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Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1·EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.
Hundreds of genes have been associated with respiratory chain disease (RCD), the most common inborn error of metabolism so far. Elimination of the respiratory electron chain by depleting the entire mitochondrial DNA (mtDNA, ρ0 cells) has therefore one of the most severe impacts on the energy metabolism in eukaryotic cells. In this study, proteomic data sets including the post-translational modifications (PTMs) phosphorylation and ubiquitination were integrated with metabolomic data sets and selected enzyme activities in the osteosarcoma cell line 143B.TK−. A shotgun based SILAC LC-MS proteomics and a targeted metabolomics approach was applied to elucidate the consequences of the ρ0 state. Pathway and protein–protein interaction (PPI) network analyses revealed a nonuniform down-regulation of the respiratory electron chain, the tricarboxylic acid (TCA) cycle, and the pyruvate metabolism in ρ0 cells. Metabolites of the TCA cycle were dysregulated, such as a reduction of citric acid and cis-aconitic acid (six and 2.5-fold), and an increase of lactic acid, oxalacetic acid (both twofold), and succinic acid (fivefold) in ρ0 cells. Signaling pathways such as GPCR, EGFR, G12/13 alpha, and Rho GTPases were up-regulated in ρ0 cells, which could be indicative for the mitochondrial retrograde response, a pathway of communication from mitochondria to the nucleus. This was supported by our phosphoproteome data, which revealed two main processes, GTPase-related signal transduction and cytoskeleton organization. Furthermore, a general de-ubiquitination in ρ0 cells was observed, for example, 80S ribosomal proteins were in average threefold and SLC amino acid transporters fivefold de-ubiquitinated. The latter might cause the observed significant increase of amino acid levels in ρ0 cells. We conclude that an elimination of the respiratory electron chain, e.g. mtDNA depletion, not only leads to an uneven down-regulation of mitochondrial energy pathways, but also triggers the retrograde response.
The yeast Rcf1 protein is a member of the conserved family of proteins termed the hypoxia-induced gene (domain) 1 (Hig1 or HIGD1) family. Rcf1 interacts with components of the mitochondrial oxidative phosphorylation system, in particular the cytochrome bc1 (complex III)-cytochrome c oxidase (complex IV) supercomplex (termed III-IV) and the ADP/ATP carrier proteins. Rcf1 plays a role in the assembly and modulation of the activity of complex IV; however, the molecular basis for how Rcf1 influences the activity of complex IV is currently unknown. Hig1 type 2 isoforms, which include the Rcf1 protein, are characterized in part by the presence of a conserved motif, (Q/I)X3(R/H)XRX3Q, termed here the QRRQ motif. We show that mutation of conserved residues within the Rcf1 QRRQ motif alters the interactions between Rcf1 and partner proteins and results in the destabilization of complex IV and alteration of its enzymatic properties. Our findings indicate that Rcf1 does not serve as a stoichiometric component, i.e. as a subunit of complex IV, to support its activity. Rather, we propose that Rcf1 serves to dynamically interact with complex IV during its assembly process and, in doing so, regulates a late maturation step of complex IV. We speculate that the Rcf1/Hig1 proteins play a role in the incorporation and/or remodeling of lipids, in particular cardiolipin, into complex IV and. possibly, other mitochondrial proteins such as ADP/ATP carrier proteins.
There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were “uncoupling” NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.
Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) are important modulators of endothelial cell homeostasis. We investigated the signaling pathway linking the activation of CYP 2C9 to enhanced endothelial cell proliferation. Overexpression of CYP 2C9 in cultured human endothelial cells markedly increased proliferation. This effect was paralleled by an up-regulation of the G1 phase regulatory protein, cyclin D1. The specific CYP 2C9 inhibitor, sulfaphenazole, prevented both the enhanced cell proliferation and up-regulation of cyclin D1. CYP 2C9 overexpression also decreased the activity of the c-Jun N-terminal kinase (JNK). Coexpression of wild type JNK with CYP 2C9 attenuated the CYP 2C9-induced increase in cyclin D1 expression and abolished the CYP 2C9-induced proliferation response. In contrast, cotransfecting dominant negative JNK with CYP 2C9 restored the CYP 2C9-mediated up-regulation of cyclin D1 and proliferation. The inactivation of JNK is linked to its dephosphorylation by dual specificity mitogen-activated protein (MAP) kinase phosphatases (MKPs). Overexpression of CYP 2C9 significantly increased the expression of MKP-1, as did incubation with 11,12-EET. These data demonstrate that the mitogenic effect of CYP 2C9 is due to the generation of EETs, which promote the MKP-1-mediated dephosphorylation and inactivation of JNK, effects ultimately culminating in the expression of cyclin D1 and endothelial cell proliferation.
Over the last decade, cases of metabolic syndrome and type II diabetes have increased exponentially. Exercise and ω-3 polyunsaturated fatty acid (PUFA)-enriched diets are usually prescribed but no therapy is effectively able to restore the impaired glucose metabolism, hypertension, and atherogenic dyslipidemia encountered by diabetic patients. PUFAs are metabolized by different enzymes into bioactive metabolites with anti- or pro-inflammatory activity. One important class of PUFA metabolizing enzymes are the cytochrome P450 (CYP) enzymes that can generate a series of bioactive products, many of which have been attributed protective/anti-inflammatory and insulin-sensitizing effects in animal models. PUFA epoxides are, however, further metabolized by the soluble epoxide hydrolase (sEH) to fatty acid diols. The biological actions of the latter are less well understood but while low concentrations may be biologically important, higher concentrations of diols derived from linoleic acid and docosahexaenoic acid have been linked with inflammation. One potential application for sEH inhibitors is in the treatment of diabetic retinopathy where sEH expression and activity is elevated as are levels of a diol of docosahexaenoic acid that can induce the destabilization of the retina vasculature.
Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27Kip1 in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induced a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 was not significantly affected. This effect on p27Kip1 protein was associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an “Akt-resistant”/constitutively active FOXO3a mutant reversed the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreased p27Kip1 expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27Kip1 expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1.
Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells
(2003)
Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-γ (IFN-γ) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-γ-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-γ and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-γ induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.
A gene trap strategy has been used to identify genes that are repressed in cells transformed by an activated epidermal growth factor (EGF)/EGF receptor signal transduction pathway. EGF receptor-expressing NIH3T3 cells (HER1 cells) were infected with a retrovirus containing coding sequences for the human CD2 antigen and for secreted alkaline phosphatase in the U3 region. By selecting for and against CD2 expression, we obtained clones in which the gene trap had integrated into genes selectively repressed by EGF. Two of these clones encoded for the secreted extracellular matrix proteins TIMP3 and COL1A2. We show here that both genes are downstream targets of RAS and are specifically repressed by EGF-induced transformation. Moreover, this strategy tags tumor suppressor genes in their normal chromosomal location, thereby improving target-specific screens for antineoplastic drugs.
Objectives: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays.
Methods: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing.
Results: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-β-lactamase (NDM) [n = 2]; Verona integron–encoded metallo-β-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17–46%)/89% (CI: 75–97%) for CARBA NP, 74% (CI: 60–85%)/82% (CI: 67–91%) for faropenem, 91% (CI: 78–97%)/82% (CI: 66–92%) for simplified CIM, and 93% (CI: 81–99%)/100% (CI: 91–100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92–100%)/100% (CI: 91–100%) on the 81 isolates, and 100% (CI: 29–100%)/100% (CI: 96–100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France.
Discussion: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of blaOXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified.