Isolation of Drosophila cyclin D, a protein expressed in the morphogenetic furrow before entry into S phase.

During Drosophila development, nuclear and cell divisions are coordinated in response to developmental signals. In yeast and mammalian cells, signals that control cell division regulate the activity of cyclin-dependent kinases (Cdks) through proteins such as cyclins that interact with the Cdks. Here we describe two Drosophila cyclins identified from a set of Cdk-interacting proteins. One, cyclin J, is of a distinctive sequence type; its exclusive maternal expression pattern suggests that it may regulate oogenesis or the early nuclear divisions of embryogenesis. The other belongs to the D class of cyclins, previously identified in mammalian cells. We show that Drosophila cyclin D is expressed in early embryos and in imaginal disc cells in a pattern that anticipates cell divisions. Expression in the developing eye disc at the anterior edge of the morphogenetic furrow suggests that cyclin D acts early, prior to cyclin E, in inducing G1-arrested cells to enter S phase. Our results also suggest that, although cyclin D may be necessary, its expression alone is not sufficient to initiate the events leading to S phase.

RecA protein stimulates homologous recombination in plants.

A number of RecA-like proteins have been found in eukaryotic organisms. We demonstrate that the prokaryotic recombination protein RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells. Resistance to the DNA crosslinking agent mitomycin C requires homologous recombination as well as excision repair activity. Tobacco protoplasts expressing a nucleus-targeted RecA protein were at least three times as efficient as wild-type cells in repairing mitomycin C-induced damage. Moreover, homologous recombination at a defined locus carrying an endogenous nuclear marker gene was stimulated at least 10-fold in transgenic plant cells expressing nucleus-targeted RecA. The increase in resistance to mitomycin C and the stimulation of intrachromosomal recombination demonstrate that Escherichia coli RecA protein is functional in genomic homologous recombination in plants, especially when targeted to the plant nucleus.

Interactions of protein antigens with antibodies.

There are now several crystal structures of antibody Fab fragments complexed to their protein antigens. These include Fab complexes with lysozyme, two Fab complexes with influenza virus neuraminidase, and three Fab complexes with their anti-idiotype Fabs. The pattern of binding that emerges is similar to that found with other protein-protein interactions, with good shape complementarity between the interacting surfaces and reasonable juxtapositions of polar residues so as to permit hydrogen-bond formation. Water molecules have been observed in cavities within the interface and on the periphery, where they often form bridging hydrogen bonds between antibody and antigen. For the most part the antigen is bound in the middle of the antibody combining site with most of the six complementarity-determining residues involved in binding. For the most studied antigen, lysozyme, the epitopes for four antibodies occupy approximately 45% of the accessible surface area. Some conformational changes have been observed to accompany binding in both the antibody and the antigen, although most of the information on conformational change in the latter comes from studies of complexes with small antigens.

Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

High-affinity binding of Escherichia coli SecB to the signal sequence region of a presecretory protein.

The Escherichia coli cytosolic homotetrameric protein SecB is known to be involved in protein export across the plasma membrane. A currently prevalent view holds that SecB functions exclusively as a chaperone interacting nonspecifically with unfolded proteins, not necessarily exported proteins, whereas a contrary view holds that SecB functions primarily as a specific signal-recognition factor--i.e., in binding to the signal sequence region of exported proteins. To experimentally resolve these differences we assayed for binding between chemically pure SecB and chemically pure precursor (p) form (containing a signal sequence) and mature (m) form (lacking a signal sequence) of a model secretory protein (maltose binding protein, MBP) that was C-terminally truncated. Because of the C-terminal truncation, neither p nor m was able to fold. We found that SecB bound with 100-fold higher affinity to p (Kd 0.8 nM) than it bound to m (Kd 80 nM). As the presence of the signal sequence in p is the only feature that distinguished p from m, these data strongly suggest that the high-affinity binding of SecB is to the signal sequence region and not the mature region of p. Consistent with this conclusion, we found that a wild-type signal peptide, but not an export-incompetent mutant signal peptide of another exported protein (LamB), competed for binding to p. Moreover, the high-affinity binding of SecB to p was resistant to 1 M salt, whereas the low-affinity binding of SecB to m was not. These qualitative differences suggested that SecB binding to m was primarily by electrostatic interactions, whereas SecB binding to p was primarily via hydrophobic interactions, presumably with the hydrophobic core of the signal sequence. Taken together our data strongly support the notion that SecB is primarily a specific signal-recognition factor.

Grb-IR: a SH2-domain-containing protein that binds to the insulin receptor and inhibits its function.

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum.

