Biomedical subjects

Just Justesen

Publications and source records attributed to Just Justesen.

10 recordsLinked to original sources

PubMed2005

Invariant amino acids essential for decoding function of polypeptide release factor eRF1.

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125-131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.

Amino Acid Sequence↗

PubMed2005

Role of HSF activation for resistance to heat, cold and high-temperature knock-down.

Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance of Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression. In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF, such as Hsps, although occurring following LTCA, are not of major importance for cold stress resistance and neither for RCH nor high-temperature knock-down resistance in D. melanogaster.

Acclimatization↗

PubMed2005

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide.

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG(beta)-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG(beta)-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG(beta)-specific and anti-hCG(beta)/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG(beta)-R68E was eliminated, mAbs reacting with hCG(beta)-specific epitopes bound to hCG(beta) and hCG(beta)-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG(beta)-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG(beta)-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.

Amino Acid Substitution↗

PubMed2005

Full genome gene expression analysis of the heat stress response in Drosophila melanogaster.

The availability of full genome sequences has allowed the construction of microarrays, with which screening of the full genome for changes in gene expression is possible. This method can provide a wealth of information about biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening (36 degrees C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved in the stress response. We identified 3 main groups of stress responsive genes that were early-upregulated, early-downregulated, and late-upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis, which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in general. The majority of stress responsive genes identified in this and other studies were downregulated, and the degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an expected exception, heat shock genes were generally found to be upregulated by stress in general.

Animals↗

PubMed2004

Interaction between the 2'-5' oligoadenylate synthetase-like protein p59 OASL and the transcriptional repressor methyl CpG-binding protein 1.

The human 2'-5' oligoadenylate synthetases (OAS) form a conserved family of interferon-induced proteins consisting of four genes: OAS1, OAS2, OAS3 and the 2'-5' oligoadenylate synthetase-like gene (OASL). When activated by double-stranded RNA, OAS1-3 polymerize ATP into 2'-5'-linked oligoadenylates; 2'-5'-linked oligoadenylates, in turn, activate a latent endoribonuclease that degrades viral and cellular RNAs. In contrast, while the p59 OASL protein is highly homologous to the OAS family (45% identity), its 350 amino acid N-terminal domain lacks 2'-5' oligoadenylate synthetase activity. A C-terminal 164 amino acid domain, which is 30% homologous to a tandem repeat of ubiquitin, further distinguishes the p59 OASL protein and suggests that it serves a biological role which is distinct from other OAS family members. To dissect the function of p59 OASL, we utilized the yeast two-hybrid system to identify interacting proteins. Methyl CpG-binding protein 1 (MBD1), which functions as a transcriptional repressor, was identified as a strong p59 OASL interactor. Interestingly, like p59 OASL, transcription of the MBD1 gene was induced by interferon, indicating that these genes are co-ordinately regulated. The interaction was confirmed in vitro and in vivo and was mapped to the ubiquitin-like domain of p59 OASL. The p59 OASL-MBD1 interaction was specific, because p59 OASL did not interact with any of the other MBD family members and MBD1 did not interact with OAS1. These findings link the p59 OASL with MBD1 transcriptional control in the context of an interferon-stimulated cell, and provide the basis for future studies to examine the functional role of this interaction.

2',5'-Oligoadenylate Synthetase↗

PubMed2004

Small ISGs coming forward.

Interferons (IFNs) were first characterized as antiviral proteins. Since then, IFNs have proved to be involved in malignant, angiogenic, inflammatory, immune, and fibrous diseases and, thus, possess a broad spectrum of pathophysiologic properties. IFNs activate a cascade of intracellular signaling pathways leading to upregulation of more than 1000 IFN-stimulated genes (ISGs) within the cell. The function of some of the IFN-induced proteins is well described, whereas that of many others remain poorly characterized. This review focuses on three families of small intracellular and intrinsically nonsecreted proteins (10-20 kDa) separated into groups according to their amino acid sequence similarity: the ISG12 group (6-16, ISG12, and ISG12-S), the 1-8 group (9-27/Leu13, 1-8U, and 1-8D), and the ISG15 group (ISG15/UCRP). These IFN-induced genes are abundantly and widely expressed and mainly induced by type I IFN. ISG15 is very well described and is a member of the ubiquitin-like group of proteins. 9-27/Leu-13 associates with CD81/TAPA-1 and plays a role in B cell development. The functions of 1-8U, 1-8D, 6-16, ISG12, and ISG12-S proteins are unknown at present.

Amino Acid Sequence↗

PubMed2003

Characterization of the 2'-5'-oligoadenylate synthetase ubiquitin-like family.

