Search

NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. Previously, we identified a negative regulator of the NEDD8 conjugation system, NEDD8 ultimate buster-1 (NUB1) , that recruits NEDD8 and its conjugates to the proteasome for degradation. Recently, we performed yeast two-hybrid screening with NUB1 as bait and isolated a ubiquitin precursor UbC1 that is composed of nine tandem repeats of a ubiquitin unit through α-peptide bonds . Interestingly, NUB1 interacted with UbC1 through its UBA domain. Further study revealed that the UBA domain interacted with α-peptide bond-linked polyubiquitin, but not with isopeptide bond-linked polyubiquitin, indicating that the UBA domain of NUB1 is a specific acceptor for the linear ubiquitin precursor. A functional study revealed that an unidentified protein that was immunoprecipitated with NUB1 served as a ubiquitin C-terminal hydrolase for UbC1. Thus, NUB1 seems to form a protein complex with the unidentified ubiquitin C-terminal hydrolase and recruit UbC1 to this complex. This might allow the ubiquitin C-terminal hydrolase to hydrolyze UbC1, in order to generate ubiquitin monomers. Northern blot analysis showed that the mRNAs of both NUB1 and UbC1 were enriched in the testis. Furthermore, in situ hybridization showed that both mRNAs were strongly expressed in seminiferous tubules of the testis. These results may imply that the UbC1 hydrolysis mediated by NUB1 is involved in cellular functions in the seminiferous tubules such as spermatogenesis. [ABSTRACT FROM AUTHOR]

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the α,β-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of α,β-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of α,β-dehydrogenation is viewed as a process in which the substrate αC-H and βC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO– as an H+, the second being transferred as a hydride to the flavin N(5) position. Hereby the p Ka of the substrate αC-H is lowered from > 20 to ≈ 8 by the effect of specific hydrogen bonds. Concomitantly, the p Ka of Glu376-COO– is also raised to 8–9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases. [ABSTRACT FROM AUTHOR]

The transcription factor v-Myb is a potent inducer of myeloid leukemias, and its cellular homologue c-Myb plays a crucial role in the regulation of hematopoiesis. Recently, Bies and coworkers (Bies, J., Markus, J. & Wolff, L. (2002) J. Biol. Chem , 277 , 8999–9009) presented evidence that murine c-Myb can be sumoylated under overexpression conditions in COS7 cells when cotransfected with FLAG-tagged SUMO-1. Here we provide independent evidence that human c-Myb is also subject to SUMO-1 conjugation under more physiological conditions as revealed by coimmunoprecipitation analysis of Jurkat cells and transfected CV-1 cells. Analysis in an in vitro conjugation system showed that modification of the two sites K503 and K527 is interdependent. A two-hybrid screening revealed that the SUMO-1 conjugase Ubc9 is one of a few major Myb-interacting proteins. The moderate basal level of sumoylation was greatly enhanced by cotransfection of PIASy, an E3 ligase for SUMO-1. The functional consequence of abolishing sumoylation was enhanced activation both of a transiently transfected reporter gene and of a resident Myb-target gene. When single and double mutants were compared, we found a clear correlation between reduction in sumoylation and increase in transcriptional activation. Enhancing sumoylation by contransfection of PIASy had a negative effect on both Myb-induced and basal level reporter activation. Furthermore, PIASy caused a shift in nuclear distribution of c-Myb towards the insoluble matrix fraction. We propose that the negative influence on transactivation properties by the negative regulatory domain region of c-Myb depends on the sumoylation sites located here. [ABSTRACT FROM AUTHOR]

For the biosynthesis of punicic acid (18:3Δ9Z ,11E ,13Z ) a (11,14)-linoleoyl desaturase activity has been proposed. To isolate this acyl-lipid-desaturase, PCR-based cloning was used. This approach resulted in the isolation of two complete cDNAs. The first isolated full-length cDNA harbors a sequence of 1350 bp encoding a protein of 395 amino acids. The second cDNA was 1415 bp long encoding a protein of 387 amino acids. For functional identification proteins encoded by the cDNAs were expressed in Saccharomyces cerevisiae , and formation of newly formed fatty acids was analyzed by gas chromatography-free induction decay (GC-FID) and GC/MS. The expression of the heterologous enzymes resulted in the first case in a significant amount of linoleic acid and in the second case, after linoleic acid supplementation, in formation of punicic acid. The results presented here identify one cDNA coding for a classical Δ12 -acyl-lipid-desaturase. The other one codes for a new type of (1,4)-acyl-lipid-desaturase that converts a cis double bond located in the Δ12 -position of linoleic acid or γ-linolenic acid, but not in α-linolenic acid, into a conjugated cis–trans double bond system. [ABSTRACT FROM AUTHOR]

Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (earlier designated as GST-2) is related to sigma class GSTs and was previously described as an indirect flight muscle-associated protein with no known catalytic properties. We now report that DmGSTS1-1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB), but has relatively high glutathione-conjugating activity for 4-hydroxynonenal (4-HNE), an electrophilic aldehyde derived from lipid peroxidation. 4-HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases. Drosophila strains carrying P-element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4-HNE. In flies with both, one, or none of the GstS1 alleles disrupted by P-element insertion, there is a linear correlation between DmGSTS1-1 protein content and 4-HNE-conjugating activity. This correlation indicates that in adult Drosophila 70 ± 6% of the capacity to conjugate 4-HNE is attributable to DmGSTS1-1. The high abundance of DmGSTS1-1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress. Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S-transferases (e.g. GSTA4-4). The independent emergence of 4-HNE-conjugating activity in more than one branch of the glutathione S-transferase superfamily suggests that 4-HNE catabolism may be essential for aerobic life. [ABSTRACT FROM AUTHOR]

We have previously cloned cDNAs encoding the N-terminally extended class III human ubiquitin-conjugating enzymes (E2s), UBE2E2 and UBE2E3, the biological functions of which are not known. In this study, we performed yeast two-hybrid screening for protein(s) interacting with UBE2E2, and two RING-finger proteins, ARA54 and RNF8, were identified. Both ARA54, a ligand-dependent androgen receptor coactivator, and RNF8 interacted with class III E2s (UBE2E2, UbcH6, and UBE2E3), but not with other E2s (UbcH5, UbcH7, UbcH10, hCdc34, and hBendless) in the yeast two-hybrid assay. The use of various deletion mutants of UBE2E2 and RING-finger proteins and two RING point mutants, ARA54 C(220)S and RNF8 C(403)S, in which the RING structure is disrupted, showed that the UBC domain of UBE2E2 and the RING domain of these RING-finger proteins were involved in this association. Wild-type ARA54 and RNF8, expressed in insect Sf9 cells, catalyzed E2-dependent autoubiquitination in vitro, whereas the point mutated proteins showed markedly reduced activity. Ubiquitination of wild-type ARA54 and RNF8, expressed in COS-7 cells, was also observed, and a proteasome inhibitor, MG132, prevented the degradation of these wild-type proteins, but was much less effective in protecting the RING mutants. Transfection of COS-7 cells with a green fluorescent protein chimera showed that RNF8 was localized in the nucleus, and ARA54 in both the cytoplasm and nucleus. Our results suggest that ARA54 and RNF8 possibly act as Ub-ligases (E3) in the ubiquitination of certain nuclear protein(s). [ABSTRACT FROM AUTHOR]

Examines the characterization of a monkey steroid UDP-glucuronosyltransferase, UGT2B19. Role of UGT2B19 in the testosterone conjugation; Relevance of using monkey model in understanding steroid glucuronidation in extrahepatic target tissue; Construction of two cyclic DNA libraries.

Examines the role of water structure in conformational changes in nucleic acids in ambient and high-pressure conditions. Discussions on the mechanism of conformational transition nucleic acids; Induction of water activity by high concentrations of salt; Stabilization on the conjugative effects of cystidine.