Two interacting heat shock cognate proteins in the lumen of the yeast endoplasmic reticulum (ER), Sec63p and BiP (Kar2p), are required for posttranslational translocation of yeast alpha-factor precursor in vitro. To investigate the role of these proteins in cotranslational translocation, we examined the import of invertase into wild-type, sec63, and kar2 mutant yeast membranes. We found that Sec63p and Kar2p are necessary for both co- and posttranslational translocation in yeast. Several kar2 mutants, one of which had normal ATPase activity, were defective in cotranslational translocation of invertase. We conclude that the requirement for BiP/Kar2p, which is not seen in a reaction reconstituted with pure mammalian membrane proteins [Görlich, D. & Rapoport, T.A. (1993) Cell 75, 615-630], is not due to a distinction between cotranslational translocation in mammalian cells and posttranslational translocation in yeast cells.

Transcription termination of RNA polymerase I due to a T-rich element interacting with Reb1p.

All transcription terminators for RNA polymerase I (pol I) that have been studied so far, ranging from yeast to humans, require a specific DNA binding protein to cause termination. In yeast, this terminator protein has been identified as Reb1p. We now show that, in addition to the binding site for Reb1p, the yeast pol I terminator also requires the presence of a T-rich region coding for the last 12 nucleotides of the transcript. Reb1p cooperates with this T-rich element, both to pause the polymerase and to effect release of the transcript. These findings have implications for the termination mechanism used by all three nuclear RNA polymerases, since all three are known to pause at this terminator.

Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences.

Elevated expression of the marORAB multiple antibiotic-resistance operon enhances the resistance of Escherichia coli to various medically significant antibiotics. Transcription of the operon is repressed in vivo by the marR-encoded protein, MarR, and derepressed by salicylate and certain antibiotics. The possibility that repression results from MarR interacting with the marO operator-promoter region was studied in vitro using purified MarR and a DNA fragment containing marO. MarR formed at least two complexes with marO DNA, bound > 30-fold more tightly to it than to salmon sperm DNA, and protected two separate 21-bp sites within marO from digestion by DNase I. Site I abuts the downstream side of the putative -35 transcription-start signal and includes 4 bp of the -10 signal. Site II begins 13 bp downstream of site I, ending immediately before the first base pair of marR. Site II, approximately 80% homologous to site I, is not required for repression since a site II-deleted mutant (marO133) was repressed in trans by wild-type MarR. The absence of site II did not prevent MarR from complexing with the site I of marO133. Salicylate bound to MarR (Kd approximately 0.5 mM) and weakened the interaction of MarR with sites I and II. Thus, repression of the mar operon, which curbs the antibiotic resistance of E. coli, correlates with the formation of MarR-site I complexes. Salicylate appears to induce the mar operon by binding to MarR and inhibiting complex formation, whereas tetracycline and chloramphenicol, which neither bind MarR nor inhibit complex formation, must induce by an indirect mechanism.

Human TAFII31 protein is a transcriptional coactivator of the p53 protein.

The p53 protein activates transcription of a target gene by binding to a specific DNA response element and interacting with the transcriptional apparatus of RNA polymerase II. The amino-terminal domain of p53 interacts with a component of the TFIID basal transcription complex. The human TATA-binding-protein-associated factor TAFII31, a component of TFIID, has been identified as a critical protein required for p53-mediated transcriptional activation. TAFII31 and p53 proteins bind to each other via amino acid residues in the amino-terminal domain of p53 that are essential for transcription. Antibodies directed against TAFII31 protein inhibit p53-activated but not basal transcription in vitro. These results demonstrate that TAFII31 is a coactivator for the p53 protein.

Stathmin interaction with a putative kinase and coiled-coil-forming protein domains.

Stathmin is a ubiquitous, cytosolic 19-kDa protein, which is phosphorylated on up to four sites in response to many regulatory signals within cells. Its molecular characterization indicates a functional organization including an N-terminal regulatory domain that bears the phosphorylation sites, linked to a putative alpha-helical binding domain predicted to participate in coiled-coil, protein-protein interactions. We therefore proposed that stathmin may play the role of a relay integrating diverse intracellular regulatory pathways; its action on various target proteins would be a function of its combined phosphorylation state. To search for such target proteins, we used the two-hybrid screen in yeast, with stathmin as a "bait." We isolated and characterized four cDNAs encoding protein domains that interact with stathmin in vivo. One of the corresponding proteins was identified as BiP, a member of the hsp70 heat-shock protein family. Another is a previously unidentified, putative serine/threonine kinase, KIS, which might be regulated by stathmin or, more likely, be part of the kinases controlling its phosphorylation state. Finally, two clones code for subdomains of two proteins, CC1 and CC2, predicted to form alpha-helices participating in coiled-coil interacting structures. Their isolation by interaction screening further supports our model for the regulatory function of stathmin through coiled-coil interactions with diverse downstream targets via its presumed alpha-helical binding domain. The molecular and biological characterization of KIS, CC1, and CC2 proteins will give further insights into the molecular functions and mechanisms of action of stathmin as a relay of integrated intracellular regulatory pathways.

