Biomedical subjects

T Dierks

Publications and source records attributed to T Dierks.

At least 37 records · Page 2Linked to original sources

PubMed1999

The iron sulfur protein AtsB is required for posttranslational formation of formylglycine in the Klebsiella sulfatase.

The catalytic residue of eukaryotic and prokaryotic sulfatases is a alpha-formylglycine. In the sulfatase of Klebsiella pneumoniae the formylglycine is generated by posttranslational oxidation of serine 72. We cloned the atsBA operon of K. pneumoniae and found that the sulfatase was expressed in inactive form in Escherichia coli transformed with the structural gene (atsA). Coexpression of the atsB gene, however, led to production of high sulfatase activity, indicating that the atsB gene product plays a posttranslational role that is essential for the sulfatase to gain its catalytic activity. This was verified after purification of the sulfatase from the periplasm of the cells. Peptide analysis of the protein expressed in the presence of AtsB revealed that half of the polypeptides carried the formylglycine at position 72, while the remaining polypeptides carried the encoded serine. The inactive sulfatase expressed in the absence of AtsB carried exclusively serine 72, demonstrating that the atsB gene is required for formylglycine modification. This gene encodes a 395-amino acid residue iron sulfur protein that has a cytosolic localization and is supposed to directly or indirectly catalyze the oxidation of the serine to formylglycine.

Alanine↗

PubMed1999

Amino acid residues forming the active site of arylsulfatase A. Role in catalytic activity and substrate binding.

Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage.

Amino Acid Sequence↗

PubMed1999

Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases.

Sulfatases carry at their catalytic site a unique post-translational modification, an alpha-formylglycine residue that is essential for enzyme activity. Formylglycine is generated by oxidation of a conserved cysteine or, in some prokaryotic sulfatases, serine residue. In eukaryotes, this oxidation occurs in the endoplasmic reticulum during or shortly after import of the nascent sulfatase polypeptide. The modification of arylsulfatase A was studied in vitro and was found to be directed by a short linear sequence, CTPSR, starting with the cysteine to be modified. Mutational analyses showed that the cysteine, proline and arginine are the key residues within this motif, whereas formylglycine formation tolerated the individual, but not the simultaneous substitution of the threonine or serine. The CTPSR motif was transferred to a heterologous protein leading to low-efficient formylglycine formation. The efficiency reached control values when seven additional residues (AALLTGR) directly following the CTPSR motif in arylsulfatase A were present. Mutating up to four residues simultaneously within this heptamer sequence inhibited the modification only moderately. AALLTGR may, therefore, have an auxiliary function in presenting the core motif to the modifying enzyme. Within the two motifs, the key residues are fully, and other residues are highly conserved among all known members of the sulfatase family.

Alanine↗

PubMed1999

Multimodal imaging of residual function and compensatory resource allocation in cortical atrophy: a case study of parietal lobe function in a patient with Huntington's disease.

In a case of Huntington's disease (HD) with dementia and pronounced parieto-frontal atrophy, the functional state of the affected regions was investigated using functional magnetic resonance imaging (fMRI) and fluorodeoxyglucose-positron emission tomography (FDG-PET). It was observed that although parietal areas showed extensive atrophy and reduced resting glucose metabolism, the patient performed with similar accuracy but with longer response time in a visuospatial task compared with healthy control subjects. At the same time, the blood oxygen level-dependent (BOLD) fMRI signal in these areas, which are involved in visuospatial processing, showed a similar task-dependent modulation as in control subjects. The signal amplitude (signal percent change) of the task-dependent activation was even higher for the HD patient than in the control group. This residual functionality of parietal areas involved in visuospatial processing could account for the patient's performance in the task concerned, which contrasted with his poor performance in other cognitive tasks. The increased percent-signal change suggests that a higher neuronal effort was necessary to reach a similar degree of accuracy as in control subjects, fitting well with the longer reaction time. We propose that fMRI should be considered as a tool for the assessment of functionality of morphologically abnormal cortex and for the investigation of compensatory resource allocation in neurodegenerative disorders.

Atrophy↗

PubMed1999

Generators of brain electrical activity in patients with Wilson's disease.

Electroencephalographic (EEG) generators were investigated in 13 patients suffering from hepatolenticular degeneration with and without neurological symptoms and in 13 healthy subjects for comparison by the use of FFT approximation. Quantitative assessment of motor deficits and psychiatric disturbances was correlated with EEG features. We found mainly an increase in delta activity, a decrease in alpha activity combined with a more posterior localisation of the EEG generators in the delta band and a more anterior one in the alpha band in patients compared with healthy controls. The localisation of the EEG generators in the patients with clinical apparent neurological symptoms were in all frequency bands more superficial compared with controls and patients without neurological symptoms. With longer duration of the disease, the lower the premorbid intelligence the more posterior was the delta EEG generator localised. Although the alpha EEG generator was more anteriorly localised with longer duration of the disease and more severe cognitive deficits, it was more superficial with more pronounced psychiatric symptoms, more severe cognitive deficits, lower premorbid intelligence and more pronounced motor disabilities. With more pronounced psychiatric symptoms and cognitive deficits, the beta EEG generator was more anteriorly localised. The present study demonstrated that a significant deviant EEG pattern exists between patients with and without clinical neurological symptoms and that stage-dependent alterations in psychiatric symptoms and cognitive ability are reflected on the EEG.

Adult↗

PubMed1999

Activation of Heschl's gyrus during auditory hallucinations.

Apart from being a common feature of mental illness, auditory hallucinations provide an intriguing model for the study of internally generated sensory perceptions that are attributed to external sources. Until now, the knowledge about the cortical network that supports such hallucinations has been restricted by methodological limitations. Here, we describe an experiment with paranoid schizophrenic patients whose on- and offset of auditory hallucinations could be monitored within one functional magnetic resonance imaging (fMRI) session. We demonstrate an increase of the blood oxygen level-dependent (BOLD) signal in Heschl's gyrus during the patients' hallucinations. Our results provide direct evidence of the involvement of primary auditory areas in auditory verbal hallucinations and establish novel constraints for psychopathological models.

Acoustic Stimulation↗

PubMed1999

The functional neuroanatomy of target detection: an fMRI study of visual and auditory oddball tasks.

The neuronal response patterns that are required for an adequate behavioural reaction to subjectively relevant changes in the environment are commonly studied by means of oddball paradigms, in which occasional 'target' stimuli have to be detected in a train of frequent 'non-target' stimuli. The detection of such task-relevant stimuli is accompanied by a parietocentral positive component of the event-related potential, the P300. We performed EEG recordings of visual and auditory event-related potentials and functional magnetic resonance imaging (fMRI) when healthy subjects performed an oddball task. Significant increases in fMRI signal for target versus non-target conditions were observed in the supramarginal gyrus, frontal operculum and insular cortex bilaterally, and in further circumscribed parietal and frontal regions. These effects were consistent over various stimulation and response modalities and can be regarded as specific for target detection in both the auditory and the visual modality. These results therefore contribute to the understanding of the target detection network in human cerebral cortex and impose constraints on attempts at localizing the neuronal P300 generator. This is of importance both from a neurobiological perspective and because of the widespread application of the physiological correlates of target detection in clinical P300 studies.

Adult↗

PubMed1998

Multimodal imaging of residual function and compensatory resource allocation in cortical atrophy: a case study of parietal lobe function in a patient with Huntington's disease.

Adult↗

PubMed1998

Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A.

Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.

Alanine↗

PubMed1998

Posttranslational formation of formylglycine in prokaryotic sulfatases by modification of either cysteine or serine.

