Plasmodium falciparum causes the most severe form of malaria and kills up to 2.7 million people annually. Despite the global importance of P. falciparum, the vast majority of its proteins have not been characterized experimentally. Here we identify P. falciparum protein-protein interactions using a high-throughput version of the yeast two-hybrid assay that circumvents the difficulties in expressing P. falciparum proteins in Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid screens with P. falciparum protein fragments, we identified 2,846 unique interactions, most of which include at least one previously uncharacterized protein. Informatic analyses of network connectivity, coexpression of the genes encoding interacting fragments, and enrichment of specific protein domains or Gene Ontology annotations were used to identify groups of interacting proteins, including one implicated in chromatin modification, transcription, messenger RNA stability and ubiquitination, and another implicated in the invasion of host cells. These data constitute the first extensive description of the protein interaction network for this important human pathogen.

Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%–4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

CD223 (LAG‐3) is an activation‐induced cell surface molecule, structurally similar to the T cell coreceptor CD4, that binds MHC class II molecules with high affinity. Little is known about theexpression and function of murine CD223. Here, we show that mRNA expression is restricted to the thymic medulla, splenic red pulp and sparse cells in the adult brain cortex. In contrast, surprisingly high expression was seen in defined tracts at the base of the cerebellum and in the choroid plexus of day 7 postnatal brain. mCD223:Ig, but not CD4:Ig, fusion proteins stained cells expressing MHC class II molecules. Analysis of mCD223 cell surface expression was performed with a new monoclonal antibody (mAb) that recognizes an epitope in the D2 domain. Although it blocked mCD223 function in vitro, it did not block binding of mCD223 to MHC class II molecules. While very few TCRα β  T cells in the spleen and thymus of naive mice express surface mCD223 (<3 %), ∼ 18 % TCR γ δ  T cells and ∼10 % NK cells are positive. This small population of TCRα β  T cells are cycling memory T cells (BrdU+, CD44hi, CD62Llo). In contrast, all T cells express mCD223 2–3 days post activation. This study and the anti‐CD223 mAb should greatly assist in the elucidation of CD223 function.

Mitogen Activated Protein Kinase (MAPK) pathways form the backbone of signal transduction within the mammalian cell. Here, we apply a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. A core network of 641 interactions is supported by multiple lines of evidence including conservation with yeast. Using siRNA knockdowns, we reveal that a significant number of novel interactors can modulate MAPK mediated signaling. We uncover the Na-H exchanger NHE1 as a scaffold for a novel set of MAPKs, link HSP90 chaperones to MAPK pathways, and identify MUC12 as the human analogue to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions along with the accompanying clone libraries. It illustrates a methodology for probing signaling networks based on functional refinement of experimentally-derived protein interaction maps.

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