonts, generated by combining blue native-polyacrylamide electrophoresis with quantitative mass spectrometry and machine learning. This integrative approach, spanning 3 species, identifies >20,000 putative protein interactions, organized into 600 protein clusters. We validate selected interactions, assigning functions in chromatin regulation to previously unannotated proteins and suggesting a role for an EELM2 domain-containing protein and a putative microrchidia protein as mechanistic links between AP2-domain transcription factors and epigenetic regulation. Our interactome represents a high-confidence map of the native organization of core cellular processes in Plasmodium parasites. The network reveals putative functions for uncharacterized proteins, provides mechanistic and structural insight, and uncovers potential alternative therapeutic targets.

Highlights

Plasmodium parasites caused 216 million new cases of malaria in 2016 and nearly 500,000 deaths (World Health Organization, 2017)
Blue native (BN)-PAGE, which separates protein complexes in native conformation based on Coomassie brilliant blue binding, has a higher resolution than gel filtration or sucrose density centrifugation and has proven to be useful in resolving membrane protein complexes (Bode et al, 2016; Heide et al, 2012; Schagger et al, 1994; Schagger and von Jagow, 1991), which are often underrepresented in high-throughput datasets
Plasmodium Complexome Profiling Using BN-PAGE Coupled to Tandem MS To produce a comprehensive and accurate protein interaction network in Plasmodium, we applied a strategy based on highthroughput biochemical fractionation using BN-PAGE coupled to quantitative tandem MS to three different species, namely P. falciparum, P. knowlesi, and P. berghei

Summary

Introduction

Plasmodium parasites caused 216 million new cases of malaria in 2016 and nearly 500,000 deaths (World Health Organization, 2017). Systematic pull-down studies have been effective in elucidating PPI networks (Gavin et al, 2002, 2006; Hein et al, 2015; Huttlin et al, 2015, 2017), but lack of proteome-scale panels of antibodies and limitations in genetic manipulation that restrict high-throughput protein tagging in Plasmodium make this costly and time-consuming approach unfeasible, to apply in several species. Highthroughput chromatographic fractionation combined with quantitative mass spectrometry has emerged recently as an alternative strategy to elucidate protein complexes at the systems level and has been applied in organisms ranging from bacteria to humans (Crozier et al, 2017; Havugimana et al, 2012; Kastritis et al, 2017; Kirkwood et al, 2013; Kristensen et al, 2012; Wan et al, 2015) This approach provides a global analysis of the interactome and does not require any genetic manipulation or affinity reagents.

Results

Discussion

Conclusion