A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase). MobB and MobC (Scherzinger, E., Lurz, R., Otto. S & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41-481. In this study we report the purification of MobA to apparent bomogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by sing nucleotides at the 5' or 3' end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3' side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5'-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3' end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex. [ABSTRACT FROM AUTHOR]

We have investigated the internalization step of an immunotoxin and its relationship with cytotoxicity, with the F(ab′)2-T101(ricin-A-chain) immunotoxin, directed against the CD5 antigen expressed on leukemic CEM cells. We first demonstrated that the biological action of the conjugate was related to its entry into the cell by an energy-dependent endocytotic process. We also found that during the first hours of cell intoxication, internalization is not the rate-limiting step of immunotoxin cytotoxicity. Internalization becomes limiting in cell intoxication only when the entry rate is low. Lastly we show that ammonium chloride, which strongly enhances immunotoxin potency, acts on internalized molecules for a very short time, suggesting that this enhancer affects an early intracellular step. [ABSTRACT FROM AUTHOR]

Recognition of substrate proteins by the ubiquitin-conjugation system is a highly specific and regulated event and involves the action of ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3). However, the E2 and E3 involved in the recognition of particular substrates have been identified in only a few cases. The ubiquitin-protein ligase E6-associated protein (E6-AP) was originally identified as a protein involved in the human papillomavirus E6-oncoprotein-induced degradation of p53. The substrate proteins of E6-AP in the absence of the E6 oncoprotein, however, have not been identified. We show here that E6-AP can target itself for ubiquitination in vitro and provide evidence that, under conditions of overexpression, E6-AP efficiently promotes its own degradation in vivo. Autoubiquitination of E6-AP is mediated mainly by intermolecular transfer of ubiquitin. In addition, highly ubiquitinated forms of E6-AP cannot bind to p53 in the presence of the E6 oncoprotein and, conversely, binding of E6-AP to p53 interferes with ubiquitination of E6-AP. These results suggest that autoubiquitination and subsequent degradation of E6-AP represents a mechanism to control intracellular E6-AP levels by inactivating E6-AP molecules that are not bound to substrate proteins. [ABSTRACT FROM AUTHOR]

Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1-chloro-2,4-dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH-transferase activity. Conjugation of 1-chloro-2,4-dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium. The results indicate the ability of rat lung to utilize extracellular GSH. [ABSTRACT FROM AUTHOR]

Kinetics of transport and metabolism of bromosulfophthalein have been studied in isolated liver cells in a dose-dependent manner obtaining the following results. The disposition of bromosulfophthalein in suspensions of isolated liver cells is similar to the turnover in the whole liver. The initial maximal rate of uptake of bromosulfophthalein is 2-3 times faster than intracellular conjugation with glutathione. Conjugation proceeds to an equilibrium between intracellutar substrate (bromosulfophthalein) and product (bromosulfophthalein-glutathione conjugate) which are both transiently accumulated in the cell. Formation of bromosulfophthaleinglutathione is accompanied by an equimolar decrease of glutathione. The bromosulfophthaleinglutathione conjugate is slowly released from the cells in an energy-dependent and saturable transport process. The maximal velocity of excretion amounts to only 6 % of the maximal velocity of uptake and to 20 % of the maximal velocity of conjugation. Excretion, therefore, represents the slowest step in the overall turnover. [ABSTRACT FROM AUTHOR]

Synthetic peptides were covalently attached to Sepharose and water-soluble dextran of high molecular weight. In these polysaccharide conjugates, the bound peptides bear a free α-amino group. The interaction of leucine aminopeptidase with its peptide ligands Thr-Phe-Pro (substrate) or Thr-(Bu¹)-Phe-Pro (competitive inhibitor) was strikingly different in soluble dextran conjugates and insoluble Sepharose conjugates of these ligands. The affinity of the enzyme to its inhibitor Thr(Bu¹)-Phe-Pro was decreased by more than 100-fold upon insolubilization of the peptide on Sepharose. When the peptide was coupled to dextran of weight-average molecular weight ≈500000, the potency of the inhibition was not impaired. Leucine aminopeptidase failed to hydrolyze the Sepharose-bound substrate Thr-Phe-Pro to a measurable extent, but it released N-terminal threonine from the soluble dextran conjugate of this peptide to an extent of at least 38%, of the theory. The interference, caused by the insoluble matrix was hardly overcome, when a new type of spacer, consisting of >80 DL-alanyl residues, was inserted between matrix and ligand. The spacer which is several enzyme subunit diameters in length, gave rise to a small fraction (about 5%) of accessible ligand but did not improve specific interaction to a sufficient extent in order to enable specific binding of leucine aminopeptidase to inhibitor-, or substrate-substituted Sepharoses. Nonspecific adsorption of the enzyme was observed in an inhibitor derivative of Sepharose with ε-aminohexanoic acid inserted as spacer. [ABSTRACT FROM AUTHOR]

