Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Coupling the phosphotransferase system and the methyl-accepting chemotaxis protein-dependent chemotaxis signaling pathways of Escherichia coli.

Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY. CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation. E. coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell. The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II. We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling. In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway. Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not. These findings suggest the following model for signal transduction in PTS-dependent chemotaxis. During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP. Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation. This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR.

Electrophile and antioxidant regulation of enzymes that detoxify carcinogens.

Detoxication (phase 2) enzymes, such as glutathione S-transferases (GSTs), NAD(P)H:(quinone-acceptor) oxidoreductase (QR), and UDP-glucuronsyltransferase, are induced in animal cells exposed to a variety of electrophilic compounds and phenolic antioxidants. Induction protects against the toxic and neoplastic effects of carcinogens and is mediated by activation of upstream electrophile-responsive/antioxidant-responsive elements (EpRE/ARE). The mechanism of activation of these enhancers was analyzed by transient gene expression of growth hormone reporter constructs containing a 41-bp region derived from the mouse GST Ya gene 5'-upstream region that contains the EpRE/ARE element and of constructs in which this element was replaced with either one or two consensus phorbol 12-tetradecanoate 13-acetate (TPA)-responsive elements (TREs). When these three constructs were compared in Hep G2 (human) and Hepa 1c1c7 (murine) hepatoma cells, the wild-type sequence was highly activated by diverse inducers, including tert-butylhydroquinone, Michael reaction acceptors, 1,2-dithiole-3-thione, sulforaphane,2,3-dimercapto-1-propanol, HgCl2, sodium arsenite, and phenylarsine oxide. In contrast, constructs with consensus TRE sites were not induced significantly. TPA in combination with these compounds led to additive or synergistic inductions of the EpRE/ARE construct, but induction of the TRE construct was similar to that induced by TPA alone. Transfection of the EpRE/ARE reporter construct into F9 cells, which lack endogenous TRE-binding proteins, produced large inductions by the same compounds, which also induced QR activity in these cells. We conclude that activation of the EpRE/ARE by electrophile and antioxidant inducers is mediated by EpRE/ARE-specific proteins.

Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

Function and Regulation of Cytochrome P450 4V2 and the Implications in Bietti’s Crystalline Dystrophy

Thesis (Ph.D.)--University of Washington, 2016-06

Diversity in the disulfide folding pathways of cystine knot peptides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides; poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Differential use of signal peptides and membrane domains is a common occurrence in the protein output of transcriptional units

Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi- spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units ( TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our highconfidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome.

Signal integration between IFN gamma and TLR signalling pathways in macrophages

Macrophages are major effector cells of the innate immune system, and appropriate regulation of macrophage function requires the integration of multiple signalling inputs derived from the recognition of host factors (e.g. interferon-gamma/IFN gamma) and pathogen products (e.g. toll-like receptor/TLR agonists). The profound effects of IFN gamma pre-treatment (priming) on TLR-induced macrophage activation have long been recognised, but many of the mechanisms underlying the priming phenotype have only recently been identified. This review summarises the known mechanisms of integration between the IFN gamma and TLR signalling pathways. Synergy occurs at multiple levels, ranging from signal recognition to convergence of signals at the promoters of target genes. In particular, the cross-talk between the IFN gamma and LPS and CpG DNA signalling pathways is discussed. (c) 2006 Elsevier GmbH. All rights reserved.

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs