Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Expression of the arginase structural gene ( agaA ) in Aspergillus nidulans is subject to complex transcriptional and post-transcriptional regulation. Arginase mRNA has a long 5′-UTR sequence. Analysis of this sequence in silico revealed its putative complex secondary structure, the presence of arginine-binding motifs (arginine aptamers) and a short intron with two potential 3′ splicing sites. In this report we present evidence that L-arginine (i) binds directly to the arginase 5′-UTR; (ii) invokes drastic changes in the secondary structure of the 5′-UTR, unlike several other L-amino acids and D-arginine; and (iii) forces the selection of one of two 3′ splice sites of an intron present in the 5′-UTR. We postulate that expression of the eukaryotic structural gene coding for arginase in A. nidulans is regulated at the level of mRNA stability, depending on riboswitch-mediated alternative splicing of the 5′-UTR intron.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana . Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris , two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 10 5 - to 10 6 -fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Mutations in the mouse ATRN gene, which encodes attractin, offer links between this protein and pigmentation, metabolism, immune status and neurodegeneration. However, the mechanisms of attractin action are not understood. The protein was first identified in humans in a circulating form in serum. A protease activity was postulated similar to the membrane-bound ectoenzyme DP4/CD26. In the last decade, both DP4/CD26 and attractin were controversially described to be the major source of human serum DP4 activity. We purified attractin from human plasma, and found that the DP4-like activity of the preparation shows nearly identical kinetic properties to that of recombinant human DP4. In contrast, the native electrophoretic behavior of this activity is clearly different from human and porcine DP4, but co-migrates with the protein band identified as attractin by Western blotting and N-terminal sequencing. Nevertheless, a DP4 impurity could be demonstrated in purified plasma attractin and the activity could be removed by ADA affinity chromatography, resulting in a homogenous attractin preparation without DP4 activity. These results are substantiated by expression of different attractin isoforms, in which no DP4 activity was found either. This indicates that the multidomain protein attractin acts as a receptor or adhesion protein rather than a protease.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
We have investigated the thermodynamic parameters and binding of a regulatory subunit of cAMP-dependent protein kinase (PKA) to its natural low-molecular-weight ligand, cAMP, and analogues thereof. For analysis of this model system, we compared side-by-side isothermal titration calorimetry (ITC) with surface plasmon resonance (SPR). Both ITC and SPR analyses revealed that binding of the protein to cAMP or its analogues was enthalpically driven and characterised by similar free energy values (Δ G =-9.4 to -10.7 kcal mol -1 ) for all interactions. Despite the similar affinities, binding of the cyclic nucleotides used here was characterised by significant differences in the contribution of entropy (- T Δ S ) and enthalpy (Δ H ) to Δ G . The comparison of ITC and SPR data for one cAMP analogue further revealed deviations caused by the method. These equilibrium parameters could be complemented by thermodynamic data of the transition state (Δ H ≠ , Δ G ≠ , Δ S ≠ ) for both association and dissociation measured by SPR. This direct comparison of ITC and SPR highlights method-specific advantages and drawbacks for thermodynamic analyses of protein/ligand interactions.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
In this study, we tested the biological activity of a novel acylated form of (Pro 3 )glucose-dependent insulinotropic polypetide [(Pro 3 )GIP] prepared by conjugating palmitic acid to Lys 16 to enhance its efficacy in vivo by promoting binding to albumin and extending its biological actions. Like the parent molecule (Pro 3 )GIP, (Pro 3 )GIPLys 16 PAL was completely stable to the actions of DPP-IV and significantly ( p <0.01 to p <0.001) inhibited GIP-stimulated cAMP production and cellular insulin secretion. Furthermore, acute administration of (Pro 3 )GIPLys 16 PAL also significantly ( p <0.05 to p <0.001) countered the glucose-lowering and insulin-releasing actions of GIP in ob/ob mice. Daily injection of (Pro 3 )GIPLys 16 PAL (25 nmol/kg bw) in 14–18-week-old ob/ob mice over 14 days had no effect on body weight, food intake or non-fasting plasma glucose and insulin concentrations. (Pro 3 )GIPLys 16 PAL treatment also failed to significantly alter the glycaemic response to an i.p. glucose load or test meal, but insulin concentrations were significantly reduced (1.5-fold; p <0.05) after the glucose load. Insulin sensitivity was enhanced (1.3-fold; p <0.05) and pancreatic insulin was significantly reduced ( p <0.05) in the (Pro 3 )GIPLys 16 PAL-treated mice. These data demonstrate that acylation of Lys 16 with palmitic acid in (Pro 3 )GIP does not improve its biological effectiveness as a GIP receptor antagonist.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
HIV-1 Nef is a small protein (approx. 25 kDa) that is posttranslationally modified by myristoylation. To explain its complex activities, a ‘Nef-cycle’ is discussed, which postulates different molecular conformations of Nef. Using recombinant full-length non-myristoylated Nef and synthetic peptides, we demonstrate by fluorescence titration experiments that a peptide representing the myristoylated N-terminus of Nef is specifically bound by Nef. A non-myristoylated N-terminal fragment of Nef or a myristoylated control peptide does not bind to Nef. These results are the first direct experimental evidence of the existence of a myristate-binding pocket in Nef, a prerequisite of the postulated ‘closed’ Nef conformation.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCζ-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. The mechanisms underlying bile salt-induced ceramide formation have remained unclear to date and thus were studied in rat hepatocytes. Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), lowered the apparent pH ves within seconds from 6.0 to 5.6 in an FITC-dextran-accessible endosomal compartment that also contains acidic sphingomyelinase. Simultaneously, a rapid decrease in N -(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence was observed, suggestive of an increase in cytosolic [Cl - ], which is known to activate vacuolar-type H + -ATPase. No vesicular acidification or increase in cytosolic [Cl - ] was found in response to the non-apoptotic bile salt taurocholate or the anti-apoptotic bile salt tauroursodesoxycholate. Inhibition of TLCS-induced endosomal acidification by bafilomycin or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid largely abolished the TLCS-induced ceramide-formation and downstream ceramide-dependent processes, such as p47 phox -serine phosphorylation, NADPH oxidase activation, CD95 activation and apoptosis. These responses were also abolished after knockdown of acidic sphingomyelinase in rat hepatocytes. In conclusion, hydrophobic, proapoptotic bile salts stimulate ceramide formation through chloride-dependent acidification of endosomes, with subsequent activation of acidic sphingomyelinase. Our data suggest that changes in ion homeostasis underlie the stimulation of ceramide formation in response to hydrophobic bile acids as an important upstream event of bile salt-induced apoptosis.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro . In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Resveratrol is a polyphenolic chemopreventive agent that has been shown to influence cellular redox reactions. As a systematic approach to elucidating the complex effects of resveratrol on eukaryotic cells, we studied its dose-dependent effects on the transcript levels of genes and activities of enzymes related to redox metabolism, cell cycle regulation, and apoptotic cascades in the cancer cell line A549. Glutathione peroxidase (GPx)1 mRNA levels, as well as GPx and thioredoxin reductase (TrxR) activities, were significantly increased after resveratrol treatment, whereas total glutathione concentrations decreased. Increased transcript levels were also detected for selenophosphate synthetase 2 and superoxide dismutase 2. However, mRNA levels of thioredoxin, TrxR, glutathione reductase, glutathione S-transferase, superoxide dismutase 1, and catalase were not altered. Among the 12 genes studied that are related to the cell cycle, differentiation and apoptosis, mRNA levels of six genes, including P53 , FAS , and BCL2 , were upregulated, while the mRNA level of survivin was reduced. The results suggest that GPx and other selenoproteins are important targets of resveratrol. Furthermore, genes supporting cell survival and differentiation, as well as genes involved in proliferation inhibition and apoptosis, are induced by resveratrol, resulting in a delicate balance that is likely to contribute to the chemopreventive effects of resveratrol.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Effects of chemical ablation of the GIP and GLP-1 receptors on metabolic aspects of obesity-diabetes were investigated using the stable receptor antagonists (Pro 3 )GIP and exendin(9–39)amide. Ob/ob mice received a daily i.p. injection of saline vehicle, (Pro 3 )GIP, exendin(9–39)amide or a combination of both peptides over a 14-day period. Non-fasting plasma glucose levels were significantly ( p <0.05) lower in (Pro 3 )GIP-treated mice compared to control mice after just 9 days of treatment. (Pro 3 )GIP-treated mice also displayed significantly lower plasma glucose concentrations in response to feeding and intraperitoneal administration of either glucose or insulin ( p <0.05 to p <0.001). The (Pro 3 )GIP-treated group also exhibited significantly ( p <0.05) reduced pancreatic insulin content. Acute administration of exendin(9–39)amide immediately prior to re-feeding completely annulled the beneficial effects of sub-chronic (Pro 3 )GIP treatment, but non-fasting concentrations of active GLP-1 were unchanged. Combined sub-chronic administration of (Pro 3 GIP) with exendin(9–39)amide revealed no beneficial effects. Similarly, daily administration of exendin(9–39)amide alone had no significant effects on any of the metabolic parameters measured. These studies highlight an important role for GIP in obesity-related forms of diabetes, suggesting the possible involvement of GLP-1 in the beneficial actions of GIP receptor antagonism.