Volume 381, Issue 5-6

Traditionally, growth factor-coupled signaling to the nucleus has been thought to be primarily directed toward transcriptional regulation. However, there are now increasing indications from a diversity of experimental systems that other aspects of RNA processing, including translation, lifetime and stability, and splicing are under strict growth factor control. In this review, we present the emerging evidence for growth factor signaling pathways that impact on these different RNA processing events. Particularly noteworthy is the realization that growth factor signaling through Ras can effect the regulation of two RNA cap-binding proteins, the cytosolic eIF-4E complex, which is necessary for initiating translation, and the nuclear capbinding complex, the CBC, which plays a role in capdependent pre-mRNA splicing, U snRNA export and 3′-end processing. This, taken together with other findings that demonstrate the ability of stress response pathways and the small G protein, Cdc42, to activate the CBC, raises some interesting possibilities regarding how signaling to the two cellular RNA cap-binding protein complexes may coordinate the growth-coupled regulation of gene expression at the level of RNA processing.

Several GTPases participate in bacterial protein biosynthesis. Initiation factor 2 controls the formation of the ribosomal initiation complex and places initiator fMet-tRNA fMet in the ribosomal P-site. Elongation factors Tu and G are responsible for codon-specific binding of the aminoacyl-tRNA to the A-site, and peptidyltRNA to the P-site, respectively, during the elongation phase of protein biosynthesis. Release factor 3, a GTPase which is not ubiquitous, is involved in termination and release of the nascent polypeptide. Other translation factors, including initiation factors 1 and 3, elongation factor Ts, release factors 1 and 2, and ribosomal release factor do not belong to the family of GTP/GDP binding proteins. The guanosine nucleotide binding domains of the GTPases involved in translation are structurally related to the Gα subunit of heterotrimeric G proteins and to the proteins of the Ras family. We have identified and sequenced all genes coding for translation factors in the extreme thermophile Thermus thermophilus . The proteins were overproduced in Escherichia coli , purified, biochemically characterised and used for crystallisation and structural analysis. Further biochemical investigations were aimed at gaining insight into the molecular mechanism underlying the regulation of the GTPase activity of the translation factors, and to elucidate the role of their ribosomal binding sites in this process.

The elongation factors (EF) Tu and G and initiation factor 2 (IF2) from bacteria are multidomain GTPases with essential functions in the elongation and initiation phases of translation. They bind to the same site on the ribosome where their low intrinsic GTPase activities are strongly stimulated. The factors differ fundamentally from each other, and from the majority of GTPases, in the mechanisms of GTPase control, the timing of P i release, and the functional role of GTP hydrolysis. EF-Tu·GTP forms a ternary complex with aminoacyl-tRNA, which binds to the ribosome. Only when a matching codon is recognized, the GTPase of EF-Tu is stimulated, rapid GTP hydrolysis and P i release take place, EF-Tu rearranges to the GDP form, and aminoacyl-tRNA is released into the peptidyltransferase center. In contrast, EF-G hydrolyzes GTP immediately upon binding to the ribosome, stimulated by ribosomal protein L7/12. Subsequent translocation is driven by the slow dissociation of P i , suggesting a mechano-chemical function of EF-G. Accordingly, different conformations of EF-G on the ribosome are revealed by cryo-electron microscopy. GTP hydrolysis by IF2 is triggered upon formation of the 70S initiation complex, and the dissociation of P i and/or IF2 follows a rearrangement of the ribosome into the elongation-competent state.

Activation of platelets plays a central role in hemostasis as well as in various thromboembolic diseases like myocardial infarction or stroke. Most platelet activating stimuli function through receptors which couple to heterotrimeric G proteins of the G i , G q and G 12 families. Recent studies have elucidated the roles of individual G proteins in the regulation of platelet functions like shape change, aggregation and granule secretion. The signaling pathways mediated by heterotrimeric G proteins operate synergistically to induce a full activation of platelets. This review summarizes recent progress in the understanding of upstream regulation of platelet activation through G protein-coupled receptors.

Among the Ras family, Ran is a unique small G protein. It does not have a lipid modification motif at the C-terminus to bind to the membrane, which is often observed within the Ras family. Ran may therefore interact with a wide range of proteins in various intracellular locations. This means that Ran could play many different roles like nucleocytoplasmic transport, microtubule assembly and so on. All of the Ran functions should be regulated by RanGEF and RanGAP. It is an interesting issue why RCC1, a RanGEF, is localized in the nucleus and RanGAP1/Ran1p in the cytoplasm. It is possible that RCC1 checks the state of chromosomal DNA replication and transfers it to the downstream events through Ran; thereby, RCC1 would be involved in coupling the spatial localization of cellular macromolecules with the cell cycle progression. In this context, Ran will be a very important cell cycle mediator. There is yet another G protein cascade, Gtr1-Gtr2, which interacts with the Ran cycle.

