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.

While it is generally recognized that misfolding of specific proteins can cause late-onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenorhabditis elegans. SDS-insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of a translated CAG repeat in the N terminus of the huntingtin (htt) protein. Here we describe the generation and characterization of a full-length HD Drosophila model to reveal a previously unknown disease mechanism that occurs early in the course of pathogenesis, before expanded htt is imported into the nucleus in detectable amounts. We find that expanded full-length htt (128Qhtt(FL)) leads to behavioral, neurodegenerative, and electrophysiological phenotypes. These phenotypes are caused by a Ca2+-dependent increase in neurotransmitter release efficiency in 128Qhtt(FL) animals. Partial loss of function in synaptic transmission (syntaxin, Snap, Rop) and voltage-gated Ca2+ channel genes suppresses both the electrophysiological and the neurodegenerative phenotypes. Thus, our data indicate that increased neurotransmission is at the root of neuronal degeneration caused by expanded full-length htt during early stages of pathogenesis.

We demonstrate that the N terminus of GCAP-2, like those of other members of the recoverin family of Ca 2؉-binding proteins, is fatty acylated. However, unlike other proteins of this family, more GCAP-2 is present in the membrane fraction at low Ca 2؉ than at high Ca 2؉ concentrations. We investigated the role of the N-terminal fatty acyl residue in the ability of GCAP-2 to regulate RetGCs. Myristoylated or nonacylated GCAP-2 forms were expressed in Escherichia coli. Wild-type GCAP-2 and the Gly 2 3 Ala 2 GCAP-2 mutant, which is unable to undergo N-terminal myristoylation, were also expressed in mammalian HEK293 cells. We found that compartmentalization of GCAP-2 in photoreceptor outer segment membranes is Ca 2؉ -and ionic strength-sensitive, but it does not require the presence of the fatty acyl group and does not necessarily directly reflect GCAP-2 interaction with RetGC. The lack of myristoylation does not significantly affect the ability of GCAP-2 to stimulate RetGC. Nor does it affect the ability of the Ca 2؉ -loaded form of GCAP-2 to compete with the GCAP-2 mutant that constitutively activates RetGC. We conclude that while Ca 2؉ binding plays a major regulatory role in GCAP-2 function, it does not operate through a calcium-myristoyl switch similar to the one found in recoverin.