Eukaryotic sulfatases carry an alpha-formylglycine residue that is essential for activity and is located within the catalytic site. This formylglycine is generated by posttranslational modification of a conserved cysteine residue. The arylsulfatase gene of Pseudomonas aeruginosa also encodes a cysteine at the critical position. This protein could be expressed in active form in a sulfatase-deficient strain of P. aeruginosa, thereby restoring growth on aromatic sulfates as sole sulfur source, and in Escherichia coli. Analysis of the mature protein expressed in E. coli revealed the presence of formylglycine at the expected position, showing that the cysteine is also converted to formylglycine in a prokaryotic sulfatase. Substituting the relevant cysteine by a serine codon in the P. aeruginosa gene led to expression of inactive sulfatase protein, lacking the formylglycine. The machinery catalyzing the modification of the Pseudomonas sulfatase in E. coli therefore resembles the eukaryotic machinery, accepting cysteine but not serine as a modification substrate. By contrast, in the arylsulfatase of Klebsiella pneumoniae a formylglycine is found generated by modification of a serine residue. The expression of both the Klebsiella and the Pseudomonas sulfatases as active enzymes in E. coli suggests that two modification systems are present, or that a common modification system is modulated by a cofactor.

Alanine↗

PubMed1998

Longitudinal changes in quantitative EEG during long-term tacrine treatment of patients with Alzheimer's disease.

Quantitative EEG is a potentially useful tool in demonstrating the effects of treatments with acetylcholinesterase (AChE) inhibitors on the progression of Alzheimer's disease (AD). In order to define the profile of EEG changes during tacrine long-term treatment, for 12 months we followed 15 AD patients receiving an optimal individually tolerable dose. After 3 months theta global field power (GFP) was significantly reduced, and after 6 months both theta and delta GFP decreased. Theta GFP was still reduced after 12 months of treatment when compared to the baseline. Significant decreases in fast activities of beta 1 and beta 2 GFP were also observed. The untreated reference group (n = 10) did not show any significant changes in GFP after 12 months follow-up, although generators of theta activity had a significant shift towards posterior regions. These findings suggest that slowing in fast EEG frequencies during chronic treatment with AChE inhibitors may provide an early indicator of declining treatment efficiency.

Aged↗

PubMed1998

Sulfatases, trapping of the sulfated enzyme intermediate by substituting the active site formylglycine.

Sulfatases contain an active site formylglycine residue that is generated by post-translational modification. Crystal structures of two lysosomal sulfatases revealed significant similarity to the catalytic site of alkaline phosphatase containing a serine at the position of formylglycine. To elucidate the catalytic mechanism of sulfate ester hydrolysis, the formylglycine of arylsulfatases A and B was substituted by serine. These mutants upon incubation with substrate were covalently sulfated at the introduced serine. This sulfated enzyme intermediate was stable at pH 5. At alkaline pH it was slowly hydrolyzed. These characteristics are analogous to that of alkaline phosphatase which forms a phosphoserine intermediate that is stable at pH 5, but is hydrolyzed at alkaline pH. In wild-type sulfatases the hydroxyl needed for formation of the sulfated enzyme intermediate is provided by the aldehyde hydrate of the formylglycine. The second, non-esterified hydroxyl of the aldehyde hydrate is essential for rapid desulfation of the enzyme at acidic pH, which most likely occurs by elimination. The lack of this second hydroxyl in the serine mutants explains the trapping of the sulfated enzyme intermediate. Thus, in acting as a geminal diol the formylglycine residue allows for efficient ester hydrolysis in an acidic milieu.

Alanine↗

PubMed1998

Arylsulfatase from Klebsiella pneumoniae carries a formylglycine generated from a serine.

Eukaryotic sulfatases share an unusual posttranslational protein modification, which converts a cysteine into alpha-formylglycine. The alpha-formylglycine is essential for the catalytic activity. Klebsiella pneumoniae expresses an inducible arylsulfatase for which the DNA predicts a serine at the position occupied by the alpha-formylglycine residue in eukaryotic sulfatases. Structural analysis showed that the majority of the arylsulfatase polypeptides from K. pneumoniae carries the alpha-formylglycine, whereas the remaining arylsulfatase polypeptides contain the predicted serine residue. This demonstrates the evolutionary conservation between prokaryotes and eukaryotes of this novel protein modification that so far has been found only in sulfatases. alpha-Formylglycine in Klebsiella is generated from a serine and not from a cysteine as in eukaryotes.

Alanine↗

PubMed1998

Conversion of cysteine to formylglycine in eukaryotic sulfatases occurs by a common mechanism in the endoplasmic reticulum.