The lack of regrowth of injured neurons in the adult central nervous system (CNS) of higher vertebrates was accepted as a fact for many decades. In the last few years a very different view emerged; regeneration of lesioned fibre tracts in vivo could be induced experimentally, and molecules that are responsible for inhibition and repulsion of growing neurites have been defined. Mechanisms that link cellular phenomena like growth cone turning or growth cone collapse to intracellular changes in second messenger systems and cytoskeletal dynamics became unveiled. This article reviews recent developments in this field, focusing especially on one of the best characterised neurite outgrowth inhibitory molecules found in CNS myelin that was recently cloned: Nogo-A. Nogo-A is a high molecular weight transmembrane protein and an antigen of the monoclonal antibody mAb IN-1 that was shown to promote long-distance regeneration and functional recovery in vivo when applied to spinal cord-injured adult rats. Nogo-A is expressed by oligodendrocytes in white matter of the CNS. With the molecular characterisation of this factor new possibilities open up to achieve structural and functional repair of the injured CNS.

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins ( e. g. , Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The ‘injected’ toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp. , respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica . These toxins deamidate/ transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp. , that acts as a GEF protein.

Serine/threonine protein kinases of the Ste20p/PAK family are highly conserved from yeast to man. These protein kinases have been implicated in the signaling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades and to cytoskeletal components such as myosin-I. In the yeast Saccharomyces cerevisiae , Ste20p is involved in transmitting the mating-pheromone signal from the βγ-subunits of a heterotrimeric G protein to a downstream MAP kinase cascade. We have previously shown that binding of the G-protein β-subunit (G β ) to a short binding site in the non-catalytic carboxy-terminal region of Ste20p is essential for transmitting the pheromone signal. In this study, we searched protein sequence databases for sequences that are similar to the G β binding site in Ste20p. We identified a sequence motif with the consensus sequence S S L ϕP L I/V x ϕϕβ (x: any residue; ϕ: A, I, L, S, or T; β: basic residues) that is solely present in members of Ste20p/PAK family protein kinases. We propose that this sequence motif, which we have designated GBB (G̱ β̱ ḇinding) motif, is specifically responsible for binding of G β to Ste20p/PAK protein kinases in response to activation of heterotrimeric G protein coupled receptors. Thus, the GBB motif is a novel type of signaling domain that serves to link protein kinases of the Ste20p/PAK family to G protein coupled receptors.

Methylation of CpG residues in mammalian genomes is a mechanism of vital importance for many cellular functions, which all relate to gene expression. In this study we describe the identification of a CpG island in the 5′-region of the gene encoding human megalin/LRP-2, a receptor capable of binding multiple ligands, which is involved in the regulation of calcium metabolism. Southern blot analysis and genomic bisulfite sequencing revealed that the CpG island is methylated in a non-expressing cell line, largely unmethylated in an expressing cell line and unmethylated in human parathyroid tissue. In addition, we show that artificial methylation of LRP-2 promoter reporter plasmids leads to strong transcriptional repression, in vitro as well as in transfected cells. No evidence for aberrant LRP-2 gene methylation in parathyroid adenomas, in which the LRP-2 protein is generally down-regulated, was found.

We present a theoretical description of the method of DNA sequencing with simultaneous exponential PCR amplification of the template (DEXAS). Based on the theory of probability, the formula determining the optimal ratio of concentrations of deoxy- and dideoxynucleotides in the reaction mixture is derived, as well as the length distribution of sequenced DNA fragments. The prediction of the number of mutations is given and the theoretically determined aspects of DEXAS are compared with the corresponding quantities of classical sequencing methods. Some other experimentally observed effects are also discussed.

Plant ribosome-inactivating proteins specifically cleave an N -glycosidic bond of a unique adenosine in the largest ribosomal RNA, releasing an adenine from ribosomes of different sources. Here, 1 H-nuclear magnetic resonance is used to analyze the enzymatic products of the A-chain of cinnamomin, a type-II ribosome-inactivating protein (RIP) acting on the nucleotides in situ . The enzymatic activities of the RIP on nine nucleotides are compared. Cinnamomin A-chain can cleave the N -glycosidic bond and release an adenine base from adenine nucleotides except 5′-ATP; however, it cannot act on 5′-GMP, 5′-CMP, and 5′-UMP. The A-chain in the mixture of cinnamomin A- and B-chain exhibits higher activity toward adenine nucleotides than the A-chain alone does, suggesting that the B-chain can conformationally stabilize the A-chain. Intact cinnamomin also exhibits lower activity toward adenine nucleotides. However, cinnamomin B-chain and heat-denatured intact cinnamomin cannot hydrolyze all the tested nucleotides. We conclude that hydrolysis of the N -C glycosidic bond of nucleotide compounds by cinnamomin A-chain has a base preference, and the negatively charged phosphate group(s) reduces the recognition ability of the A-chain to adenine nucleotide.

