Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

BackgroundEukaryotic cells have evolved various response mechanisms to counteract the deleterious consequences of oxidative stress. Among these processes, metabolic alterations seem to play an important role.ResultsWe recently discovered that yeast cells with reduced activity of the key glycolytic enzyme triosephosphate isomerase exhibit an increased resistance to the thiol-oxidizing reagent diamide. Here we show that this phenotype is conserved in Caenorhabditis elegans and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H) pool. Remarkably, another key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to be inactivated in response to various oxidant treatments, and we show that this provokes a similar redirection of the metabolic flux.ConclusionThe naturally occurring inactivation of GAPDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract oxidative stress. As a consequence, altering the homoeostasis of cytoplasmic metabolites is a fundamental mechanism for balancing the redox state of eukaryotic cells under stress conditions.

P olyglutamine (polyQ) expansions in several unrelated proteins are responsible for at least eight inherited neurodegenerative diseases. These include Huntington's disease (HD), spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and spinocerebellar ataxia types 1, 2, 3, 6, and 7 (1-3). Perhaps the most baffling aspect of these diseases is that the proteins are expressed widely in brain and other tissues, yet each is toxic in a different, highly specific group of neurons and produces a distinct pathology (2).The major characteristic of HD is a selective loss of neurons in the striatum and cortex leading to movement disorders, dementia, and eventually, death (4, 5). The causative agent is a 350-kDa protein, huntingtin (Ht), with glutamine expansions in the N-terminal region (6). The toxicity of Ht in specific neurons correlates with the length of the glutamine expansion, but the mechanism of toxicity is unknown (7,8).A central event in HD is the production of an N-terminal fragment of Ht that aggregates in affected neurons during the natural progression of the disease in humans (9). In transgenic animal models an N-terminal fragment is sufficient to produce an HD-like phenotype, which also depends on the length of the Q repeat (10, 11). Aggregates are found in both the nucleus and/or the cytoplasm of affected neurons in human patients, transgenic animals, and cell lines (12). It is by no means clear, however, whether the aggregates are themselves pathogenic, simply benign byproducts (and thereby markers) of other pathogenic polyQ misfolding events, or a defense mechanism whose purpose is to reduce the interaction of toxic misfolded polyQ proteins with other proteins.Indeed, even the mechanisms underlying the aggregation of these fragments are unknown. A further complexity is that several other proteins interact with Ht. These include HAP1, HIP1, Hsp35, WW domain-containing proteins, the ubiquitin-conjugating enzyme hE2-25k, the SH3GL3 protein, cystathione ␤-synthase, and calmodulin (12)(13)(14).These problems are inherently difficult to study. Although several mammalian cell-line and transgenic-animal models exist for studying Ht, none is as readily amenable to genetic analysis as yeast. We demonstrate that the aggregation of N-terminal fragments of Ht in yeast cells depends on the length of its polyQ repeat and that this aggregation depends on the balance of chaperone protein activities in the cell. Furthermore, N-terminal fragments with up to 103 glutamines have little or no toxicity in either the aggregated or the soluble state. Thus, yeast cells should provide a valuable system for investigating general factors that affect aggregation and for determining what neuronal cell-type specific factors might inf luence toxicity. Materials and MethodsPlasmid Construction. Plasmids encoding fusions between the N-terminal region of Ht and green fluorescent protein (GFP) were the kind gift of G. Lawless (Univ. of California, Los Angeles).To create yeast expression plasmids for HtQ25 or HtQ103, DNAs were d...