The interferon-induced 2'-5'-oligoadenylate synthetases (OAS) are important for the antiviral activity of interferons. The human and murine OAS gene families each contain four genes: OAS1, OAS2, OAS3 and OASL, all having one or more conserved OAS units composed of five translated exons. The OASL gene has both an OAS unit and a C-terminus of two ubiquitin-like repeats. In this study, we demonstrate that murine Oasl1 protein is inactive while murine Oasl2 is active as an OAS. Further more, murine Oasl2 requires double-stranded RNA as co-factor. The affinity of murine Oasl2 for the double-stranded RNA activator is higher than that of human OAS1 (p42 isoform). We propose a model for the evolutionary origin of the murine Oasl1 and Oasl2 genes. The identification of a human orthologue (hOASL2) to the murine Oasl2 gene establishes that the OASL gene was duplicated prior to the radiation of the rodent and primate groups. We suggest that murine Oasl2, which has both enzymatic activity and a ubiquitin-like domain, is a functional intermediate between the active OAS species and the inactive human OASL1/murine Oasl1 proteins. In addition, we propose that murine Oasl1 appears to have gained a hitherto uncharacterized function independent of 2'-5'-linked oligoadenylate synthesis.

2',5'-Oligoadenylate Synthetase↗

PubMed2003

Redox modulation of NO-dependent induction of interleukin 8 gene in monocytic U937 cells.

We have examined the effects of various antioxidants and inhibitors of redox-sensitive signal transduction pathways on induction of interleukin 8 (IL-8) gene by NO in monocytic U937 cells. We have observed that nitrosoglutathione or another NO-generating compound spermine NONOate caused significant accumulation of IL-8 mRNA. Pretreatment of cells with pyrrolidine dithiocarbamate or with antioxidants, which scavenge hydroxyl radical, dimethyl sulfoxide (DMSO), or dimetylthiourea (DMTU) completely abrogated NO-dependent induction of IL-8 gene expression. The transcriptional activation of IL-8 gene was not affected by sodium formate or sodium salicylate, suggesting that suppression of the IL-8 gene induction is specific to the class of hydroxyl radical scavenger used. Furthermore, we have shown that IL-8 induction was not inhibited by catalase and the iron chelator deferoxamine, indicating that the inhibitory actions of DMSO and DMTU are not related to scavenging of reactive oxygen species produced from hydrogen peroxide in the iron-catalyzed reactions. Finally, we have not observed any significant inhibition of NO-dependent IL-8 gene induction by superoxide scavengers such as N-acetyl cysteine, uric acid, and superoxide dismutase. Therefore, it seems likely that in U937 cells, hydroxyl radicals or species with reactivity similar to hydroxyl radicals contribute to NO-mediated IL-8 gene induction.

Gene Expression Regulation↗

PubMed2003

Crystal structure of the 2'-specific and double-stranded RNA-activated interferon-induced antiviral protein 2'-5'-oligoadenylate synthetase.

2'-5'-oligoadenylate synthetases are interferon-induced, double-stranded RNA-activated antiviral enzymes which are the only proteins known to catalyze 2'-specific nucleotidyl transfer. This crystal structure of a 2'-5'-oligoadenylate synthetase reveals a structural conservation with the 3'-specific poly(A) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2'- and 3'-specific nucleotidyl transferases. Comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. The 2'-5'-oligoadenylate synthetases are activated by viral double-stranded RNA in infected cells and initiate a cellular response by synthesizing 2'-5'-oligoadenylates, which in turn activate RNase L. This crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsRNA activation site that is probed by mutagenesis, thus providing structural insight into cellular recognition of viral double-stranded RNA.

2',5'-Oligoadenylate Synthetase↗

PubMed2002

Elimination of luteinizing hormone cross-reactive epitopes from human chorionic gonadotropin.

The beta-chain of human chorionic gonadotropin (hCG) has been shown to have efficacy in clinical trials when used as a contraceptive vaccine. This hormone is a heterodimer, the alpha-chain being shared with the other members of the glycoprotein hormone family but the beta-chain being unique to hCG. Nevertheless, there is sequence homology between the hCG beta-chain and the beta-chain of human luteinizing hormone (hLH) which results in cross-reactive antibodies being produced following immunization with wild-type hCGbeta. To reduce or eliminate such cross-reactions we generated a number of mutants of the hCGbeta-chain. One mutant (hCGbeta(R68E)), containing an arginine to glutamic acid replacement at position 68, has been expressed as a recombinant protein in High Five insect cells. The recombinant BAChCGbeta(R68E) form of this molecule was used to immunize rabbits and the antibody response compared to the response following immunization with the recombinant wild-type protein BAChCGbeta and with the native hCGalphabeta heterodimer isolated from pregnancy urine. The mutant elicited the production of antibodies which avidly recognize native hCG. Compared to immunization with wild-type hCG, the response showed very little cross reactivity with hLH. This is demonstrated to be due to a radically altered epitope usage in the response to the mutant, which now focuses mainly upon the C-terminal region of the beta-chain.

Animals↗