[From gene to disease: cystinosis]. / Van gen naar ziekte; cystinose.

Cystinosis is an autosomal recessive disorder caused by an impaired transport of cystine out of lysosomes. The most severe infantile form of cystinosis starts with Fanconi syndrome at the age of 3-6 months. Untreated patients develop renal failure before the age of 10. The cystinosis gene (CTNS) maps to chromosome 17p13, spans 23 kb and is composed of 12 exons. CTNS encodes a 367 amino acid protein, cystinosin, which is a H(+)-driven lysosomal cystine transporter. It is enigmatic how lysosomal cystine accumulation induces the clinical symptoms. ATP depletion was demonstrated in an experimental model consisting of loading lysosomes with cystine dimethylester. The amino-thiol cysteamine depletes lysosomal cystine content by a disulfide-exchange reaction with cystine. Therapy with cysteamine should be administered as early as possible and continued after a renal transplantation as the extra renal damage still progresses. Improved life expectancy of cystinotic patients requires the attention of internists with a special interest for this rare disorder.

Decrease in S-adenosylmethionine synthesis by 6-mercaptopurine and methylmercaptopurine ribonucleoside in Molt F4 human malignant lymphoblasts.

6-Mercaptopurine (6-MP) and methylmercaptopurine ribonucleoside (Me-MPR) are purine anti-metabolites which are both metabolized to methylthio-IMP (Me-tIMP), a strong inhibitor of purine synthesis de novo. Me-MPR is converted directly into Me-tIMP by adenosine kinase. 6-MP is converted into tIMP, and thereafter it is methylated to Me-tIMP by thiopurine methyltransferase, an S-adenosylmethionine (S-Ado-Met)-dependent conversion. S-Ado-Met is formed from methionine and ATP by methionine adenosyltransferase, and is a universal methyl donor, involved in methylation of several macromolecules, e.g. DNA and RNA. Therefore, depletion of S-Ado-Met could result in an altered methylation state of these macromolecules, thereby affecting their functionality, leading to dysregulation of cellular processes and cytotoxicity. In this study the effects of 6-MP and Me-MPR on S-Ado-Met, S-adenosylhomocysteine (S-Ado-Hcy), homocysteine and methionine concentrations are determined. Both drugs cause a decrease in intracellular S-Ado-Met concentrations and an increase in S-Ado-Hcy and methionine concentrations in Molt F4 human malignant lymphoblasts. The effects of both 6-MP and Me-MPR can be ascribed to a decreased conversion of methionine into S-Ado-Met, due to the ATP depletion induced by the inhibition of purine synthesis de novo by Me-tIMP. Both 6-MP and Me-MPR thus affect the methylation state of the cells, and this may result in dysregulation of cellular processes and may be an additional mechanism of cytotoxicity for 6-MP and Me-MPR.

Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli.

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must apply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization. The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (tau c > 3 micros). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.

Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.