bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone
Authors: DOLINSKY Vernon W. , DOUGLAS Donna N. , LEHNER Richard , VANCE Dennis E.
Published: April 2004

Hepatic VLDL (very-low-density lipoprotein) assembly is a complex process that is largely regulated by the provision of lipid for apolipoprotein B assembly. Intracellular stored TAG (triacylglycerol) undergoes an initial lipolysis followed by re-esterification of the lipolytic products to form TAG prior to their incorporation into a VLDL particle. TGH (TAG hydrolase) is a lipase that hydrolyses intracellular TAG within the hepatocyte. We have utilized both dexamethasone-injected mouse and primary hepatocyte models to address whether stimulation of TAG biosynthesis by the synthetic glucocorticoid, dexamethasone, altered hepatic lipolysis and re-esterification and the provision of stored TAG for lipoprotein secretion. Dexamethasone treatment resulted in decreased TGH expression, primarily due to a dexamethasone-induced decrease in TGH mRNA stability. The expression and activities of diacylglycerol acyltransferases 1 and 2 were stimulated by dexamethasone. The combination of reduced intracellular TAG lipolysis and increased TAG biosynthesis contributed to the accumulation of TAG within the livers of dexamethasone-injected mice. The rate of hepatic TAG secretion in dexamethasonetreated mice was maintained at similar levels as in control mice. Our data demonstrate that stimulation of de novo TAG synthesis by dexamethasone increased the proportion of secreted TAG that was derived from de novo sources, while the utilization of stored TAG for secretion was reduced. The results show that, during markedly increased TAG synthesis, some TAGs are diverted from the cytosolic storage pool and are utilized directly for VLDL assembly within the endoplasmic reticulum lumen.
Characterization of a cis-acting regulatory element in the protein-coding region of human dihydrofolate reductase mRNA
Authors: TAI Ningwen , SCHMITZ John C. , CHEN Tian-min , CHU Edward
Published: April 2004

Previous studies have shown that human DHFR (dihydrofolate reductase), in addition to its critical role in DNA biosynthesis, functions as an RNA-binding protein. The interaction between DHFR and its own mRNA results in translational repression. In this study, we characterized the cis-acting elements on human DHFR mRNA that are required for the DHFR mRNA–DHFR protein interaction. Using a series of gel-shift and nitrocellulose filter-binding assays, a 164 nt RNA sequence, corresponding to nt 401–564, was identified within the coding region that binds to DHFR protein with an affinity similar to that of full-length DHFR mRNA. To document in vivo biological activity, various DHFR sequences contained within the coding region were cloned on to the 5´ end of a luciferase reporter plasmid, and transient transfection experiments were performed using human colon cancer RKO cells. In cells transfected with p644/DHFR:401–564, luciferase activity was decreased by 50% when compared with cells transfected with the p644 plasmid alone. Luciferase mRNA levels were identical under each of these conditions, as determined by Northern-blot analysis. In cells transfected with p644/DHFR:401–564, luciferase activity was restored to almost 100% of control when cells were treated with the antifolate analogue methotrexate or with a short-interfering RNA targeting DHFR mRNA. These findings provide evidence that the DHFR 401–564 sequence is a DHFR-response element. In vitro and in vivo studies further localized this cis-element to an 82 nt sequence corresponding to nt 401–482. This work provides new insights into critical elements that mediate RNA–protein interactions.
TGF-beta1 (transforming growth factor-beta1)-mediated adhesion of gastric carcinoma cells involves a decrease in Ras/ERKs (extracellular-signal-regulated kinases) cascade activity dependent on c-Src activity
Authors: KIM Hwang-Phill , LEE Mi-Sook , YU Jiyon , PARK Jin-Ah , JONG Hyun-Soon , KIM Tae-You , LEE Jung Weon , BANG Yung-Jue
Published: April 2004

Signalling by integrin-mediated cell anchorage to extracellular matrix proteins is co-operative with other receptor-mediated signalling pathways to regulate cell adhesion, spreading, proliferation, survival, migration, differentiation and gene expression. It was observed that an anchorage-independent gastric carcinoma cell line (SNU16) became adherent on TGF-β1 (transforming growth factor β1) treatment. To understand how a signal cross-talk between integrin and TGF-β1 pathways forms the basis for TGF-β1 effects, cell adhesion and signalling activities were studied using an adherent subline (SNU16Ad, an adherent variant cell line derived from SNU16) derived from the SNU16 cells. SNU16 and SNU16Ad cells, but not integrin α5-expressing SNU16 cells, showed an increase in adhesion on extracellular matrix proteins after TGF-β1 treatment. This increase was shown to be mediated by an integrin α3 subunit, which was up-regulated in adherent SNU16Ad cells and in TGF-β1-treated SNU16 cells, compared with the parental SNU16 cells. After TGF-β1 treatment of SNU16Ad cells on fibronectin, Tyr-416 phosphorylation of c-Src was increased, but Ras-GTP loading and ERK1/ERK2 (extracellular-signal-regulated kinases 1 and 2) activity were decreased, which showed a dependence on c-Src family kinase activity. Studies on adhesion and signalling activities using pharmacological inhibitors or by transient-transfection approaches showed that inhibition of ERK1/ERK2 activity increased TGF-β1-mediated cell adhesion slightly, but not the basal cell adhesion significantly, and that c-Src family kinase activity and decrease in Ras/ERKs cascade activity were required for the TGF-β1 effects. Altogether, the present study indicates that TGF-β1 treatment causes anchorage-independent gastric carcinoma cells to adhere by an increase in integrin α3 level and a c-Src family kinase activity-dependent decrease in Ras/ERKs cascade activity.
A synergistic relationship between three regions of stathmin family proteins is required for the formation of a stable complex with tubulin
Authors: JOURDAIN Isabelle , LACHKAR Sylvie , CHARBAUT Elodie , GIGANT Benoit , KNOSSOW Marcel , SOBEL André , CURMI Patrick A.
Published: April 2004

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3SLD were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer.
Glycoproteins VI and Ib-IX-V stimulate tyrosine phosphorylation of tyrosine kinase Syk and phospholipase Cgamma2 at distinct sites
Authors: SUZUKI-INOUE Katsue , WILDE Jonathan I. , ANDREWS Robert K. , AUGER Jocelyn M. , SIRAGANIAN Reuben P. , SEKIYA Fujio , RHEE Sue Goo , WATSON Steve P.
Published: April 2004

Glycoproteins GPVI and GPIb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCγ2 (phospholipase Cγ2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPIb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk activity. In addition, GPVI stimulates tyrosine phosphorylation of PLCγ2 at Tyr753 and Tyr759, whereas GPIb-IX-V only induces significant phosphorylation at Tyr753. Both receptors stimulate tyrosine phosphorylation of Btk at the regulatory Tyr223 and Tyr551. Syk and Btk phosphorylate peptides from PLCγ2 containing Tyr753 and Tyr759 respectively, suggesting that they may stimulate phosphorylation at these sites in phospholipase. Studies using PLCγ2-deficient platelets demonstrated that phospholipase is not required for the activation of integrin αIIbβ3 by GPIb-IX-V. Our results demonstrate fundamental differences between GPVI and GPIb-IX-V in the regulation of tyrosine phosphorylation of Syk and PLCγ2 consistent with the functional impairment of phospholipase in signalling by GPIb-IX-V.
New insights in dihydropyrimidine dehydrogenase deficiency: a pivotal role for beta-aminoisobutyric acid?
Authors: van KUILENBURG André B. P. , STROOMER Alida E. M. , van LENTHE Henk , ABELING Nico G. G. M. , van GENNIP Albert H.
Published: April 2004

DPD (dihydropyrimidine dehydrogenase) constitutes the first step of the pyrimidine degradation pathway, in which the pyrimidine bases uracil and thymine are catabolized to β-alanine and the R-enantiomer of β-AIB (β-aminoisobutyric acid) respectively. The S-enantiomer of β-AIB is predominantly derived from the catabolism of valine. It has been suggested that an altered homoeostasis of β-alanine underlies some of the clinical abnormalities encountered in patients with a DPD deficiency. In the present study, we demonstrated that only a slightly decreased concentration of β-alanine was present in the urine and plasma, whereas normal levels of β-alanine were present in the cerebrospinal fluid of patients with a DPD deficiency. Therefore the metabolism of β-alanine-containing peptides, such as carnosine, may be an important factor involved in the homoeostasis of β-alanine in patients with DPD deficiency. The mean concentration of β-AIB was approx. 2–3-fold lower in cerebrospinal fluid and urine of patients with a DPD deficiency, when compared with controls. In contrast, strongly decreased levels (10-fold) of β-AIB were present in the plasma of DPD patients. Our results demonstrate that, under pathological conditions, the catabolism of valine can result in the production of significant amounts of β-AIB. Furthermore, the observation that the R-enantiomer of β-AIB is abundantly present in the urine of DPD patients suggests that significant cross-over exists between the thymine and valine catabolic pathways.
sigma54-dependent transcription activator phage shock protein F of Escherichia coli: a fragmentation approach to identify sequences that contribute to self-association
Authors: BORDES Patricia , WIGNESHWERARAJ Siva R. , ZHANG Xiaodong , BUCK Martin
Published: April 2004

Proteins that belong to the AAA (ATPases associated with various cellular activities) superfamily of mechanochemical enzymes are versatile and control a wide array of cellular functions. Many AAA proteins share the common property of self-association into oligomeric structures and use nucleotide binding and hydrolysis to regulate their biological output. The Escherichia coli transcription activator PspF (phage shock protein F) is a member of the σ54-dependent transcriptional activators that belong to the AAA protein family. Nucleotide interactions condition the functional state of PspF, enabling it to self-associate and interact with its target, the σ54–RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of σ54-dependent activators remain poorly characterized. In the present study, we have used a fragment of the AAA domain of PspF as a probe to study the nucleotide-conditioned self-association of PspF. Results show that the PspF fragment acts in trans to inhibit specifically self-association of PspF. The PspF fragment prevented efficient binding of nucleotides to PspF, consistent with the observation that the site for nucleotide interactions within an oligomer of AAA proteins is created between two protomers. Using proximity-based footprinting and cross-linking techniques, we demonstrate that the sequences represented in this fragment are close to one protomer–protomer interface within a PspF oligomer. As the sequences represented in this PspF fragment also contain a highly conserved motif that interacts with the σ54–RNAP closed complex, we suggest that PspF may be organized to link nucleotide interactions and self-association to σ54–RNAP binding and transcription activation.
Interferon-gamma inhibits interferon-alpha signalling in hepatic cells: evidence for the involvement of STAT1 induction and hyperexpression of STAT1 in chronic hepatitis C
Authors: RADAEVA Svetlana , JARUGA Barbara , KIM Won-Ho , HELLER Theo , LIANG T. Jake , GAO Bin
Published: April 2004

IFN-γ (interferon-γ) modulates IFN-α therapy in chronic hepatitis C infection; however, the underlying mechanism remains unclear. Here we demonstrate that long-term (3–6 days) but not short-term (up to 1 day) IFN-γ treatment of human hepatoma Hep3B cells attenuates IFN-α activation of STAT1 (signal transducers and activators of transcription factor 1), STAT2 and STAT3, but enhances IFN-γ and interleukin 6 activation of STATs. Prolonged exposure to IFN-γ also significantly induces STAT1 protein expression without affecting STAT2, STAT3 and ERK (extracellular-signal-regulated kinase) 1/2 protein expression. To determine the role of STAT1 protein overexpression in regulation of IFN-α signalling, Hep3B cells were stably transfected with wild-type STAT1. Overexpression of STAT1 via stable transfection enhances IFN-γ activation of STAT1, but surprisingly attenuates IFN-α activation of STAT1, STAT2 and STAT3 without affecting Janus kinase activation. This STAT1-mediated inhibition does not require STAT1 tyrosine phosphorylation because overexpression of dominant-negative STAT1 with a mutation on tyrosine residue 701 also blocks IFN-α activation of STAT1, STAT2 and STAT3. Moreover, overexpression of STAT1 blocks IFN-α-activated STAT2 translocation from IFN-α receptor 2 to IFN-α receptor 1, a critical step in IFN-α signalling activation. Finally, significantly higher levels of STAT1 protein expression, which is probably induced by IFN-γ, are detected in the majority of hepatitis C virus-infected livers compared with healthy controls. In conclusion, long-term IFN-γ treatment inhibits IFN-α-activated signals most probably, at least in part, through the induction of STAT1 protein expression, which could partly contribute to IFN-α treatment failure in hepatitis C patients.
Fructoselysine 3-epimerase, an enzyme involved in the metabolism of the unusual Amadori compound psicoselysine in Escherichia coli
Authors: WIAME Elsa , VAN SCHAFTINGEN Emile
Published: April 2004

The frl (fructoselysine) operon encodes fructoselysine 6-kinase and fructoselysine 6-phosphate deglycase, allowing the conversion of fructoselysine into glucose 6-phosphate and lysine. We now show that a third enzyme encoded by this operon catalyses the metal-dependent reversible interconversion of fructoselysine with its C-3 epimer, psicoselysine. The enzyme can be easily assayed through the formation of tritiated water from [3-3H]fructoselysine. Psicoselysine supports the growth of Escherichia coli, causing the induction of the three enzymes of the frl operon. No growth on fructoselysine or psicoselysine was observed with Tn5 mutants in which the putative transporter (FrlA) or fructoselysine 6-phosphate deglycase (FrlB) had been inactivated, indicating the importance of the frl operon for the metabolism of both substrates. The ability of E. coli to grow on psicoselysine suggests the occurrence of this unusual Amadori compound in Nature.
The lipid composition of high-density lipoprotein affects its re-absorption in the kidney by proximal tubule epithelial cells
Authors: BREZNAN Dalibor , VEERESWARAN Vasanthi , VIAU France J. , NEVILLE Tracey A.-M. , SPARKS Daniel L.
Published: April 2004

The kidney is believed to play a major role in the clearance of apoA-I (apolipoprotein A-I) and HDL (high-density lipoprotein) particles from the bloodstream. Proximal tubule epithelial cells of the kidney appear to prevent the loss of these proteins in the urine by re-absorbing them from the urinary filtrate. Experiments were undertaken to investigate the factors that regulate the renal re-absorption of apoA-I and small HDL in a transformed human proximal tubule epithelial (HKC-8) cell line. Fluorescent microscopic studies show that HKC-8 cells can readily bind and take up HDL particles. Intracellular localization of fluorescently labelled native HDL shows its accumulation in endocytotic vesicles, in a perinuclear region after 1 h. Binding studies reveal a saturable cell association of 125I-HDL with the HKC-8 cell surface after 2 h. HKC-8 cells do not degrade apoA-I or other HDL-apoproteins. The specific cell association of lipid-free apoA-I is approx. 2-fold less than that observed for native HDL. Similarly, reconstituted HDL prepared from HDL-apoproteins and pure phospholipids also exhibits a low cell association with the HKC-8 cells. In contrast, reconstituted HDL prepared with the extracted lipids of HDL and pure apoA-I exhibits an even higher cell association than that observed with the native lipoprotein. A detailed characterization of the major lipid classes in reconstituted HDL shows that only cholesteryl ester increases the cell association of the recombinant particles. These results show that the cholesteryl ester content of HDL may play an important role in the re-absorptive salvage of HDL by the proximal tubule cells of the kidney.
Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases
Authors: DARNET Sylvain , RAHIER Alain
Published: April 2004

In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4α-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO. The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation. To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana. SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N. benthamiana inoculated with the viral transcripts. After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9β,19-cyclopropylsterols (i.e. 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4α-methylsterols were not affected. In the case of silencing with SMO2, a large accumulation of 4α-methyl-Δ7-sterols (i.e. 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols. These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4α-methylsterol precursors respectively.
Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1
Authors: QADRI Ishtiaq , IWAHASHI Mieko , CAPASSO Juan M. , HOPKEN Matthew W. , FLORES Sonia , SCHAACK Jerome , SIMON Francis R.
Published: April 2004

Activation of cellular kinases and transcription factors mediates the early phase of the cellular response to chemically or biologically induced stress. In the present study we investigated the oxidant/antioxidant balance in Huh-7 cells expressing the HCV (hepatitis C virus) subgenomic replicon, and observed a 5-fold increase in oxidative stress during HCV replication. We used MnSOD (manganese-superoxide dismutase) as an indicator of the cellular antioxidant response, and found that its activity, protein levels and promoter activity were significantly increased, whereas Cu/ZnSOD was not affected. The oxidative stress-induced protein kinases p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) were activated in the HCV repliconcontaining cells and in Huh-7 cells transduced with Ad-NS5A [a recombinant adenovirus encoding NS5A (non-structural protein 5A)], coupled with a 4–5-fold increase in AP-1 (activator protein-1) DNA binding. Ava.1 cells, which encode a replication-defective HCV replicon, showed no significant changes in MnSOD, p38 MAPK or JNK activity. The AP-1 inhibitors dithiothreitol and N-acetylcysteine, as well as a dominant negative AP-1 mutant, significantly reduced AP-1 activation, demonstrating that this activation is oxidative stress-related. Exogenous NS5A had no effect on AP-1 activation in vitro, suggesting that NS5A acts at the upstream targets of AP-1 involving p38 MAPK and JNK signalling cascades. AP-1-dependent gene expression was increased in HCV subgenomic replicon-expressing Huh-7 cells. MnSOD activation was blocked by inhibitors of JNK (JNKI1) and p38 MAPK (SB203580), but not by an ERK (extracellular-signal-regulated kinase) inhibitor (U0126), in HCV-replicating and Ad-NS5A-transduced cells. Our results demonstrate that cellular responses to oxidative stress in HCV subgenomic replicon-expressing and Ad-NS5A-transduced cells are regulated by two distinct signalling pathways involving p38 MAPK and JNK via AP-1 that is linked to increased oxidative stress and therefore to an increased antioxidant MnSOD response.
Identification of an orphan guanylate cyclase receptor selectively expressed in mouse testis
Authors: KUHN Michaela , NG Chi Kin Domingos , SU Yueh-Hsing , KILIĆ Ana , MITKO Danuta , BIEN-LY Nga , KÖMÜVES László G. , YANG Ruey-Bing
Published: April 2004

We have identified a novel membrane form of guanylate cyclase (GC) from a mouse testis cDNA library and termed it mGC-G (mouse GC-G) based on its high sequence homology to rat GC-G. It encodes a potential type I transmembrane receptor, with the characteristic domain structure common to all members of the family of membrane GCs, including an extracellular, putative ligand-binding domain, a single membrane-spanning segment and cytoplasmic protein kinase-like and cyclase catalytic domains. Real-time quantitative reverse transcriptase–PCR and Northern-blot analyses showed that mGC-G is highly and selectively expressed in mouse testis. Phylogenetic analysis based on the extracellular protein sequence revealed that mGC-G is closely related to members of the subfamily of natriuretic peptide receptor GCs. When overexpressed in HEK-293T cells (human embryonic kidney 293T cells) or COS-7 cells, mGC-G manifests as a membrane-bound glycoprotein, which can form either homomeric or heteromeric complexes with the natriuretic peptide receptor GC-A. It exhibits marked cGMP-generating GC activity; however, notably, all ligands known to activate other receptor GCs failed to stimulate enzymic activity. The unique testis-enriched expression of mGC-G, which is completely different from the broader tissue distribution of rat GC-G, suggests the existence of as-yet-unidentified ligands and unappreciated species-specific physiological functions mediated through mGC-G/cGMP signalling in the testis.
AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes
Authors: MALBON Craig C. , TAO Jiangchuan , WANG Hsien-yu
Published: April 2004

Cell signalling mediated via GPCRs (G-protein-coupled receptors) is a major paradigm in biology, involving the assembly of receptors, G-proteins, effectors and downstream elements into complexes that approach in design ‘solid-state’ signalling devices. Scaffold molecules, such as the AKAPs (A-kinase anchoring proteins), were discovered more than a decade ago and represent dynamic platforms, enabling multivalent signalling. AKAP79 and AKAP250 were the first to be shown to bind to membrane-embedded GPCRs, orchestrating the interactions of various protein kinases (including tyrosine kinases), protein phosphatases (e.g. calcineurin) and cytoskeletal elements with at least one member of the superfamily of GPCRs, the prototypical β2-adrenergic receptor. In this review, the multivalent interactions of AKAP250 with the cell membrane, receptor, cytoskeleton and constituent components are detailed, providing a working model for AKAP-based GPCR signalling complexes. Dynamic regulation of the AKAP–receptor complex is mediated by ordered protein phosphorylation.
Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine
Authors: TOMSIG Jose Luis , SOHMA Hitoshi , CREUTZ Carl E.
Published: April 2004

The role of copines in regulating signalling from the TNF-α (tumour necrosis factor-α) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-κB (nuclear factor-κB) by TNF-α. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-α-dependent activation of NF-κB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-α was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-α signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-α-dependent degradation of IκB, a regulator of NF-κB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IκB through the ubiquitin ligase enzyme complex SCFβTrCP. Therefore the copine dominant-negative construct might inhibit TNF-α signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa.
Identification of a negative regulatory cis-element in the enhancer core region of the prostate-specific antigen promoter: implications for intersection of androgen receptor and nuclear factor-kappaB signalling in prostate cancer cells
Authors: CINAR Bekir , YEUNG Fan , KONAKA Hiroyuki , MAYO Marty W. , FREEMAN Michael R. , ZHAU Haiyen E. , CHUNG Leland W. K.
Published: April 2004

The NF-κB (nuclear factor-κB) transcription factors mediate activation of a large number of gene promoters containing diverse κB-site sequences. Here, PSA (prostate-specific antigen) was used as an AR (androgen receptor)-responsive gene to examine the underlying mechanism by which the NF-κB p65 transcription factor down-regulates the transcriptional activity of AR in cells. We observed that activation of NF-κB by TNFα (tumour necrosis factor α) inhibited both basal and androgen-stimulated PSA expression, and that this down-regulation occurred at the promoter level, as confirmed by the super-repressor IκBα (S32A/S36A), a dominant negative inhibitor of NF-κB. Using a linker-scanning mutagenesis approach, we identified a cis-element, designated XBE (X-factor-binding element), in the AREc (androgen response element enhancer core) of the PSA promoter, which negatively regulated several AR-responsive promoters, including that of PSA. When three copies of XBE in tandem were juxtaposed to GRE4 (glucocorticoid response element 4), a 4–6-fold reduction of inducible GRE4 activity was detected in three different cell lines, LNCaP, ARCaP-AR and PC3-AR. Bioinformatics and molecular biochemical studies indicated that XBE is a κB-like element that binds specifically to the NF-κB p65 subunit; consistent with these observations, only NF-κB p65, but not the NF-κB p50 subunit, was capable of inhibiting AR-mediated PSA promoter transactivation in LNCaP cells. In addition, our data also showed that AR binds to XBE, as well as to the κB consensus site, and that the transfection of AR inhibits the κB-responsive promoter in transient co-transfection assays. Collectively, these data indicate that cross-modulation between AR and NF-κB p65 transcription factors may occur by a novel mechanism involving binding to a common cis-DNA element.
Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution
Authors: AGRIMI G. , Di NOIA M. A. , MAROBBIO C. M. T. , FIERMONTE G. , LASORSA F. M. , PALMIERI F.
Published: April 2004

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.
The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis
Authors: WU Xueji , YANO Mihiro , WASHIDA Hiroyo , KIDO Hiroshi
Published: April 2004

The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP–ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403–414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis.
Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria
Authors: ESTEVES Telma C. , ECHTAY Karim S. , JONASSEN Tanya , CLARKE Catherine F. , BRAND Martin D.
Published: April 2004

Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609–613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526, 63–65]. In the present study, yeast (Saccharomyces cerevisiae) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wild-type yeast strain and two mutant strains (CENΔCOQ3 and CENΔCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria.
Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif
Authors: KIMPLE Randall J. , WILLARD Francis S. , HAINS Melinda D. , JONES Miller B. , NWEKE Gift K. , SIDEROVSKI David P.
Published: April 2004

GoLoco (‘Gαi/o–Loco’ interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein α subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Gα subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Gαi1 subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5´-[γ-thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Gαi1. Only the first and third GoLoco motifs within G18 are capable of interacting with Gα subunits, and these bind with low micromolar affinity only to Gαi1 in the GDP-bound form, and not to Gαo, Gαq, Gαs or Gα12. Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidic-glutamine-arginine tripeptide that forms critical contacts with Gα and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878–881], results in gain-of-function with respect to Gα binding and GDI activity.
N-Cyclohexanecarbonylpentadecylamine: a selective inhibitor of the acid amidase hydrolysing N-acylethanolamines, as a tool to distinguish acid amidase from fatty acid amide hydrolase
Authors: TSUBOI Kazuhito , HILLIGSMANN Christine , VANDEVOORDE Séverine , LAMBERT Didier M. , UEDA Natsuo
Published: April 2004

Anandamide (N-arachidonoylethanolamine) and other bioactive N-acylethanolamines are degraded to their corresponding fatty acids and ethanolamine. This hydrolysis is mostly attributed to catalysis by FAAH (fatty acid amide hydrolase), which exhibits an alkaline pH optimum. In addition, we have identified another amidase which catalyses the same reaction exclusively at acidic pH values [Ueda, Yamanaka and Yamamoto (2001) J. Biol. Chem. 276, 35552–35557]. In attempts to find selective inhibitors of this acid amidase, we screened various derivatives of palmitic acid, 1-hexadecanol, and 1-pentadecylamine with N-palmitoylethanolamine as substrate. Here we show that N-cyclohexanecarbonylpentadecylamine inhibits the acid amidase from rat lung with an IC50 of 4.5 µM, without inhibiting FAAH at concentrations up to 100 µM. The inhibition was reversible and non-competitive. This compound also inhibited the acid amidase in intact alveolar macrophages. With the aid of this inhibitor, it was revealed that rat basophilic leukaemia cells possess the acid amidase as well as FAAH. Thus the inhibitor may be a useful tool to distinguish the acid amidase from FAAH in various tissues and cells and to elucidate the physiological role of the enzyme.
Characterization of iron binding in IscA, an ancient iron-sulphur cluster assembly protein
Authors: DING Huangen , CLARK Robert J.
Published: April 2004

Iron–sulphur clusters are one of the most common types of redox centre in biology. At least six proteins (IscS, IscU, IscA, HscB, HscA and ferredoxin) have been identified as being essential for the biogenesis of iron–sulphur proteins in bacteria. It has been shown that IscS is a cysteine desulphurase that provides sulphur for iron–sulphur clusters, and that IscU is a scaffold for the IscS-mediated assembly of iron–sulphur clusters. The iron donor for iron–sulphur clusters, however, remains elusive. Here we show that IscA is an iron binding protein with an apparent iron association constant of 3.0×1019 M−1, and that iron-loaded IscA can provide iron for the assembly of transient iron–sulphur clusters in IscU in the presence of IscS and l-cysteine in vitro. The results suggest that IscA is capable of recruiting intracellular iron and delivering iron for iron–sulphur clusters in proteins.
The inhibitor ABIN-2 disrupts the interaction of receptor-interacting protein with the kinase subunit IKKgamma to block activation of the transcription factor NF-kappaB and potentiate apoptosis
Authors: LIU Wei-Kuang , YEN Pei-Fen , CHIEN Chia-Yi , FANN Ming-Ji , SU Jin-Yuan , CHOU Chen-Kung
Published: April 2004

NF-κB (nuclear factor κB) proteins are key transcription factors that regulate gene expression in response to various extracellular stimuli. The pathway leading to the activation of NF-κB involves a complicated network that includes a number of signalling molecules. The recent identification of a wide range of negative regulators of NF-κB has given another layer of complexity in NF-κB activation. We and others have previously identified the protein ABIN-2 (A20 binding inhibitor of NF-κB 2) as an inhibitor of NF-κB activation. In the present paper, we demonstrate that ABIN-2 exerts its inhibitory function by blocking the interaction of RIP (receptor-interacting protein) with the downstream effector IKKγ, a non-kinase component of the IκB (inhibitory κB) kinase complex. When overexpressed in cells, ABIN-2 bound to IKKγ and prevented the association of IKKγ with RIP. By a deletion mapping, a stretch of 50 amino acids on ABIN-2 is found to be essential for its interaction with IKKγ. The ABIN-2 mutant that lacked these 50 amino acids did not interact with IKKγ and, consequently, failed to inhibit NF-κB activation. Strikingly, a portion of RIP, which is similar to this 50-residue domain of ABIN-2, is also essential for RIP interaction with IKKγ. The RIP mutant with deletion of this similar region did not associate with IKKγ and had substantial reduction of its ability to mediate NF-κB activation. Taken together, these conserved 50 residues of ABIN-2 and RIP define a novel structural domain in mediating a key step in the NF-κB signalling pathway through the interaction with IKKγ. Finally, the signalling pathway of NF-κB activation is known to promote survival in many cellular events. The mechanism for decision between cell death and survival is under fine regulation. In the present paper, we demonstrated further that the expression of ABIN-2 could promote the RIP-mediated apoptosis by presumably suppressing the anti-apoptotic effect of NF-κB.
The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase
Authors: EGGERS Christopher T. , MURRAY Iain A. , DELMAR Valerie A. , DAY Anthony G. , CRAIK Charles S.
Published: April 2004

Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 µM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.
14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking
Authors: RUBIO Mercedes POZUELO , GERAGHTY Kathryn M. , WONG Barry H. C. , WOOD Nicola T. , CAMPBELL David G. , MORRICE Nick , MACKINTOSH Carol
Published: April 2004

14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3–Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-d-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.
Relevance of NAC-2, an Na+-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans
Authors: FEI You-Jun , LIU Jin-Cai , INOUE Katsuhisa , ZHUANG Lina , MIYAKE Katsuya , MIYAUCHI Seiji , GANAPATHY Vadivel
Published: April 2004

We have cloned and functionally characterized an Na+-coupled citrate transporter from Caenorhabditis elegans (ceNAC-2). This transporter shows significant sequence homology to Drosophila Indy and the mammalian Na+-coupled citrate transporter NaCT (now known as NaC2). When heterologously expressed in a mammalian cell line or in Xenopus oocytes, the cloned ceNAC-2 mediates the Na+-coupled transport of various intermediates of the citric acid cycle. However, it transports the tricarboxylate citrate more efficiently than dicarboxylates such as succinate, a feature different from that of ceNAC-1 (formerly known as ceNaDC1) and ceNAC-3 (formerly known as ceNaDC2). The transport process is electrogenic, as evidenced from the substrate-induced inward currents in oocytes expressing the transporter under voltage-clamp conditions. Expression studies using a reporter-gene fusion method in transgenic C. elegans show that the gene is expressed in the intestinal tract, the organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Functional knockdown of the transporter by RNAi (RNA interference) not only leads to a significant increase in life span, but also causes a significant decrease in body size and fat content. The substrates of ceNAC-2 play a critical role in metabolic energy production and in the biosynthesis of cholesterol and fatty acids. The present studies suggest that the knockdown of these metabolic functions by RNAi is linked to an extension of life span and a decrease in fat content and body size.
Evolutionary families of peptidase inhibitors
Authors: RAWLINGS Neil D. , TOLLE Dominic P. , BARRETT Alan J.
Published: April 2004

The proteins that inhibit peptidases are of great importance in medicine and biotechnology, but there has never been a comprehensive system of classification for them. Some of the terminology currently in use is potentially confusing. In the hope of facilitating the exchange, storage and retrieval of information about this important group of proteins, we now describe a system wherein the inhibitor units of the peptidase inhibitors are assigned to 48 families on the basis of similarities detectable at the level of amino acid sequence. Then, on the basis of three-dimensional structures, 31 of the families are assigned to 26 clans. A simple system of nomenclature is introduced for reference to each clan, family and inhibitor. We briefly discuss the specificities and mechanisms of the interactions of the inhibitors in the various families with their target enzymes. The system of families and clans of inhibitors described has been implemented in the MEROPS peptidase database (http://merops.sanger.ac.uk/), and this will provide a mechanism for updating it as new information becomes available.