European journal of biochemistry最新文献

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.

The interferon-beta promoter has been studied extensively as a model system for combinatorial transcriptional regulation. In virus-infected cells the transcription factors ATF-2, c-Jun, interferon regulatory factor (IRF)-3, IRF-7 and NF-kappaB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters. It remains unclear, however, why most other combinations of AP-1, IRF and Rel proteins fail to activate the interferon-beta gene. Here we have explored how different IRFs may cooperate with other factors to activate transcription. First we showed in undifferentiated embryonic carcinoma cells that ectopic expression of either IRF-3 or IRF-7, but not IRF-1, was sufficient to allow virus-dependent activation of the interferon-beta promoter. Moreover, the activity of IRF-3 and IRF-7 was strongly affected by promoter context, with IRF-7 preferentially being recruited to the natural interferon-beta promoter. We fully reconstituted activation of this promoter in insect cells. Maximal synergy required IRF-3 and IRF-7 but not IRF-1, and was strongly dependent on the presence of p300/CBP, even when these coactivators only modestly affected the activity of each factor by itself. These results suggest that specificity in activation of the interferon-beta gene depends on a unique promoter context and on the role played by coactivators as architectural factors.

Several oncogenic proteins are known to influence cellular glycosylation. In particular, transfection of codon 12 point mutated H-Ras increases CMP-Neu5Ac: Galbeta1,4GlcNAc alpha2,6-sialyltransferase I (ST6Gal I) activity in rodent fibroblasts. Given that Ras mediates its effects through at least three secondary effector pathways (Raf, RalGEFs and PI3K) and that transcriptional control of mouse ST6Gal I is achieved by the selective use of multiple promoters, we attempted to identify which of these parameters are involved in linking the Ras signal to ST6Gal I gene transcription in mouse fibroblasts. Transformation by human K-Ras or H-Ras (S12 and V12 point mutations, respectively) results in a 10-fold increase in ST6Gal I mRNA, but no alteration in the expression of related sialyltransferases. Using an inducible H-RasV12 expression system, a direct causal link between activated H-Ras expression and elevated ST6Gal I mRNA was demonstrated. The accumulation of the ST6Gal I transcript in response to activated Ras was accompanied by an increase of alpha2,6-sialyltransferase activity and of Neu5Acalpha2,6Gal at the cell surface. Results obtained with H-RasV12 partial loss of function mutants H-RasV12S35 (Raf signal only), H-RasV12C40 (PI3-kinase signal only) and H-RasV12G37 (RalGEFs signal only) suggest that the H-Ras induction of the mouse ST6Gal I gene (Siat1) transcription is primarily routed through RalGEFs. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase in ST6Gal I mRNA upon H-RasV12 or K-RasS12 transfection is mediated by the Siat1 housekeeping promoter P3-associated 5' untranslated exons.

The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.

The tissue-specific transcription factors HNF1alpha and HNF1beta are closely related homeodomain proteins conserved in vertebrate evolution. Heterozygous mutations in human HNF1beta but not in HNF1alpha genes are associated with kidney malformations. Overexpression of HNF1beta in Xenopus embryos leads to defective pronephros development, while HNF1alpha has no effect. We have defined the regions responsible for this functional difference between HNF1beta and HNF1alpha in transfected HeLa cells as well as in injected Xenopus embryos. Using domain swapping experiments, we located a nuclear localization signal in the POUH domain of HNF1beta, and showed that the POUS and POUH domains of HNF1beta mediate a high transactivation potential in transfected cells. In injected Xenopus embryos three HNF1beta domains are involved in nephrogenesis. These include the dimerization domain, the 26 amino acid segment specific for splice variant A as well as the POUH domain. As HNF1beta together with Pax8 and lim1 constitute the earliest regulators in the pronephric anlage, it is possible that they cooperate during early nephrogenesis. We have shown here that HNF1beta can overcome the enlargement and the induction of an ectopic pronephros mediated by overexpression of Pax8 and lim1. However, the phenotype induced by Pax8 and lim1 overexpression and characterized by cyst-like structures and thickening of the pronephric tubules was not altered by HNF1beta overexpression. Taken together, HNF1beta acts antagonistically to Pax8 and lim1 in only some processes during nephrogenesis, and a simple antagonistic relationship does not completely describe the functions of these genes. We conclude that HNF1beta has some distinct morphogenetic properties during nephrogenesis.