46 research outputs found
From folding to function through compartmentalisation : influence of aminoacid changes and pyridoxal-5'-phosphate on the cell biology of alanine:glyoxylate aminotransferase.
From folding to function through compartmentalisation - influence of aminoacid changes and roles of pyridoxal-5'-phosphate on alanine:glyoxylate aminotransferase. Primary hyperoxaluria type 1 (PH1), is a rare inherited disease caused by a deficiency in the liver-specific, pyridoxal-5’-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT). AGT is normally localized to the peroxisomes where it catalyses the conversion of the intermediary metabolite glyoxylate to glycine. In a subset of patients with PH1, linked with the Gly170Arg mutation, AGT is mistargeted to mitochondria and patients may respond to pharmacological doses of pyridoxine, a precursor of PLP. Using a model system of stably transformed CHO cells expressing different AGT variants, we have studied four mutations – Gly170Arg, Phe152Ile, Ile244Thr, Gly41Arg - on the background of a common polymorphism, Pro11Leu, characteristic of a minor AGXT allele. All four mutations unexpectedly result in the peroxisome-to-mitochondrion mistargeting of AGT. However, significant differences were found on AGT quaternary status and catalytic activity. These results emphasize the synergistic effect of the Pro11Leu polymorphism and disease-causing mutations and its potential to act as a mitochondrial targeting signal. Varying the concentration of pyridoxine in cells had a biphasic effect on AGT expression, activity and compartmentalization that differed between constructs. Where some mutants had improved function with additional pyridoxine, normal AGT was inhibited at higher levels. The results provide an explanation for the therapeutic effect of pyridoxine in some patients. Overall, PLP was shown to have both chaperone and prosthetic group effects on mutant AGTs. Using a specially developed cell-based assay of indirect glycolate toxicity, we have shown the lower ability of mutant AGTs to detoxify glyoxylate. This is the basis for a functional assay of AGT in mammalian cells which could be used to screen drugs targeted at AGT
Disease recurrence in paediatric renal transplantation
Renal transplantation (Tx) is the treatment of choice for end-stage renal disease. The incidence of acute rejection after renal Tx has decreased because of improving early immunosuppression, but the risk of disease recurrence (DR) is becoming relatively high, with a greater prevalence in children than in adults, thereby increasing patient morbidity, graft loss (GL) and, sometimes, mortality rate. The current overall graft loss to DR is 7–8%, mainly due to primary glomerulonephritis (70–80%) and inherited metabolic diseases. The more typical presentation is a recurrence of the full disease, either with a high risk of GL (focal and segmental glomerulosclerosis 14–50% DR, 40–60% GL; atypical haemolytic uraemic syndrome 20–80% DR, 10–83% GL; membranoproliferative glomerulonephritis 30–100% DR, 17–61% GL; membranous nephropathy ∼30% DR, ∼50% GL; lipoprotein glomerulopathy ∼100% DR and GL; primary hyperoxaluria type 1 80–100% DR and GL) or with a low risk of GL [immunoglobulin (Ig)A nephropathy 36–60% DR, 7–10% GL; systemic lupus erythematosus 0–30% DR, 0–5% GL; anti-neutrophilic cytoplasmic antibody (ANCA)-associated glomerulonephritis]. Recurrence may also occur with a delayed risk of GL, such as insulin-dependent diabetes mellitus, sickle cell disease, endemic nephropathy, and sarcoidosis. In other primary diseases, the post-Tx course may be complicated by specific events that are different from overt recurrence: proteinuria or cancer in some genetic forms of nephrotic syndrome, anti-glomerular basement membrane antibodies-associated glomerulonephritis (Alport syndrome, Goodpasture syndrome), and graft involvement as a consequence of lower urinary tract abnormality or human immunodeficiency virus (HIV) nephropathy. Some other post-Tx conditions may mimic recurrence, such as de novo membranous glomerulonephritis, IgA nephropathy, microangiopathy, or isolated specific deposits (cystinosis, Fabry disease). Adequate strategies should therefore be added to kidney Tx, such as donor selection, associated liver Tx, plasmatherapy, specific immunosuppression protocols. In such conditions, very few patients may be excluded from kidney Tx only because of a major risk of DR and repeated GL. In the near future the issue of DR after kidney Tx may benefit from alternatives to organ Tx, such as recombinant proteins, specific monoclonal antibodies, cell/gene therapy, and chaperone molecules