Ypt/Rab proteins of the Ras superfamily are regulators of protein transport in exo- and endocytosis. Like Ras and Rho proteins, they have a slow intrinsic GTPase activity that can be accelerated by several orders of magnitude by GTPase-activating proteins (GAP). Here we describe a new member of a family of Ypt/Rab-specific GAPs, Msb4p/Gyp4p, that shares with other Gyp family members significant homology in the catalytic domain, recently identified in Gyp1p and Gyp7p. Purified Msb4p/Gyp4p acts primarily on Sec4p, Ypt6p and Ypt7p and might have a role in polarized secretion.

AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi-phosphorylation domain in the PhK α subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them. The aim of this study was to determine whether phosphorylation by cAMPK or AMPK would influence subsequent phosphorylation by the other kinase. Surprisingly, employing four different PhK substrates, it could be demonstrated that, in contradiction to previous reports, PhK is not phosphorylated by AMPK.

In prion diseases the endogenous prion protein (PrP C ) is converted into an abnormally folded isoform, denoted PrP Sc , which represents the major component of infectious scrapie prions. The mechanism of the conversion is largely unknown, but the conversion is thought to occur after PrP C has reached the plasma membrane. Here we show that exogenous adminstration of the cationic lipopolyamine DOSPA interfered with the accumulation of PrP Sc in scrapie-infected neuroblastoma cells. Structural analysis of the compounds tested revealed that inhibition of PrP Sc was specific for lipids with a headgroup composed of the polyamine spermine and a quarternary ammonium ion between the headgroup and the lipophilic tail. The cationic lipopolyamine DOSPA induced the cellular degradation of preexisting PrP Sc aggregates within 12 hours and interfered with the de novo synthesis of PrPSc. Biosynthesis of PrP C , or the assembly of sphingolipid-cholesterol microdomains (rafts) on the plasma membrane, were not affected by this inhibitor. After removal of DOSPA and replating into normal medium propagation of PrP Sc commenced, although initially at a reduced rate. Incubation of ScN2a cells in free spermidine had no inhibitory effect on the accumulation of PrP Sc . Our results indicate that membrane targeting of a small polyamine molecule creates a potent inhibitor of PrP Sc propagation and offers the possibility to degrade preexisting PrP Sc aggregates in living cells.

The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6000-fold purified enzyme. This was obtained in 100 μg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n -butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) cochromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n -butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.

Oxidative stress is known to induce cholestasis, but the underlying mechanisms are poorly understood. In this study we have characterized the short-term effects of tert -butyl hydroperoxide ( t -BOOH)- and 1-chloro-2,4-dinitrobenzene (CDNB) on the mrp2 gene encoded canalicular export pump (Mrp2). The effects of t -BOOH and CDNB on bile formation, tissue GSH levels and subcellular Mrp2 localization were studied in perfused rat liver. Both, t -BOOH (0.5 mM) and CDNB (0.1mM) induced within 60 min a decrease of hepatic GSH levels by more than 90% and an almost complete cessation of bile flow. As revealed by confocal laser scanning microscopy, this cholestasis was accompanied by a loss of immunoreactive MRP2 from the canalicular membrane and its appearance inside the hepatocytes in putative intracellular vesicles. On the other hand, the intracellular distribution of dipeptidyl peptidase IV (DPPIV), another canalicular protein, and of zonula occludens associated polypeptide (ZO-1) remained unaffected, indicating selectivity of the Mrp2 retrieval pattern. Both, t -BOOH and CDNB induced a rapid net K + efflux from the liver and a significant decrease of liver cell hydration. We conclude that severe glutathione depletion induces cholestasis by a retrieval of Mrp2, but not of DPPIV from the canalicular membrane. The underlying mechanism is unclear; however, a decrease in liver cell hydration, which occurs under these conditions, may contribute to this effect.

In order to investigate the correlations between constitutive proteinase expression and the degree of tumorigenicity of cancer cells we have studied a model system of three keratinocyte cell lines. RT-PCR studies showed that the cell lines express the genes of matrix metalloproteinase-2, -3, -7, -9, -10 and -11, indicating that they are able to synthesize the corresponding enzymes. Actual MMP synthesis was proven by zymography and Western blotting. In conditioned media gelatinolytic activities or immunoreactive forms of MMP-2, -3, -7, -9, -10 and -11 were detected. The signal intensities showed that MMP secretion increases in the order HaCaT < A5 ≤ II-4RT, whereas only MMP-11 is secreted by all cell lines in equal amounts. Intracellularly, enhanced levels of one or both of the tumorigenic variants were only found for MMP-3, -9 and -10, suggesting special functions of these intracellular MMP pools for the tumorigenic cell lines. For MMP-11 exclusive expression in stromal fibroblasts of tumor tissues is widely accepted; however, our results and three other recent reports demonstrate that this concept is not generally valid. In conclusion, the three keratinocyte cell lines investigated here represent an excellent model for studying constitutive expression and secretion of MMPs in correlation to the degree of in vivo tumorigenicity.

We investigated cells and conditioned media of the three human keratinocyte cell lines HaCaT (nontumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic) with regard to production and secretion of the collagenases-1 to -3 (MMP-1, MMP-8 and MMP-13) and TIMP-1 using seminested RTPCR, Western blots, ELISA, immunocytochemistry and casein zymography. Transcripts of MMP-1, -8, -13 and TIMP-1 were detected in all cell lines by RTPCR and the corresponding proteins were found in the cytoplasm of all three cell lines by Western blot analysis and/or immunocytochemistry. The conditioned media of the malignant II-4RT cells contain significantly more MMP-1 and MMP-8 than those of HaCaT or A5 as evidenced by immunoblotting and ELISA. In addition to the presence of latent MMP-1, zymography also detected the active form of this enzyme. TIMP-1 was found only in extracts of all three cell lines, predominantly in A5. This study clearly indicates that the epithelial tumor cells synthesize different collagenases and TIMP-1. The malignant clone secretes increased amounts of distinct collagenases compared to the nontumorigenic cell line, thereby verifying a correlation between biological behaviour and the amount of collagenases. In addition, we provide clear evidence that MMP-8 is not exclusively found in polymorphonuclear granulocytes, but also in keratinocyte cell lines.

An inducible Streptomyces expression cassette utilising the Ptra promoter and a temperaturesensitive allele of the TraR repressor from S. nigrifaciens plasmid pSN22 was tested in Mycobacterium smegmatis. Using reporter assays and Northern blot analysis, a marked increase of Ptradirected transcription was observed upon a temperature shift from 28 to 37C. These results show that the S. nigrifaciens promoterrepressor cassette is functional in M. smegmatis. However, comparison of the level of induced Ptradirected transcription with the level of transcription directed by the strong mycobacterial promoter Phsp60 indicated that the relative strength of the Ptra promoter was low. Considering the severe limitation of inducible expression systems for mycobacteria, this Streptomyces cassette might be a useful starting point for the development of a compact and fully portable inducible mycobacterial expression cassette.

We describe the shortest prion protein allele known to date. Surprisingly, it is found as a polymorphism exactly in a species (prosimian lemurs) which seems highly susceptible to oral infection with BSEderived prions. The truncation of the prion protein we found raises several questions. First, is the truncated octarepeat structure we describe, consisting of two octarepeats, still functional in copper binding? A second question is whether this truncation is related to the remarkable oral infectibility of lemurs with BSEderived prions. And finally, one could argue that this genotype alone might favour development of a prion disease, even in the absence of exogenous infection.

Inhibition of prostaglandin synthesis by the drug indomethacin suppresses the synthesis of the cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), and leads to a metabolic state comparable to type II diabetes. It was of interest whether prostaglandindeficiency likewise causes sensitization of adenylyl cyclase, as this has been reported for the diabetic state. In liver plasma membranes of indomethacintreated male rats, basal and forskolinstimulated cyclic AMP synthesis remained unchanged when compared to untreated control rats. In control rats, stimulation of cyclic AMP synthesis by fluoride (2.2-fold) or glucagon (3.5-fold) was much lower than stimulation by forskolin (6.6-fold). In contrast, in indomethacin treated rats, stimulation of cAMP synthesis by fluoride (4.6-fold) or glucagon (5.2-fold) nearly matched the stimulation by forskolin (6.4-fold). The level of α[1]adrenergic receptors was slightly reduced, from 450 to 320 fmol/mg protein, by the indomethacin treatment. Independent of the treatment by indomethacin, stimulation of cyclic AMP synthesis by adrenaline failed, in agreement with the low density of adrenergic βreceptors. In conclusion, PGE deficiency sensitizes adenylyl cyclase in rat liver for G proteincoupled receptors (glucagon) and also for fluoride.