Analysing protein-protein interactions with the yeast two-hybrid system

Plant research is moving into the post-genomic era. Proteomic-based strategies are now being developed to study functional aspects of the genes predicted from the various genome-sequencing initiatives. All biological processes depend on interactions formed between proteins and the mapping of such interactions on a global scale is providing interesting functional insights. One of the techniques that has proved itself invaluable in the mapping of protein-protein interactions is the yeast two-hybrid system. This system is a sensitive molecular genetic approach for studying protein-protein interactions in vivo. In this review we will introduce the yeast two-hybrid system, discuss modifications of the system that may be of interest to the plant science community and suggest potential applications of the technology.     

Cellular patterning of the vertebrate embryo

Recent studies show that cell dispersal is a widespread phenomenon in the development of early vertebrate embryos. These cell movements coincide with major decisions for the spatial organization of the embryo, and they parallel genetic patterning events. For example, in the central nervous system, cell dispersal is first mainly anterior–posterior and subsequently dorsal–ventral. Thus, genes expressed in signaling centers of the embryo probably control cell movements, tightly linking cellular and genetic patterning. Cell dispersal might be important for the correct positioning of cells and tissues involved in intercellular signaling. The emergence of cell dispersal at the onset of vertebrate evolution indicates a shift from early, lineage-based cellular patterning in small embryos to late, movement-based cellular patterning of polyclones in large embryos. The conservation of the same basic body plan by invertebrate and vertebrate chordates suggests that evolution of the embryonic period preceding the phylotypic stage was by intercalary co-option of basic cell activities present in the ancestral metazoan cell.     

Membrane responses of the leech giant glial cell to the peptide transmitter myomodulin

A myomodulin peptide has been suggested to mediate the response of the giant glial cells to stimulation of the Leydig interneuron in the central nervous system of the leech Hirudo medicinalis [Eur. J. Neurosci. 11 (1999) 3125]. We have now studied the glial response to the endogenous leech MM peptide (GMGALRL-NH(2), MMHir). The peptide evokes a membrane outward current (EC(50) approximately 2 microM), which neither desensitizes nor shows any sign of run-down, and elicits a K(+) conductance increase of the glial cell membrane. The peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF) enhances the glial current response, suggesting the presence of endogenous extracellular peptidases.     

Identification of TH1 as an interaction partner of A-Raf kinase

A-Raf is an important intermediate of the growth factor Ras-MAP kinase pathway. In a two-hybrid screen of human fetal liver cDNA library, TH1 was detected as a new interaction partner of A-Raf. TH1 is a highly conserved and widely expressed protein, which was recently cloned by Bonthron DT group. The binding between A-Raf and TH1 was specific, as no binding between TH1 and B-Raf or c-Raf was observed, and the amino-terminal 162 amino acids in the A-Raf regulatory domain were found to be sufficient for this interaction. This specific interaction may have played a critical role in the activation of A-Raf.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.     

Isolation of the interacting molecules with GEX-3 by a novel functional screening

To screen for important molecules that interact with a gene of interest in Caenorhabditis elegans (C. elegans), we established a novel functional screening system using the yeast two-hybrid system with the RNA interference technique. Our screening system makes it possible to identify the molecular machinery involved in the function of a gene of interest starting with the cDNA of this gene. As a model case, we examined the molecular machinery involved in the function of GEX-3, an essential factor of tissue morphogenesis. We identified many interacting molecules by yeast two-hybrid screening and could detect some functional interactions using this novel functional screening system.     

Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase

Maspin is a 42kDa tumor suppressor protein that belongs to the serine protease inhibitor (serpin) family. It inhibits cell motility and invasion in vitro, and tumor growth and metastasis in nude mice; however, maspin's molecular mechanism of action has remained elusive. Maspin contains several tyrosine residues and we hypothesized that phosphorylation of maspin could play a role in its biological function. Our study reveals that maspin is phosphorylated on tyrosine moiety(ies) in normal mammary epithelial cells endogenously expressing maspin. In addition, transfection of the maspin gene, using either a stable or inducible system into maspin-deficient breast cancer cell lines, yields a protein product that is phosphorylated on tyrosine residue(s). Furthermore, recombinant maspin protein can be tyrosine-phosphorylated by the kinase domain from the epidermal growth factor receptor in vitro. These novel observations suggest that maspin, which deviates from the classical serpin, may be an important signal transduction molecule in its phosphorylated form.     

The EGF-like homeotic protein dlk affects cell growth and interacts with growth-modulating molecules in the yeast two-hybrid system

Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.     

Contact System. New Concepts on Activation Mechanisms and Bioregulatory Functions

This review considers the data of recent years concerning the contact system initiating the activation of blood plasma proteolytic systems, such as hemocoagulation, fibrinolysis, kininogenesis, and also complement and angiotensinogenesis. The main proteins of the contact system are the factors XII and XI, prekallikrein, and high-molecular-weight kininogen. The data on the structure, functions, and biosynthesis of these proteins and on their genes are presented. Studies in detail on the protein–protein interactions during formation of the ensemble of the contact system components on the anionic surface resulted in the postulation of the mechanism of activation of this system associated with generation of the XIIa factor and of kallikrein. This mechanism is traditionally considered a trigger of processes for the internal pathway of the hemocoagulating cascade. However, the absence of direct confirmation of such activation in vivo and the absence of hemorrhagia in the deficiency of these components stimulated the studies designed to find another mechanism of their activation and physiological role outside of the hemostasis system. As a result, a new concept on the contact system activation on the endothelial cell membrane was proposed. This concept is based on the isolation of a complex of proteins, which in addition to the above-mentioned proteins includes cytokeratin 1 and the receptors of the urokinase-like plasminogen activator and of the complement q-component. The ideas on the role of this system in the biology of vessels are developed. Some of our findings on the effect of leukocytic elastase on the key components of the contact system are also presented.