The structure of the lipopolysaccharide in two hybrid strains formed between a Salmonella donor of O group B and a Sahnonella recipient of O group C2 was analyzed. Both the hybrids had the core-determining rfa genes from the C2 parent, and the lfb genes, which determine the biosynthesis of the O-units, from group B. The core was found to be modified by alkali-labile substituents in a way not found in group B and probably typical of group C2. As expected, the O side chain had O-acetylabequose, (O factor 5) when the group B oafA genes were present, and D-glucose linked to C-4 of D-galactose (O factor 122) when the group B (or D) oafR, E,F genes were present. Furthermore the terminal D-glucose was O-acetylated at C-2. The L-rhamnose of the side chain as well as its D-galactose were found in both strains to carry an alkali-labile group apparently corresponding to group C2-type modifications. It was inferred that the genes determining these modifications were not alleles of the known oaf genes of group B. The previous conclusion, that the lipopolysaccharide-modifying genes function independently to modify, e.g. by O-acetylation or glucosylation, any sort of lipopolysaccharide offered to them, provided this has suitable attachment sites for the substituents, gets further support from these results. [ABSTRACT FROM AUTHOR]

The structure of the lipopolysaccharide in two hybrid strains formed between a Salmonella donor of O group B and a Sahnonella recipient of O group C2 was analyzed. Both the hybrids had the core-determining rfa genes from the C2 parent, and the lfb genes, which determine the biosynthesis of the O-units, from group B. The core was found to be modified by alkali-labile substituents in a way not found in group B and probably typical of group C2. As expected, the O side chain had O-acetylabequose, (O factor 5) when the group B oafA genes were present, and D-glucose linked to C-4 of D-galactose (O factor 122) when the group B (or D) oafR, E,F genes were present. Furthermore the terminal D-glucose was O-acetylated at C-2. The L-rhamnose of the side chain as well as its D-galactose were found in both strains to carry an alkali-labile group apparently corresponding to group C2-type modifications. It was inferred that the genes determining these modifications were not alleles of the known oaf genes of group B. The previous conclusion, that the lipopolysaccharide-modifying genes function independently to modify, e.g. by O-acetylation or glucosylation, any sort of lipopolysaccharide offered to them, provided this has suitable attachment sites for the substituents, gets further support from these results. [ABSTRACT FROM AUTHOR]

A hybrid between two Salmonella groups, B and C2, was constructed by conjugation, and its cell-wall lipopolysaccharide analyzed. This hybrid had from group B the rfb gene cluster, which deterges the synthesis of the O-specific units with the structure abequosyl-mannosylrhamnosyl-galactose corresponding to O antigens 4,12. Other genes affecting lipopolysaccharide synthesis were of group C2 origin, and thus the lipopolysaccharide consisted of C2-determined core with single B-determined O-specific units, possibly modified by modification genes of group C2. It turned out that the B-type O-specific unit of the hybrid was modified by an O-acetyl group attached to its L-rhamnose, a modification seen in the group C2 but not in the group-B parent. [ABSTRACT FROM AUTHOR]

A hybrid between two Salmonella groups, B and C2, was constructed by conjugation, and its cell-wall lipopolysaccharide analyzed. This hybrid had from group B the rfb gene cluster, which deterges the synthesis of the O-specific units with the structure abequosyl-mannosylrhamnosyl-galactose corresponding to O antigens 4,12. Other genes affecting lipopolysaccharide synthesis were of group C2 origin, and thus the lipopolysaccharide consisted of C2-determined core with single B-determined O-specific units, possibly modified by modification genes of group C2. It turned out that the B-type O-specific unit of the hybrid was modified by an O-acetyl group attached to its L-rhamnose, a modification seen in the group C2 but not in the group-B parent. [ABSTRACT FROM AUTHOR]