Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

We determined the nucleotide sequence of the Shiga-like toxin-1 (SLT-1) genes carried by the toxinconverting bacteriophage H-19B. Two open reading frames were identified; these were separated by 12 base pairs and encoded proteins of 315 (A subunit) and 89 (B subunit) amino acids. The predicted protein subunits had N-terminal hydrophobic signal sequences of 22 and 20 amino acids, respectively. The predicted amino acid sequence of the B subunit was identical to that of the B subunit of Shiga toxin. The A chain of ricin was found to be significantly related to the predicted Al fragment of the SLT-1 A subunit. SI nuclease protection experiments showed that the two cistrons formed a single transcriptional unit, with the A subunit being proximal to the promoter. A probable promoter was identified by primer extension, and transcription was found to increase dramatically under conditions of iron starvation. A 21-base-pair sequence with dyad symmetry was found in the region of the SLT-1 -10 sequence, which was found to be 68% homologous to a region of dyad symmetry found in the -35 region of the promoter of the iucA gene op plasmid ColV-K30, which specifies the 74,000-dalton ferric-aerobactin receptor protein. Shigella dysenteriae 1 produces a toxin which is cytotoxic to eucaryotic cell lines (8). Binding to the glycolipid globotriaosylceramide (Gb3) membrane receptor is mediated by a pentamer of 7-kilodalton (kDa) B subunits, while the 31-kDa A subunit, after proteolytic nicking and reduction, inhibits protein synthesis by catalytic inactivation of the 60S ribosomal subunit (8,20,34,35). O'Brien and LaVeck (30,31) have shown that some Escherichia coli strains produce large amounts of a cytotoxin which appeared very similar to Shiga toxin with respect to its subunit structure and mechanism of action. It was completely neutralized by antiserum raised against Shiga toxin and was named Shiga-like toxin 1 (SLT-1) (28, 42). Recently, Strockbine et al. (42) have characterized a second cytotoxin, also produced by E. coli, which is related to SLT-1 at the DNA sequence level but is not neutralized by antiserum raised against Shiga toxin or SLT-

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.

Double-stranded oligodeoxyribonucleotides or single-stranded oligoribonucleotides with specific secondary structure have been proposed as potential antagonists to target nucleic acid-binding proteins (the sense approach). A major limitation of this strategy is that these derivatives are generally considered to be too large for pharmaceutical applications. We have developed a synthetic linker approach whereby nucleic acid duplexes of a much smaller size (miniduplexes) can be generated directly from a standard oligonucleotide synthesis. In this approach, four synthetic linkers (derivatized respectively from 1,9-nonanediol, triethylene glycol, 1,3-propanediol, and hexaethylene glycol) of different length and hydrophobicity were designed and incorporated into a model RNA molecule based on the TAR stem-loop structure of HIV-1. Their thermal stabilities were evaluated by measuring denaturation profiles (Tm measurements). These linker-derivatized RNA molecules were then assessed for their ability to bind to either a full-length protein (HIV-1 Tat protein) or a short peptide (Tat-derived peptide) through RNA mobility shift assays. Results from this study indicate that such modified miniduplex structures retain full binding activity relative to that of the wild-type sequence (Kd values), while Tm values were increased by 24-31 degrees C compared to an open duplex of the same length. This system provides a new direction in the use of nucleic acid miniduplexes as a novel class of oligonucleotide analogues for both fundamental research and possible therapeutic applications.