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PR - Sustainable Management Of Varietal Resistance To Blackleg In Rapeseed
Blackleg, a major disease in rapeseed, can be managed using varietal resistance. However, to minimize resistance breakdown, other management techniques must also be used. We studied current agricultural systems used to manage rapeseed and blackleg on farms by means of a survey in two French regions. The data obtained were summarized and eight main systems were identified with an associated risk level of resistance breakdown. Rapeseed return time and varietal diversity were not the only techniques used by farmers to manage blackleg. Each system was also characterised for its capability of integrating current agronomic advice. Risky types were not always found in areas where blackleg was relatively rare. Less risky types would be able to improve their management whereas risky types may not have any leeway to include agronomic advice in their system. These data are now used to develop farming systems integrating agronomic advice and farmer’s objectives and resources
The N-terminal extension is essential for the formation of the active dimeric structure of liver peroxisomal alanine:glyoxylate aminotransferase
Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation
Pyridoxamine and pyridoxal are more effective than pyridoxine in rescuing folding-defective variants of human alanine:glyoxylate aminotransferase causing primary hyperoxaluria type I.
Vitamin B6 in the form of pyridoxine (PN) is one of the most widespread pharmacological therapies for inherited diseases involving pyridoxal phosphate (PLP)-dependent enzymes, including primary hyperoxaluria type I (PH1). PH1 is caused by a deficiency of liver-peroxisomal alanine: glyoxylate aminotransferase (AGT), which allows glyoxylate oxidation to oxalate leading to the deposition of insoluble calcium oxalate in the kidney. Only a minority of PH1 patients, mostly bearing the F152I and G170R mutations, respond to PN, the only pharmacological treatment currently available. Moreover, excessive doses of PN reduce the specific activity of AGT in a PH1 cellular model. Nevertheless, the possible effect(s) of other B6 vitamers has not been investigated previously. Here, we compared the ability of PN in rescuing the effects of the F152I and G170R mutations with that of pyridoxamine (PM) and PL. We found that supplementation with PN raises the intracellular concentration of PN phosphate (PNP), which competes with PLP for apoenzyme binding leading to the formation of an inactive AGT-PNP complex. In contrast, PNP does not accumulate in the cell upon PM or PL supplementation, but higher levels of PLP and PM phosphate (PMP), the two active forms of the AGT coenzyme, are found. This leads to an increased ability of PM and PL to rescue the effects of the F152I and G170R mutations compared with PN. A similar effect was also observed for other folding-defective AGT variants. Thus, PM and PL should be investigated as matter of importance as therapeutics for PH1 patients bearing folding mutations
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PR - Comparing Maize Management And Coexistence Means Between GM And Non GM Maize In Two French Regions (p103-110)
We compared how coexistence in maize at the farm level was i) envisioned in the east of France where GM have never been really cropped, and ii) managed in the south of France where GM maize was cropped in 2006 and 2007. Surveys in respectively 27 and 23 contrasted farms were made in maize-rich sub-regions with high maize pest pressure. Sampling farms for diversity, we used semi-directive interviews with two different protocols for the 2 regions. After data analysis, we identified factors explaining the choice of GM or NGM maize cropping like: presence of maize specialty crops (no GM), presence of other technical scope of progress for maize yield (no GM), perception of corn borer as a risk (more GM) or return time of maize (more GM when maize is frequent). We also identified the different coexistence means preferred according to the region: buffer zone in the South of France with its large fields and sowing period shifts in the East of France where available sowing days are plenty in the spring. We also found out that the results of the first year of testing the GM crop may be the main factor explaining GM maize adoption or abandonment. We will use these results to build a multi-criteria model of maize allocation in a small region