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Staphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipocalin-like fold and a unique mechanism of action. Each of these cytoplasmic proteins is co-expressed from one operon, together with a corresponding target extracellular cysteine protease (staphopain). To cast more light on staphostatin/staphopain interaction and the evolution of the encoding operons, we have cloned and characterized a staphopain (StpA2 aur CH-91 ) and its inhibitor (StpinA2 aur CH-91 ) from a novel staphylococcal thiol protease operon ( stpAB2 CH-91 ) identified in S . aureus strain CH-91. Furthermore, we have expressed a staphostatin from Staphylococcus warneri (StpinB war ) and characterized its target protease (StpB war ). Analysis of the reciprocal interactions among novel and previously described members of the staphostatin and staphopain families demonstrates that the co-transcribed protease is the primary target for each staphostatin. Nevertheless, the inhibitor derived from one species of Staphylococcus can inhibit the staphopain from another species, although the K i values are generally higher and inhibition only occurs if both proteins belong to the same subgroup of either S. aureus staphopain A/staphostatin A (α group) or staphopain B/staphostatin B (β group) orthologs. This indicates that both subgroups arose in a single event of ancestral allelic duplication, followed by parallel evolution of the protease/inhibitor pairs. The tight coevolution is likely the result of the known deleterious effects of uncontrolled staphopain action.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
The amino acid sequence His-Pro-Phe as N-terminal residues 6–8 of the natural renin substrate, angiotensinogen, is conserved among species. We investigated whether this His-Pro-Phe motif functions as the determinant of the substrate specificity of renin. Mutant angiotensinogens in which the Ile-His-Pro-Phe-His-Leu sequence at positions 5–10 of wild-type angiotensinogen was replaced by either His-Pro-Phe-His-Leu-Leu or Ala-Ile-His-Pro-Phe-His were cleaved by renin at the C-terminal side of residues 9 and 11, respectively, while wild-type angiotensinogen was cleaved at residue 10. A triple Ala substitution for the His-Pro-Phe motif of angiotensinogen prevented its cleavage by renin. In contrast, triple Ala substitution for residues 9–11, including the natural site of cleavage by renin, allowed cleavage between the two Ala residues at positions 10 and 11. Furthermore, the 33-residue C-terminal peptide of human megsin, which carries a naturally occurring His-Pro-Phe sequence, was cleaved by renin at the C-terminal side of the His-Pro-Phe-Leu-Phe sequence. These results indicate that the His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin. By binding to a corresponding pocket on renin, the His-Pro-Phe motif may act as a molecular anchor to recruit the scissile peptide bond to a favorable site for catalysis.
Unable to retrieve citations for this document
Retrieving citations for document...
Requires Authentication
Unlicensed
Licensed
January 29, 2007
Abstract
Abnormal accumulation of neurotoxic β-amyloid peptides (Aβ) in brain represents a key factor in the progression of Alzheimer's disease (AD). Identification of small molecules that effectively reduce brain levels of Aβ is important for development of Aβ-lowering agents for AD. In this study, we demonstrate that in vivo Aβ levels in brain are significantly reduced by the cysteine protease inhibitor E64d and the related CA074Me inhibitor, which inhibits cathepsin B. Direct infusion of these inhibitors into brains of guinea pigs resulted in reduced levels of Aβ by 50–70% after 30 days of treatment. Substantial decreases in Aβ also occurred after only 7 days of inhibitor infusion, with a reduction in both Aβ40 and Aβ42 peptide forms. A prominent decrease in Aβ peptides was observed in brain synaptosomal nerve terminal preparations after CA074Me treatment. Analyses of APP-derived proteolytic fragments showed that CA074Me reduced brain levels of the CTFβ fragment, and increased amounts of the sAPPα fragment. These results suggest that CA074Me inhibits Aβ production by modulating APP processing. Animals appeared healthy after treatment with these inhibitors. These results, showing highly effective in vivo decreases in brain Aβ levels by these cysteine protease inhibitors, indicate the feasibility of using related compounds for lowering Aβ in AD.