Small molecules that mimic components of bioactive protein surfaces

Small molecules designed to mimic specific structural components of a protein (peptide strands, sheets, turns, helices, or amino acids) can be expected to display agonist or antagonist biological responses by virtue of interacting with the same receptors that recognize the protein. Here we describe some minimalist approaches to structural mimetics of amino acids and of strand, turn, or helix segments of proteins. The designed molecules show potent and selective inhibition of protease, transferase, and phospholipase enzymes, or antagonism of G-protein coupled or transcriptional receptors, and have potent anti-tumour, anti-inflammatory, or antiviral activity.

Changes in neuronal protein 22 expression and cytoskeletal association in the alcohol-dependent and withdrawn rat brain

The action of alcohol on neuronal pathways has been an issue of increasing research focus, with numerous findings contradicting the previously accepted idea that its effect is nonspecific. The human NP22 (hNP22) gene was revealed by its elevated expression in the frontal cortex of the human alcoholic. The sequences of hNP22 and the rat orthologue rNP22 contain a number of domains consistent with those of cytoskeletal-interacting proteins. Localization of rNP22 is restricted to the cytoplasm and processes of neurons and it colocalizes with elements of the microfilament and microtubule matrices including filamentous actin (F-actin), alpha-tubulin, tau, and microtubule-associated protein 2 (MAP2). Withdrawal of Wistar rats after alcohol dependence induced by alcohol vapor produced elevated levels of rNP22 mRNA and protein in the cortex, CA2, and dentate gyrus regions of the hippocampus. In contrast, there was decreased rNP22 expression in the striatum after chronic ethanol exposure. Chronic ethanol exposure did not markedly alter rNP22 colocalization with F-actin, alpha-tubulin, or MAP2, although colocalization at the periphery of the neuronal soma with F-actin was observed only after chronic ethanol exposure and withdrawal. Rat NP22 colocalization with MAP2 was reduced during withdrawal, whereas association with alpha-tubulin and actin was maintained. These findings suggest that the effect of chronic ethanol exposure and withdrawal on rNP22 expression is region selective. Rat NP22 may affect microtubule or microfilament function, thereby regulating the neuroplastic changes associated with the development of alcohol dependence and physical withdrawal.

TG2, a novel extracellular protein with multiple functions

TG2 is multifunctional enzyme which can be secreted to the cell surface by an unknown mechanism where its Ca(2+)-dependent transamidase activity is implicated in a number of events important to cell behaviour. However, this activity may only be transient due to the oxidation of the enzyme in the extracellular environment including its reaction with NO probably accounting for its many other roles, which are transamidation independent. In this review, we discuss the novel roles of TG2 at the cell surface and in the ECM acting either as a transamidating enzyme or as an extracellular scaffold protein involved in cell adhesion. Such roles include its ability to act as an FN co-receptor for ß integrins or in a heterocomplex with FN interacting with the cell surface heparan sulphate proteoglycan syndecan-4 leading to activation of PKCa. These different properties of TG2 involve this protein in various physiological processes, which if not regulated appropriately can also lead to its involvement in a number of diseases. These include metastatic cancer, tissue fibrosis and coeliac disease, thus increasing its attractiveness as both a therapeutic target and diagnostic marker.

Uncovering changes in the redox protein interactome of PTEN

Phosphatase and tensin homolog (PTEN) is a redox-sensitive, dual-specificity protein phosphatase involved in regulating a number of cellular processes including metabolism, apoptosis, cell proliferation and survival. It acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of a redox regulation of PTEN downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on the PTEN interactome is still poorly defined. To investigate this, PTEN-GST fusion protein was prepared in its reduced form and an H2O2-oxidized form that was reversible by DTT treatment, and these were immobilized on a glutathione-sepharose-based support. The immobilized protein was incubated with cell lysate to capture interacting proteins. Captured proteins were eluted from the beads, analyzed by LC-MSMS and comparatively quantified using label-free methods. After subtraction of interactors that were also present in the resin and GST controls, 97 individual protein interactors were identified, including several that are novel. Fourteen interactors that varied significantly with the redox status of PTEN were identified, including thioredoxin and peroxiredoxin-1. Except for one interactor, their binding was higher for oxidized PTEN. Using western blotting, altered binding to PTEN was confirmed for 3 selected interactors (Prdx1, Trx, and Anxa2) and DDB1 was validated as a novel interactor with unaltered binding. Our results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome which is important for the cellular function of PTEN. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.