Sulfatases undergo an unusual protein modification leading to conversion of a specific cysteine residue into alpha-formylglycine. This conversion is essential for catalytic activity. In arylsulfatase A the alpha-formylglycine is generated inside the endoplasmic reticulum at a late stage of protein translocation. Using in vitro translation in the presence of transport-competent microsomes we found that arylsulfatase B is also modified in a similar way by the formylglycine-generating machinery. Modification depended on protein transport and on the correct position of the relevant cysteine. Arylsulfatase A and B did not compete for modification, as became apparent in co-expression experiments. This could argue for an association of the modification machinery with the protein translocation apparatus.

Alanine↗

PubMed1998

EEG slowing and cerebrospinal fluid tau levels in patients with cognitive decline.

We explored the relationship between cerebrospinal fluid (CSF) tau levels as indirect markers of tau-related pathology in Alzheimer's disease (AD) and EEG slowing, a typical neurophysiological finding in the disease. A positive correlation between CSF tau levels and ratio of alpha/delta global field power was found in 14 AD patients (r = 0.65, p = 0.01). This relationship was better approximated by polynomial fit of 2nd degree (p = 0.002). A subgroup of AD patients (n = 7) with higher tau levels and shorter duration of illness showed a strong relationship between CSF tau levels and alpha/theta (r = 0.83, p = 0.02), and alpha/delta (r = 0.87, p = 0.01) ratios of the global field power. There were no significant correlations between EEG slowing and CSF tau levels in 12 patients with mild cognitive dysfunction or in 14 healthy control subjects. That a strong inverse linear correlation exists in AD patients with higher levels of tau and shorter duration of illness may imply that with longer illness duration CSF tau levels decrease due to neuronal death.

Aged↗

PubMed1998

A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease.

In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides. Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family. This novel protein modification generates a 2-amino-3-oxopropanoic acid (C alpha-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases. The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded. The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate. One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate. The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group. In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine. Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the C alpha-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine.

Aldehydes↗

PubMed1998

Mutations in a polycistronic nuclear gene associated with molybdenum cofactor deficiency.

All molybdoenzymes other than nitrogenase require molybdopterin as a metal-binding cofactor. Several genes necessary for the synthesis of the molybdenum cofactor (MoCo) have been characterized in bacteria and plants. The proteins encoded by the Escherichia coli genes moaA and moaC catalyse the first steps in MoCo synthesis. The human homologues of these genes are therefore candidate genes for molybdenum cofactor deficiency, a rare and fatal disease. Using oligonucleotides complementary to a conserved region in the moaA gene, we have isolated a human cDNA derived from liver mRNA. This transcript contains an open reading frame (ORF) encoding the human moaA homologue and a second ORF encoding a human moaC homologue. Mutations can be found in the majority of MoCo-deficient patients that confirm the functional role of both ORFs in the corresponding gene MOCS1 (for 'molybdenum cofactor synthesis-step 1'). Northern-blot analysis detected only full-length transcripts containing both consecutive ORFs in various human tissues. The mRNA structure suggests a translation reinitiation mechanism for the second ORF. These data indicate the existence of a eukaryotic mRNA, which as a single and uniform transcript guides the synthesis of two different enzymatic polypeptides with disease-causing potential.

Amino Acid Sequence↗

PubMed1997

Conversion of cysteine to formylglycine: a protein modification in the endoplasmic reticulum.

In sulfatases a Calpha-formylglycine residue is found at a position where their cDNA sequences predict a cysteine residue. In multiple sulfatase deficiency, an inherited lysosomal storage disorder, catalytically inactive sulfatases are synthesized which retain the cysteine residue, indicating that the Calpha-formylglycine residue is required for sulfatase activity. Using in vitro translation in the absence or presence of transport competent microsomes we found that newly synthesized sulfatase polypeptides carry a cysteine residue and that the oxidation of its thiol group to an aldehyde is catalyzed in the endoplasmic reticulum. A linear sequence of 16 residues surrounding the Cys-69 in arylsulfatase A is sufficient to direct the oxidation. This novel protein modification occurs after or at a late stage of cotranslational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure.

Alanine↗