Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2

PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain three catalytic motifs, conserved within the haloacid dehalogenase superfamily. Mutation of Asp32 and Asp203, key residues within two motifs, abolish PHOSPHO1 activity and confirm it as a member of this superfamily. We also show that Asp43 and Asp123, residues that line the substrate-binding site in our PHOSPHO1 model, are important for substrate hydrolysis. Further comparative modelling reveals that the active sites of PHOSPHO1 and PHOSPHO2 are very similar, but surprisingly, recombinant PHOSPHO2 hydrolyses phosphoethanolamine and phosphocholine relatively poorly. Instead, PHOSPHO2 shows high specific activity toward pyridoxal-5-phosphate (V(max) of 633 nmol min(-1) mg(-1) and K(m) of 45.5 microM). Models of PHOSPHO2 and PHOSPHO1 suggest subtle differences in the charge distributions around the putative substrate entry site and in the location of potential H-bond donors.     

Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.     

Mutations in the "lid" region affect chain length specificity and thermostability of a Pseudomonas fragi lipase

The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chain-length preference profile, increasing the relative activity towards C8 substrates. Moreover, mutations conferred to PFL higher temperature stability. On the other hand, replacement of the serine at position 141 by glycine destabilized the protein.     

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.     

Cytogenetic Analysis of Experimental Hybrids in Species of Triatominae (Hemiptera-Reduviidae)

A cytogenetic analysis was performed in experimental hybrids between species of Chagas disease transmitting bugs with remarkable differences in the amount and distribution of heterochromatin. Using C-banding technique, we identified the parental species chromosomes and analysed the meiotic behaviour in the male hybrids between Triatoma platensis and T. infestans, T. platensis and T. delpontei, and T. infestans and T. rubrovaria. The two former hybrids have an entirely normal meiotic behaviour despite the extensive differences in C-banded karyotypes observed in the parental species, indicating that heterochromatin differences between homeologous chromosomes are not a barrier that influences meiotic synapsis and recombination. On the contrary, the experimental hybrids between T. infestans and T. rubrovaria show failures in pairing of homeologous chromosomes that lead to the production of abnormal spermatids and hybrid sterility. Our data suggest that karyotypic repatterning within triatomines has involved at least two different pathways. Among closely related species, chromosomal changes have largely involved addition or deletion of heterochromatic regions. In more distant species, chromosomal rearrangements (i.e. inversions and translocations) have also arisen. Hybridisation data also allow to hypothesize about the origin and divergence of this taxonomic group, as well as the mechanisms that maintain species isolation.     

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.