A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2.
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Jasmonate (JA) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites. The induction of the secondary metabolite biosynthetic gene Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by elicitor requires JA as a second messenger. A 42 bp region in the Str promoter is both necessary and sufficient for JA- and elicitor-responsive expression. This region is unlike other previously identified JA-responsive regions, and contains a GCC-box-like element. Yeast one-hybrid screening identified cDNAs encoding two AP2-domain proteins. These octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) proteins bind in a sequence-specific manner the JA- and elicitor-responsive element. ORCA2 trans-activates the Str promoter and its expression is rapidly inducible with JA and elicitor, whereas Orca1 is expressed constitutively. The results indicate that a GCC-box-like element and ORCA2 play key roles in JA- and elicitor-responsive expression of the terpenoid indole alkaloid biosynthetic gene Str. (+info)
Destabilase from the medicinal leech is a representative of a novel family of lysozymes.
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Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein. (+info)
Sequencing, tissue distribution and chromosomal assignment of a novel ubiquitin-specific protease USP23.
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We have identified human and mouse cDNAs encoding a novel ubiquitin-specific protease designated USP23. Both cDNAs encode a 62-kDa protein containing the highly conserved His and Cys domains characteristic of the C19 cysteine protease family of ubiquitin-specific processing proteases (UCH-2). Human tissue Northern blots revealed USP23 to be ubiquitously expressed, whereas USP12, its closest human paralogue, displayed a more restricted expression pattern. The human USP23 gene mapped to chromosome 1q22. (+info)
Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins.
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Covalent conjugation of proteins by ubiquitin or ubiquitin-like molecules is an important form of post-translational modification and plays a critical role in many cellular processes. Similar to the concept of phosphorylation and dephosphorylation, these conjugates are regulated by a large number of deconjugating enzymes. Here, we report the cloning of a 2,141-base pair DNA fragment from human placenta cDNA library by a strategy that involves expressed sequence tag data base searching, polymerase chain reaction, and rapid amplification of cDNA ends. Nucleotide sequence analysis revealed that the cloned cDNA contains an open reading frame of 1,143 base pairs encoding a novel protease, USP21, which is composed of 381 residues with a calculated molecular mass of 43 kDa. The human USP21 gene is located on chromosome 1q21 and encodes a member of the ubiquitin-specific protease family with highly conserved Cys and His domains. The activity and specificity of USP21 were determined by using a COS cell expression system in vivo. We showed that USP21 is capable of removing ubiquitin from ubiquitinated proteins as expected. Furthermore, USP21 is capable of removing NEDD8 from NEDD8 conjugates but has no effect on Sentrin-1 conjugates. As expected from its biochemical activity, overexpression of USP21 has a profound growth inhibitory effect on U2OS cells. Thus, USP21 is the first ubiquitin-specific protease shown to have dual specificity for both ubiquitin and NEDD8 and may play an important role in the regulation of cell growth. (+info)
Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site.
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BACKGROUND: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. RESULTS: We have determined the crystal structure of cyanase at 1.65 A resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. CONCLUSIONS: The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding alpha-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed. (+info)
Microbial thiocyanate utilization under highly alkaline conditions.
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Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS-) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO-) to ammonia and CO2. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a "cyanate pathway" in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate. (+info)
Control of ubiquitination of proteins in rat tissues by ubiquitin conjugating enzymes and isopeptidases.
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The activity of the ubiquitin-dependent proteolytic system in differentiated tissues under basal conditions remains poorly explored. We measured rates of ubiquitination in rat tissue extracts. Accumulation of ubiquitinated proteins increased in the presence of ubiquitin aldehyde, indicating that deubiquitinating enzymes can regulate ubiquitination. Rates of ubiquitination varied fourfold, with the highest rate in the testis. We tested whether ubiquitin-activating enzyme (E1) or ubiquitin-conjugating enzymes (E2s) could be limiting for conjugation. Immunodepletion of the E2s UBC2 or UBC4 lowered rates of conjugation similarly. Supplementation of extracts with excess UBC2 or UBC4, but not E1, stimulated conjugation. However, UBC2-stimulated rates of ubiquitination still differed among tissues, indicating that tissue differences in E3s or substrate availability may also be rate controlling. UBC2 and UBC4 stimulated conjugation half-maximally at concentrations of 10-50 and 28-44 nM, respectively. Endogenous tissue levels of UBC2, but not UBC4, appeared saturating for conjugation, suggesting that in vivo modulation of UBC4 levels can likely control ubiquitin conjugation. Thus the pool of ubiquitin conjugates and therefore the rate of degradation of proteins by this system may be controlled by E2s, E3s, and isopeptidases. The regulation of the ubiquitin pathway appears complex, but precise. (+info)
Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1.
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The COOH-terminal A168-170 region of the giant sarcomeric protein titin interacts with muscle-specific RING finger-1 (MURF-1). To investigate the functional significance of this interaction, we expressed green fluorescent protein fusion constructs encoding defined fragments of titin's M-line region and MURF-1 in cardiac myocytes. Upon expression of MURF-1 or its central region (containing its titin-binding site), the integrity of titin's M-line region was dramatically disrupted. Disruption of titin's M-line region also resulted in a perturbation of thick filament components, but, surprisingly, not of the NH2-terminal or I-band regions of titin, the Z-lines, or the thin filaments. This specific phenotype also was caused by the expression of titin A168-170. These data suggest that the interaction of titin with MURF-1 is important for the stability of the sarcomeric M-line region.MURF-1 also binds to ubiquitin-conjugating enzyme-9 and isopeptidase T-3, enzymes involved in small ubiquitin-related modifier-mediated nuclear import, and with glucocorticoid modulatory element binding protein-1 (GMEB-1), a transcriptional regulator. Consistent with our in vitro binding data implicating MURF-1 with nuclear functions, endogenous MURF-1 also was detected in the nuclei of some myocytes. The dual interactions of MURF-1 with titin and GMEB-1 may link myofibril signaling pathways (perhaps including titin's kinase domain) with muscle gene expression. (+info)