Identification and molecular analysis of cable pilus biosynthesis genes in Burkholderia cepacia

Burkholderia cepacia is an opportunistic respiratory pathogen in cystic fibrosis patients. One highly transmissible and virulent clone belonging to genomovar IIIa expresses pili with unique cable morphology, which enable the bacterium to bind cytokeratin 13 in epithelial cells. The cblA gene, encoding the major pilin subunit, is often used as a DNA marker to identify potentially virulent isolates. The authors have now cloned and sequenced four additional genes, cblB, cblC, cblD and cblS, in the pilus gene cluster. This work shows that the products of the first four genes of the cbl operon, cblA, cblB, cblC and cblD, are sufficient for pilus assembly on the bacterial surface. Deletion of cblB abrogated pilus assembly and compromised the stability of the CblA protein in the periplasm. In contrast, deletion of cblD resulted in no pili, but there was no effect on expression and stability of the CblA protein subunit. These results, together with protein sequence homologies, predicted structural analyses, and the presence of typical amino acid motifs, are consistent with the assignment of functional roles for CblB as a chaperone that stabilizes the major pilin subunit in the periplasm, and CblD as the initiator of pilus biogenesis. It is also shown that expression of Cbl pili in Escherichia coli is not sufficient to mediate the binding of bacteria to the epithelial cell receptor cytokeratin 13, and that B. cepacia still binds to cytokeratin 13 in the absence of Cbl pili, suggesting that additional bacterial components are required for effective binding.

STIMULATION OF PHOSPHOLIPASE C-BETA(2) BY RECOMBINANT GUANINE-NUCLEOTIDE-BINDING PROTEIN BETA-GAMMA DIMERS PRODUCED IN A BACULOVIRUS/INSECT CELL EXPRESSION SYSTEM - REQUIREMENT OF GAMMA-SUBUNIT ISOPRENYLATION FOR STIMULATION OF PHOSPHOLIPASE-C

Recombinant wild-type beta(1) gamma(1) dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta(1) gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta(1) gamma(1)C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta(1) gamma(1) dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta(1) gamma(1) preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta(1) gamma(1) dimers. Soluble wild-type and mutant beta(1) gamma(1) dimers and native beta(1) gamma(1) dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta(2). Only isoprenylated beta(1) gamma(1) dimers were capable of stimulating phospholipase C-beta(2). The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.

Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C

We have previously shown that isoprenylation and/or additional pest-translational processing of the G protein gamma(1) subunit carboxyl terminus is required for beta(1) gamma(1) subunit stimulation of phospholipase C-beta(2) (PLC beta(2)) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma(1) subunit alone is sufficient for beta(1) gamma(1)-mediated PLC beta(2) stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta(1) gamma(1) dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta(1) gamma(1) dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma(1) subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta(1) gamma(1) dimers with a recombinant PLC beta(2) isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta(1) gamma(1) dimers capable of stimulating PLC beta(2) and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta(1) gamma(1) dimers than for fully modified native beta(1) gamma(1) purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

Increased expression of the RIα subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer

The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIα subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIα expression in patients with ovarian cancer. We have evaluated the expression of RIα in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RIα mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RIα mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RIα mRNA expression between different ovarian histological subtypes in this study. No associations were found between RIα mRNA expression and differentiation state. RIα mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RIα mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RIα mRNA expression is associated with advanced stage disease. RIα expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2sigma;2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2sigma;2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca<inf>o</inf>²⁺) homeostasis. To elucidate the role of AP2sigma;2 in Ca<inf>o</inf>²⁺ regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2sigma;2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2sigma;2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2sigma;2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2sigma;2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2sigma;2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the -subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel–Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP).

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

Identification of the p28 subunit of eukaryotic initiation factor 3(eIF3k) as a new interaction partner of cyclin D3.

Cyclin D3 is found to play a crucial role not only in progression through the G1 phase as a regulatory subunit of cyclin-dependent kinase 4 (CDK 4) and CDK 6, but also in many other aspects such as cell cycle, cell differentiation, transcriptional regulation and apoptosis. In this work, we screened a human fetal liver cDNA library using human cyclin D3 as bait and identified human eukaryotic initiation factor 3 p28 protein (eIF3k) as a partner of cyclin D3. The association of cyclin D3 with eIF3k was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscopic analysis. We found that cyclin D3 specifically interacted with eIF3k through its C-terminal domain. Immunofluorescence experiments showed that eIF3k distributed both in nucleus and cytoplasm and colocalized with cyclin D3. In addition, the cellular translation activity in HeLa cells was upregulated by cyclin D3 overexpression and the mRNA levels are constant. These data provide a new clue to our understanding of the cellular function of cyclin D3.

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER)(1). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes(2). Controversy exists, however, concerning whether ER has a role outside the nucleus(3), particularly in mediating the cardiovascular protective effects of oestrogen(4). Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ERa are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85 alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ERa with PI(3)K.

Ribosomal 18S RNA processing by the IGF-I-responsive WDR3 protein is integrated with p53 function in cancer cell proliferation.

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G1 phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

Up-regulation of GABA transporters and GABAA Receptor α1 subunit in tremor rat hippocampus

The loss of GABAergic neurotransmission has been closely linked with epileptogenesis. The modulation of the synaptic activity occurs both via the removal of GABA from the synaptic cleft and by GABA transporters (GATs) and by modulation of GABA receptors. The tremor rat (TRM; tm/tm) is the parent strain of the spontaneously epileptic rat (SER; zi/zi, tm/tm), which exhibits absence-like seizure after 8 weeks of age. However, there are no reports that can elucidate the effects of GATs and GABAA receptors (GABARs) on TRMs. The present study was conducted to detect GATs and GABAR a1 subunit in TRMs hippocampus at mRNA and protein levels. In this study, total synaptosomal GABA content was significantly decreased in TRMs hippocampus compared with control Wistar rats by high performance liquid chromatography (HPLC); mRNA and protein expressions of GAT-1, GAT-3 and GABAR a1 subunit were all significantly increased in TRMs hippocampus by real time PCR and western blot, respectively; GAT-1 and GABAR a1 subunit proteins were localized widely in TRMs and control rats hippocampus including CA1, CA3 and dentate gyrus (DG) regions whereas only a wide distribution of GAT-3 was observed in CA1 region by immunohistochemistry. These data demonstrate that excessive expressions of GAT-1 as well as GAT-3 and GABAR a1 subunit in TRMs hippocampus may provide the potential therapeutic targets for genetic epilepsy.

Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.