Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network.

Collecting and harvesting biological data: the GPCRDB and NucleaRDB information systems

The amount of genomic and proteomic data that is entered each day into databases and the experimental literature is outstripping the ability of experimental scientists to keep pace. While generic databases derived from automated curation efforts are useful, most biological scientists tend to focus on a class or family of molecules and their biological impact. Consequently, there is a need for molecular class-specific or other specialized databases. Such databases collect and organize data around a single topic or class of molecules. If curated well, such systems are extremely useful as they allow experimental scientists to obtain a large portion of the available data most relevant to their needs from a single source. We are involved in the development of two such databases with substantial pharmacological relevance. These are the GPCRDB and NucleaRDB information systems, which collect and disseminate data related to G protein-coupled receptors and intra-nuclear hormone receptors, respectively. The GPCRDB was a pilot project aimed at building a generic molecular class-specific database capable of dealing with highly heterogeneous data. A first version of the GPCRDB project has been completed and it is routinely used by thousands of scientists. The NucleaRDB was started recently as an application of the concept for the generalization of this technology. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm/ and the NucleaRDB at http://www.receptors.org/NR/.

Structure and function of declarative and nondeclarative memory systems

This article reviews recent studies of memory systems in humans and nonhuman primates. Three major conclusions from recent work are that (i) the capacity for nondeclarative (nonconscious) learning can now be studied in a broad array of tasks that assess classification learning, perceptuomotor skill learning, artificial grammar learning, and prototype abstraction; (ii) cortical areas adjacent to the hippocampal formation, including entorhinal, perirhinal, and parahippocampal cortices, are an essential part of the medial temporal lobe memory system that supports declarative (conscious) memory; and (iii) in humans, bilateral damage limited to the hippocampal formation is nevertheless sufficient to produce severe anterograde amnesia and temporally graded retrograde amnesia covering as much as 25 years.

Symmetry, stability, and dynamics of multidomain and multicomponent protein systems

Symmetry is commonly observed in many biological systems. Here we discuss representative examples of the role of symmetry in structural molecular biology. Point group symmetries are observed in many protein oligomers whose three-dimensional atomic structures have been elucidated by x-ray crystallography. Approximate symmetry also occurs in multidomain proteins. Symmetry often confers stability on the molecular system and results in economical usage of basic components to build the macromolecular structure. Symmetry is also associated with cooperativity. Mild perturbation from perfect symmetry may be essential in some systems for dynamic functions.

Cross-talk between the insulin and angiotensin signaling systems.

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.

Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx.