bluuebunny/crossref_metadata_2025 huggingface dataset:
Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I
Authors: KITZMÜLLER Claudia , CAPRINI Andrea , MOORE Stuart E. H. , FRÉNOY Jean-Pierre , SCHWAIGER Eva , KELLERMANN Odile , IVESSA N. Erwin , ERMONVAL Myriam
Published: December 2003

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI332, which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive α1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI332 in the ERAD pathway. In a first step, the Man8 isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI332 intermediates. Some other trimmed N-glycan species, in particular Glc1Man7GlcNAc2, were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI332 accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.
Ceramide and glutathione define two independently regulated pathways of cell death initiated by p53 in Molt-4 leukaemia cells
Authors: EL-ASSAAD Wissal , KOZHAYA Lina , ARAYSI Sawsan , PANJARIAN Shoghag , BITAR Fadi F. , BAZ Elizabeth , EL-SABBAN Marwan E. , DBAIBO Ghassan S.
Published: December 2003

The tumour suppressor p53 induces cell death by launching several pathways that are either dependent on or independent of gene transcription. Accumulation of the sphingolipid ceramide and reactive oxygen species are among these pathways. Crossregulation of these two pathways is possible owing to the demonstrated inhibition of neutral sphingomyelinase by glutathione, the predominant cellular antioxidant, and has been observed in some cytokine-dependent cell-death models. In a model of irradiation-induced cell death of Molt-4 leukaemia cells, it was found that ceramide accumulation and glutathione depletion were dependent on p53 up-regulation. The loss of p53 owing to expression of the papilloma virus E6 protein inhibited both pathways after irradiation. However, in this model, these two pathways appeared to be independently regulated on the basis of the following observations: (1) glutathione supplementation or depletion did not alter irradiation-induced ceramide accumulation, (2) exogenous ceramide treatment did not induce glutathione depletion, (3) glutathione depletion was dependent on new protein synthesis, whereas ceramide accumulation was independent of it and (4) caspase activation was required for ceramide accumulation but not for glutathione depletion. Furthermore, caspase 9 activation, which is dependent on the release of mitochondrial cytochrome c, was not required for ceramide accumulation. This suggested that a caspase, other than caspase 9, was necessary for ceramide accumulation. Interestingly, Bcl-2 expression inhibited these pathways, indicating a possible role for mitochondria in regulating both pathways. These findings indicate that these two pathways exhibit cross-regulation in cytokine-dependent, but not in p53-dependent, cell-death models.
Galactose-specific asialoglycoprotein receptor is involved in lipoprotein (a) catabolism
Authors: HRZENJAK Andelko , FRANK Sasa , WO Xingde , ZHOU Yonggang , van BERKEL Theo , KOSTNER Gert M.
Published: December 2003

Lp(a) [lipoprotein (a)] is a highly atherogenic plasma lipoprotein assembled from low-density lipoprotein and the glycoprotein apolipoprotein (a). The rate of Lp(a) biosynthesis correlates significantly with plasma Lp(a) concentrations, whereas the fractional catabolic rate does not have much influence. So far, little is known about Lp(a) catabolism. To study the site and mode of Lp(a) catabolism, native or sialidase-treated Lp(a) was injected into hedgehogs or ASGPR (asialoglycoprotein receptor)-knockout (ASGPR−) mice or wild-type (ASGPR+) mice, and the decay of the plasma Lp(a) concentration was followed. COS-7 cells were transfected with high- (HL-1) and low-molecular-mass ASGPR subunits (HL-2), and binding and degradation of intact or desialylated Lp(a) were measured. In hedgehogs, one of the few species that synthesize Lp(a), most of the Lp(a) was taken up by the liver, followed by kidney and spleen. Lp(a) and asialo-Lp(a) were catabolized with apparent half-lives of 13.8 and 0.55 h respectively. Asialo-orosomucoide increased both half-lives significantly. In mice, the apparent half-life of Lp(a) was 4–6 h. Catabolism of native Lp(a) by wild-type mice was significantly faster compared with ASGPR− mice and there was a significantly greater accumulation of Lp(a) in the liver of ASGPR+ mice compared with ASGPR− mice. The catabolism of asialo-Lp(a) in ASGPR− mice was 8-fold faster when compared with native Lp(a) in wild-type mice. Transfected COS-7 cells expressing functional ASGPR showed approx. 5-fold greater binding and 2-fold faster degradation of native Lp(a) compared with control cells. Our results for the first time demonstrate a physiological function of ASGPR in the catabolism of Lp(a).
Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6′-O-fluorescein-atractyloside
Authors: SMITH Vernon R. , FEARNLEY Ian M. , WALKER John E.
Published: December 2003

Atractyloside (ATR) is a high-affinity specific inhibitor of the mitochondrial ADP/ATP translocase (AAT). The binding of a fluorescent derivative, 6´-O-fluorescein-ATR (FATR), to mitochondria has been characterized. The binding constants obtained are in agreement with previously published values for ATR, demonstrating that FATR is a suitable probe of the AAT. AAT inhibited by FATR (FATR-AAT) was solubilized in dodecyl maltoside and purified by two separate ion-exchange chromatography steps at different pHs, which allowed FATR-AAT to be purified to homogeneity. The presence of the bound fluorescent probe enabled the inhibited AAT to be distinguished from the unliganded protein during chromatography, as they were markedly different in their chromatographic behaviour. The purified FATR-AAT was dimeric and in a single major conformation containing 1 mole FATR per mole of AAT dimer. In contrast, uninhibited AAT was monomeric and conformationally unstable. Use of the fluorescent ATR derivative in the development of the protocol enabled the stable dimeric AAT to be monitored directly and purified more effectively. The purification protocol was repeated using non-derivatized ATR, and highly pure AAT was obtained that was devoid of other members of the mitochondrial carrier family.
Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage
Authors: JACQUES Suzanne L. , KULIOPULOS Athan
Published: December 2003

Thrombin activation of human platelets is mediated by the high-affinity PAR1 (protease-activated receptor-1) and the low-affinity PAR4 receptor. PAR1 and PAR4 exhibit markedly disparate kinetics of activation that likely reflect differences in the macromolecular association of thrombin with their respective N-terminal extracellular domains (exodomains). Here we examine the mechanism of initial thrombin binding and cleavage of the high- and low-affinity PAR exodomains using steady-state kinetic analyses. We showed that the PAR4 exodomain lacks the functional hirudin-like sequence found in PAR1 and does not bind exosite I to cause allosteric activation or inhibition of thrombin. Instead, PAR4 contains an anionic cluster, Asp57…Asp59…Glu62…Asp65 (DDED), in its exodomain, which slows the dissociation of PAR4 from the cationic thrombin. The analogous anionic residues in the PAR1 exodomain do not influence affinity for thrombin. Although PAR4 is cleaved more slowly than PAR1 on the cell surface, peptides containing the PAR4 P4-P1 active-site-interacting sequence, Pro45-Ala-Pro-Arg (PAPR), are efficiently cleaved due to the optimal placement of dual prolines at positions P4 and P2. In comparison, thrombin has low affinity and slow cleavage rates for peptides that have a P3 proline as occurs in human PAR3. Thus, to compensate for the lack of exosite I binding, PAR4 utilizes proline residues in its P4-P1 sequence to provide high-affinity interactions with the active site and an anionic cluster to slow dissociation from the cationic thrombin.
Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus
Authors: LI Juan , YAO Yong-Neng , LIU Mo-Fang , WANG En-Duo
Published: December 2003

ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNAArgs with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS (argS´) was amplified by PCR and the gene encoding tRNAArg (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNAArg (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNAArgs from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys382 and Lys385 of the KMSK sequence and at Gly136 upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys385 and Gly136 for the amino acid-activation activity of B. stearothermophilus ArgRS.
Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates
Authors: MALLÉA Monique , MAHAMOUD Abdallah , CHEVALIER Jacqueline , ALIBERT-FRANCO Sandrine , BROUANT Pierre , BARBE Jacques , PAGÈS Jean-Marie
Published: December 2003

Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections. Several Gram-negative bacteria efflux pumps have been described. These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens. Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria. In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters. We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria. Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-[2´-(piperidino)ethyl]aminoquinoline acts as an inhibitor of the AcrAB–TolC efflux pump and restores a high level of intracellular drug concentration. Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes.
Proton conductance and fatty acyl composition of liver mitochondria correlates with body mass in birds
Authors: BRAND Martin D. , TURNER Nigel , OCLOO Augustine , ELSE Paul L. , HULBERT A. J.
Published: December 2003

The proton conductance of isolated liver mitochondria correlates significantly with body mass in mammals, but not in ectotherms. To establish whether the correlation in mammals is general for endotherms or mammal-specific, we measured proton conductance in mitochondria from birds, the other main group of endotherms, using birds varying in mass over a wide range (nearly 3000-fold), from 13 g zebra finches to 35 kg emus. Respiratory control ratios were higher in mitochondria from larger birds. Mitochondrial proton conductance in liver mitochondria from birds correlated strongly with body mass [respiration rate per mg of protein driving proton leak at 170 mV being 44.7 times (body mass in g)−0.19], thus suggesting a general relationship between body mass and proton conductance in endotherms. Mitochondria from larger birds had the same or perhaps greater surface area per mg of protein than mitochondria from smaller birds. Hence, the lower proton conductance was caused not by surface area changes but by some change in the properties of the inner membrane. Liver mitochondria from larger birds had phospholipid fatty acyl chains that were less polyunsaturated and more monounsaturated when compared with those from smaller birds. Phospholipid fatty acyl polyunsaturation correlated positively and monounsaturation correlated negatively with proton conductance. These correlations echo those seen in mammalian liver mitochondria, suggesting that they too are general for endotherms.
Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multidrug-resistance-associated protein 1: evidence for interaction between ATP and Trp653
Authors: RAMAEN Odile , MASSCHELEYN Sandrine , DUFFIEUX Francis , PAMLARD Olivier , OBERKAMPF Marine , LALLEMAND Jean-Yves , STOVEN Véronique , JACQUET Eric
Published: December 2003

Multidrug-resistance-associated protein 1 (MRP1/ABCC1) is a human ATP-binding cassette transporter that confers cell resistance to antitumour drugs. Its NBDs (nucleotide-binding domains) bind/hydrolyse ATP, a key step in the activation of MRP1 function. To relate its intrinsic functional features to the mechanism of action of the full-size transporter, we expressed the N-terminal NBD1 domain (Asn642 to Ser871) in Escherichia coli. NBD1 was highly purified under native conditions and was characterized as a soluble monomer. 15N-labelling allowed recording of the first two-dimensional NMR spectra of this domain. The NMR study showed that NBD1 was folded, and that Trp653 was a key residue in the NBD1–ATP interaction. Thus, interaction of NBD1 with ATP/ADP was studied by intrinsic tryptophan fluorescence. The affinity for ATP and ADP were in the same range (Kd(ATP)=118 µM and Kd(ADP)=139 µM). Binding of nucleotides did not influence the monomeric state of NBD1. The ATPase activity of NBD1 was magnesium-dependent and very low [Vmax and Km values of 5×10−5 pmol of ATP·(pmol NBD1)−1·s−1 and 833 µM ATP respectively]. The present study suggests that NBD1 has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1–NBD2 heterodimer formation.
The PAX6 gene is activated by the basic helix–loop–helix transcription factor NeuroD/BETA2
Authors: MARSICH Eleonora , VETERE Amedeo , DI PIAZZA Matteo , TELL Gianluca , PAOLETTI Sergio
Published: December 2003

PAX6 is a transcription factor that plays an important role during pancreatic morphogenesis. The aim of the present study is to identify the upstream activator(s) of the PAX6 gene possibly involved in the early stages of pancreatic differentiation. Recently, individual elements regulating PAX6 gene activity in the pancreas have been identified in a 1100 bp Spe/HincII fragment 4.6 kb upstream of exon 0. Preliminary sequence analysis of this region revealed some potential DNA-binding sites (E boxes) specific for the binding of basic helix–loop–helix transcription factors. By using electrophoretic mobility shift assays, we demonstrated that both nuclear protein extracts from insulin-secreting RINm5F cells and in vitro-translated NeuroD/BETA2 can bind specifically to these E boxes. Furthermore, by transient transfection experiments we demonstrated that the expression of basic helix–loop–helix transcription factor NeuroD/BETA2 can induce activation of the PAX6 promoter in the NIH-3T3 cell line. Thus we show that NeuroD/BETA2 is involved in the activation of the expression of PAX6 through E boxes in the PAX6 promoter localized in a 1.1 kb sequence within the 4.6 kb untranslated region upstream of exon 0.
Accelerated ubiquitination and proteasome degradation of a genetic variant of inducible nitric oxide synthase
Authors: YING Wei-Zhong , SANDERS Paul W.
Published: December 2003

Biochemical and pharmacological studies have suggested that NOS2 (inducible nitric oxide synthase) has a functional role in the blood pressure response to increases in dietary salt intake. On a high-salt diet, the Dahl/Rapp salt-sensitive (S) strain of rat, a genetic model of salt-sensitive hypertension, did not show increased nitric oxide production. NOS2 from S rats possesses a point mutation that results in substitution of proline for serine at position 714. In the present study, rat NOS2 was shown to be ubiquitinated in vitro and in vivo and to be degraded by the proteasome; this process was accelerated for the S714P mutant. Accelerated degradation of the S714P mutant enzyme accounted for the diminished enzyme activity of this mutant. Hsp90 (heat-shock protein 90) associated with NOS2 and modulated degradation, but was not responsible for the accentuated degradation of the S714P mutant enzyme. The combined findings demonstrate the integral role of ubiquitination and degradation by the proteasome in the regulation of NO production by rat NOS2. Demonstrating that this process is responsible for the abnormal function of the S714P mutant NOS2 in S rats confirms the physiological importance of the proteasome in NOS2 function.
The tomato lectin consists of two homologous chitin-binding modules separated by an extensin-like linker
Authors: PEUMANS Willy J. , ROUGÉ Pierre , van DAMME Els J. M.
Published: December 2003

A cDNA encoding a putative lectin expressed in tomato leaves was identified and analysed. The lectin consists of two homologous chitin-binding modules interconnected by a short proline-rich domain containing a single Ser[Pro]n repetitive motif. Each module comprises two in-tandem-arrayed hevein domains separated by a tetrapeptide linker. Besides the chitin-binding modules and proline-rich domain, the lectin contains two short unrelated domains located at the N- and C-termini of the protein respectively. Eventual elucidation of the molecular structure of the tomato lectin confirms the presumed chimaeric nature of the Solanaceae lectins but also indicates that all previously proposed models need to be revised.
Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells
Authors: BÄCKSTRÖM Malin , LINK Thomas , OLSON Fredrik J. , KARLSSON Hasse , GRAHAM Rosalind , PICCO Gianfranco , BURCHELL Joy , TAYLOR-PAPADIMITRIOU Joyce , NOLL Thomas , HANSSON Gunnar C.
Published: December 2003

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1–IgG-producing CHO-K1 clones were generated and were found to secrete MUC1–IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galβ1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1–IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1–IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.
Bioinformatic analysis of the nucleolus
Authors: LEUNG Anthony K. L. , ANDERSEN Jens S. , MANN Matthias , LAMOND Angus I.
Published: December 2003

The nucleolus is a plurifunctional, nuclear organelle, which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication and tumour suppression. Our knowledge of the human nucleolar proteome has been expanded dramatically by the two recent MS studies on isolated nucleoli from HeLa cells [Andersen, Lyon, Fox, Leung, Lam, Steen, Mann and Lamond (2002) Curr. Biol. 12, 1–11; Scherl, Coute, Deon, Calle, Kindbeiter, Sanchez, Greco, Hochstrasser and Diaz (2002) Mol. Biol. Cell 13, 4100–4109]. Nearly 400 proteins were identified within the nucleolar proteome so far in humans. Approx. 12% of the identified proteins were previously shown to be nucleolar in human cells and, as expected, nearly all of the known housekeeping proteins required for ribosome biogenesis were identified in these analyses. Surprisingly, approx. 30% represented either novel or uncharacterized proteins. This review focuses on how to apply the derived knowledge of this newly recognized nucleolar proteome, such as their amino acid/peptide composition and their homologies across species, to explore the function and dynamics of the nucleolus, and suggests ways to identify, in silico, possible functions of the novel/uncharacterized proteins and potential interaction networks within the human nucleolus, or between the nucleolus and other nuclear organelles, by drawing resources from the public domain.
Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris
Authors: SOARES-SILVA Isabel , SCHULLER Dorit , ANDRADE Raquel P. , BALTAZAR Fátima , CÁSSIO Fernanda , CASAL Margarida
Published: December 2003

In Saccharomyces cerevisiae the activity for the lactate–proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a Vmax of 2.1 nmol·s−1·mg of dry weight−1. Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest Vmax (0.84 nmol·s−1·mg of dry weight−1) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.
Characterization of a novel binding partner of the melanocortin-4 receptor: attractin-like protein
Authors: HAQQ Andrea M. , RENÉ Patricia , KISHI Toshiro , KHONG Kathy , LEE Charlotte E. , LIU Hongyan , FRIEDMAN Jeffrey M. , ELMQUIST Joel K. , CONE Roger D.
Published: December 2003

The gene dosage effect of the MC4-R (melanocortin 4 receptor) on obesity suggests that regulation of MC4-R expression and function is critically important to the central control of energy homoeostasis. In order to identify putative MC4-R regulatory proteins, we performed a yeast two-hybrid screen of a mouse brain cDNA library using the mouse MC4-R intracellular tail (residues 303–332) as bait. We report here on one positive clone that shares 63% amino acid identity with the C-terminal part of the mouse attractin gene product, a single-transmembrane-domain protein characterized as being required for agouti signalling through the melanocortin 1 receptor. We confirmed a direct interaction between this ALP (attractin-like protein) and the C-terminus of the mouse MC4-R by glutathione S-transferase pulldown experiments, and mapped the regions involved in this interaction using N- and C-terminal truncation constructs; residues 303–313 in MC4-R and residues 1280–1317 in ALP are required for binding. ALP is highly expressed in brain, but also in heart, lung, kidney and liver. Furthermore, co-localization analyses in mice showed co-expression of ALP in cells expressing MC4-R in a number of regions known to be important in the regulation of energy homoeostasis by melanocortins, such as the paraventricular nucleus of hypothalamus and the dorsal motor nucleus of the vagus.
Analysis of the role of protein kinase B (cAKT) in insulin-dependent induction of glucokinase and sterol regulatory element-binding protein 1 (SREBP1) mRNAs in hepatocytes
Authors: RIBAUX Pascale G. , IYNEDJIAN Patrick B.
Published: December 2003

Previous work showed that acute stimulation of a conditionally active protein kinase B (PKB or cAKT) was sufficient to elicit insulin-like induction of GCK (glucokinase) and SREBP1 (sterol regulatory element-binding protein 1) in hepatocytes [Iynedjian, Roth, Fleischmann and Gjinovci (2000) Biochem. J. 351, 621–627; Fleischmann and Iynedjian (2000) Biochem. J. 349, 13–17]. The objective of the present study was to determine whether activation of PKB during insulin stimulation of hepatocytes was a necessary condition for the induction of the two genes. Activation of PKB by insulin was inhibited by pretreatment of the hepatocytes with C2 ceramide. This resulted in the inhibition of insulin-dependent increases in GCK and SREBP1 mRNAs. A triple mutant of PKB failed to interfere with insulin activation of PKB in hepatocytes even at high overexpression levels achieved after adenovirus transduction. A PKB–CaaX fusion protein, which can act as a dominant-negative inhibitor of PKB activation in other cells, was shown to be constitutively activated in hepatocytes and to trigger insulin-like induction of GCK and SREBP1. In addition, constitutive PKB–CaaX activity caused refractoriness of the hepatocytes to insulin signalling at an upstream step resulting in the inhibition of both extracellular-signal-regulated kinase 1/2 and endogenous PKB activation. The stimulation of gene expression by constitutively active PKB–CaaX and inhibition of the insulin effect by ceramide are compatible with a role for PKB in the insulin-dependent induction of GCK and SREBP1.
Emerging and diverse roles of protein kinase C in immune cell signalling
Authors: TAN Seng-Lai , PARKER Peter J.
Published: December 2003

Members of the protein kinase C (PKC) family are expressed in many different cell types, where they are known to regulate a wide variety of cellular processes that impact on cell growth and differentiation, cytoskeletal remodelling and gene expression in the response to diverse stimuli. The broad tissue distribution and redundancy of in vitro function have often hampered the identification of definitive roles for each PKC family member. However, recent in vivo studies of PKC isoenzyme-selective knockout and transgenic mice have highlighted distinct functions of individual PKCs in the immune system. These genetic analyses, along with biochemical studies utilizing PKC isoenzyme-specific cDNA (wild-type, constitutively active and dominant-negative), antisense oligonucleotides (ASO), RNA interference (RNAi), and pharmacological inhibitors, indicate that PKC-regulated signalling pathways play a significant role in many aspects of immune responses, from development, differentiation, activation and survival of lymphocytes to macrophage activation. The importance of PKCs in cellular immune responses suggests that improved understanding of the molecular events that govern their actions could point to new avenues for development of treatments for immune disorders.
Ras activation in response to lysophosphatidic acid requires a permissive input from the epidermal growth factor receptor
Authors: RUBIO Ignacio , RENNERT Knut , WITTIG Ute , WETZKER Reinhard
Published: December 2003

The topology of the signalling pathway linking the G-protein-coupled receptor agonist lysophosphatidic acid (LPA) to extracellular-signal-regulated kinase activation remains undeciphered. In the present study, we report that analysis of LPA signals at the level of Ras-GTP formation and Ras nucleotide exchange discriminates true mediatory signals from permissive activities that do not participate in signal relay. Hence, whereas pertussis toxin (PTX) treatment impairs stimulation of nucleotide exchange, epidermal growth factor receptor (EGFR) inhibition does not compromise LPA-induced acceleration of nucleotide exchange, but instead attenuates basal nucleotide turnover on Ras. Our data indicate that LPA activation of Ras proceeds via PTX-sensitive Gi/o-proteins and requires a permissive input from basal EGFR activity.
Evidence for the transport of glutathione through ryanodine receptor channel type 1
Authors: BÁNHEGYI Gábor , CSALA Miklós , NAGY Gábor , SORRENTINO Vincenzo , FULCERI Rosella , BENEDETTI Angelo
Published: December 2003

In the present study, we have investigated the role of RyR1 (ryanodine receptor calcium channel type 1) in glutathione (GSH) transport through the sarcoplasmic reticulum (SR) membrane of skeletal muscles. Lanthanum chloride, a prototypic blocker of cation channels, inhibited the influx and efflux of GSH in SR vesicles. Using a rapid-filtration-based assay and lanthanum chloride as a transport blocker, an uptake of radiolabelled GSH into SR vesicles was observed. Pretreatment of SR vesicles with the RyR1 antagonists Ruthenium Red and ryanodine as well as with lanthanum chloride blocked the GSH uptake. An SR-like GSH uptake appeared in microsomes obtained from an HEK-293 (human embryonic kidney 293) cell line after transfection of RyR1. These observations strongly suggest that RyR1 mediates GSH transport through the SR membranes of skeletal muscles.
Attractin' more attention – new pieces in the obesity puzzle?
Authors: YEO Giles S. H. , SIDDLE Kenneth
Published: December 2003

Genetic, biochemical and pharmacological studies in humans and rodents have established that signalling through the G-protein-coupled melanocortin-4 receptor (MC4R) by pro-opiomelanocortin (POMC)-derived ligands plays a critical role in the central suppression of appetite. As a consequence, malfunction of this signalling system leads to the development of obesity. It has been shown previously that melanocortin signalling can be modulated by the type 1 transmembrane protein attractin, apparently acting as a co-receptor for the inhibitory ligand agouti. Work reported in this issue of Biochemical Journal (Haqq et al.) demonstrates that the cytosolic tail of an attractin-like protein (ALP) binds directly and specifically to the C-terminal region of MC4R, raising the possibility that proteins of the attractin family influence melanocortin receptor function through multiple mechanisms.
Role of receptor tyrosine kinases in G-protein-coupled receptor regulation of Ras: transactivation or parallel pathways?
Authors: DOWNWARD Julian
Published: December 2003

Ras protein regulation by G-protein-coupled receptors has been thought to occur through transactivation of receptor tyrosine kinases. New evidence suggests that these two receptor types independently control different pathways leading to Ras activation in response to lysophosphatidic acid (LPA). Epidermal growth factor receptor function is needed for basal nucleotide exchange on Ras, whereas the LPA receptor controls an inducible exchange activity.
Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice
Authors: Moores Carolyn A. , Hekmat-Nejad Mohammad , Sakowicz Roman , Milligan Ronald A.
Published: December 2003

KinI kinesins are important in regulating the complex dynamics of the microtubule cytoskeleton. They are unusual in that they depolymerize, rather than move along microtubules. To determine the attributes of KinIs that distinguish them from translocating kinesins, we examined the ATPase activity, microtubule affinity, and three-dimensional microtubule-bound structure of a minimal KinI motor domain. Together, the kinetic, affinity, and structural data lead to the conclusion that on binding to the microtubule lattice, KinIs release ADP and enter a stable, low-affinity, regulated state, from which they do not readily progress through the ATPase cycle. This state may favor detachment, or diffusion of the KinI to its site of action, the microtubule ends. Unlike conventional translocating kinesins, which are microtubule lattice–stimulated ATPases, it seems that with KinIs, nucleotide-mediated modulation of tubulin affinity is only possible when it is coupled to protofilament deformation. This provides an elegant mechanistic basis for their unique depolymerizing activity.
The ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with Gβγ
Authors: N'Diaye Elsa-Noah , Brown Eric J.
Published: December 2003

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1α and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1α–induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1α receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gβγ-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gβγ function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gβγ in pull downs. Interaction with Gβγ did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1α activation of phospholipase C, indicating that PLIC-1's inhibition of Gβγ did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gβγ; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration.
IGF-II transcription in skeletal myogenesis is controlled by mTOR and nutrients
Authors: Erbay Ebru , Park In-Hyun , Nuzzi Paul D. , Schoenherr Christopher J. , Chen Jie
Published: December 2003

Insulin-like growth factors (IGFs) are essential for skeletal muscle development, regeneration, and hypertrophy. Although autocrine actions of IGF-II are known to initiate myoblast differentiation, the regulatory elements and upstream signaling pathways for myogenic expression of IGF-II remain elusive. Here, we report the regulation of IGF-II transcription by mTOR, as well as by amino acid sufficiency, through the IGF-II promoter 3 and a downstream enhancer during C2C12 myoblast differentiation. Furthermore, we present evidence that IGF production, and not IGF signaling, is the primary target for mTOR's function in the initiation of differentiation. Moreover, myogenic signaling by mTOR is independent of its kinase activity and mediated by the PI3K–Akt pathway. Our findings represent the first identification of a signaling pathway that regulates IGF-II expression in myogenesis and implicate the mTOR–IGF axis as a molecular link between nutritional levels and skeletal muscle development.
Meiotic condensin is required for proper chromosome compaction, SC assembly, and resolution of recombination-dependent chromosome linkages
Authors: Yu Hong-Guo , Koshland Douglas E.
Published: December 2003

Condensin is an evolutionarily conserved protein complex that helps mediate chromosome condensation and segregation in mitotic cells. Here, we show that condensin has two activities that contribute to meiotic chromosome condensation in Saccharomyces cerevisiae. One activity, common to mitosis, helps mediate axial length compaction. A second activity promotes chromosome individualization with the help of Red1 and Hop1, two meiotic specific components of axial elements. Like Red1 and Hop1, condensin is also required for efficient homologue pairing and proper processing of double strand breaks. Consistent with these functional links condensin is necessary for proper chromosomal localization of Red1 and Hop1 and the subsequent assembly of the synaptonemal complex. Finally, condensin has a Red1/Hop1-independent role in the resolution of recombination-dependent linkages between homologues in meiosis I. The existence of distinct meiotic activities of condensin (axial compaction, individualization, and resolution of recombination-dependent links) provides an important framework to understand condensin's role in both meiotic and mitotic chromosome structure and function.
Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells
Authors: Hoshino Mitsunobu , Nakamura Shun
Published: December 2003

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal–binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin–CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.
Yeast homotypic vacuole fusion requires the Ccz1–Mon1 complex during the tethering/docking stage
Authors: Wang Chao-Wen , Stromhaug Per E. , Kauffman Emily J. , Weisman Lois S. , Klionsky Daniel J.
Published: December 2003

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1–Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1–Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1–Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1–Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.
The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate
Authors: Rao Mala V. , Campbell Jabbar , Yuan Aidong , Kumar Asok , Gotow Takahiro , Uchiyama Yasuo , Nixon Ralph A.
Published: December 2003

The phosphorylated carboxyl-terminal “tail” domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681–693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail–deleted (NF-MtailΔ) mutant mice using an embryonic stem cell–mediated “gene knockin” approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailΔ mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail–mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.
NF-M is an essential target for the myelin-directed “outside-in” signaling cascade that mediates radial axonal growth
Authors: Garcia Michael L. , Lobsiger Christian S. , Shah Sameer B. , Deerinck Tom J. , Crum John , Young Darren , Ward Christopher M. , Crawford Thomas O. , Gotow Takahiro , Uchiyama Yasuo , Ellisman Mark H. , Calcutt Nigel A. , Cleveland Don W.
Published: December 2003

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an “outside-in” signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.
Reversed polarized delivery of an aquaporin-2 mutant causes dominant nephrogenic diabetes insipidus
Authors: Kamsteeg Erik-Jan , Bichet Daniel G. , Konings Irene B.M. , Nivet Hubert , Lonergan Michelle , Arthus Marie-Françoise , van Os Carel H. , Deen Peter M.T.
Published: December 2003

Vasopressin regulates body water conservation by redistributing aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical surface of renal collecting ducts, resulting in water reabsorption from urine. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine. Here, we report a frame-shift mutation in AQP2 causing dominant NDI. This AQP2 mutant is a functional water channel when expressed in Xenopus oocytes. However, expressed in polarized renal cells, it is misrouted to the basolateral instead of apical plasma membrane. Additionally, this mutant forms heterotetramers with wild-type AQP2 and redirects this complex to the basolateral surface. The frame shift induces a change in the COOH terminus of AQP2, creating both a leucine- and a tyrosine-based motif, which cause the reversed sorting of AQP2. Our data reveal a novel cellular phenotype in dominant NDI and show that dominance of basolateral sorting motifs in a mutant subunit can be the molecular basis for disease.
SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase
Authors: Marsh H. Nicholas , Dubreuil Catherine I. , Quevedo Celia , Lee Anna , Majdan Marta , Walsh Gregory S. , Hausdorff Sharon , Said Farid Arab , Zoueva Olga , Kozlowski Maya , Siminovitch Katherine , Neel Benjamin G. , Miller Freda D. , Kaplan David R.
Published: December 2003

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.