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Biomedical subjects

T Langer

Publications and source records attributed to T Langer.

At least 19 recordsLinked to original sources
PubMed1996

Regulated protein degradation in mitochondria.

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of the Escherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, the m- and the i-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, the m-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.

Adenosine Triphosphate↗
PubMed1996

Molecular basis of peripheral vs central benzodiazepine receptor selectivity in a new class of peripheral benzodiazepine receptor ligands related to alpidem.

Alpidem (1), the anxiolytic imidazopyridine, has nanomolar binding affinity for both the central benzodiazepine receptor (CBR) and the peripheral benzodiazepine receptor (PBR). A novel class of PBR ligands related to alpidem has been designed by comparing the interaction models of alpidem with PBR and CBR. Several compounds in this class have shown high selectivity for PBR vs CBR, and the selectivity has been discussed in terms of interaction models. The binding behavior of the three selected compounds was extensively studied by competition and saturation assays, and the results suggest that they are capable of recognizing two sites labeled by [3H]PK11195. The molecular structure of one of the most active compounds (4e) has been determined by X-ray diffraction and compared with that of alpidem. Molecular modeling studies suggest that the bioactive conformation of 4e is likely to be very similar to the conformation found in the crystal.

Animals↗
PubMed1996

On the bioisosteric potential of diazines: diazine analogues of the combined thromboxane A2 receptor antagonist and synthetase inhibitor Ridogrel.

In this SAR study the bioisosteric potential of diazines in the field of combined antithrombotic thromboxane A2 synthetase inhibitors and receptor antagonists was investigated. In this context, two series of (E)- and (Z)-omega-[[(aryldiazinylmethylene)amino]oxy]alkanoic acids were synthesized of which pentanoic acid derivatives with a 2-pyrazinyl, 4-pyridazinyl, or 5-pyrimidinyl group were found to exhibit this dual activity, while 4-pyrimidinyl as well as 3-pyridazinyl analogues showed only receptor antagonistic activity and 2-pyrimidinyl congeners were inactive. In the series of diazine analogues of Ridogrel (1), replacement of the 3-pyridyl group by a 2-pyrazinyl, 4-pyridazinyl, or 5-pyrimidinyl moiety led to compounds that inhibit thromboxane A2 synthetase in gel-filtered human platelets comparable to 1 (IC50 of 0.006, 0.016, and 0.039 microM, respectively, versus 0.007 microM). Radioligand-binding studies with [3H]SQ 29,548 in washed human platelets revealed that these diazine analogues block the thromboxane A2 receptor with an IC50 of 11, 6.0, and 1.5 microM, respectively. This compares well with the IC50 = 1.7 microM of 1. Finally, testing of inhibition of collagen-induced platelet aggregation in human platelet aggregation in human platelet-rich plasma with 2-pyrazinyl, 4-pyridazinyl, or 5-pyrimidinyl congeners of Ridogrel indicated that these heteroaromatic moieties may serve as bioisosteric substitutes of a 3-pyridyl group in dual-acting antiplatelet agents.

Blood Platelets↗
PubMed1996

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m-AAA protease, a protease complex consisting of multiple copies of the ATP-dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP-dependent metallopeptidase activity. While the m-AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed 'i-AAA protease'. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane-spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.

ATP-Dependent Proteases↗
PubMed1996

The YTA10-12 complex, an AAA protease with chaperone-like activity in the inner membrane of mitochondria.

The mitochondrial members of the highly conserved AAA family, Yta10p and Yta12p, constitute a membrane-embedded complex of about 850 kDa. As an ATP dependent metallopeptidase (AAA protease), the YTA10-12 complex mediates the degradation of nonassembled inner membrane proteins. In contrast to nucleotide-dependent complex formation and substrate binding, proteolysis of bound polypeptides depends on the hydrolysis of ATP and the metallopeptidase activity of both subunits. Independent of its proteolytic function, the chaperone-like activity of the YTA10-12 complex is required for assembly of the membrane-associated ATP synthase. We propose that proteolytic and chaperone-like activities in the YTA10-12 complex mediate assembly and degradation processes of membrane protein complexes and thereby exert key functions in the maintenance of membrane integrity.

Adenosine Triphosphatases↗
PubMed1996

Substitution of PIM1 protease in mitochondria by Escherichia coli Lon protease.

PIM1 protease in mitochondria belongs to a conserved family of ATP-dependent proteases, which includes the Escherichia coli Lon protease. Yeast cells lacking PIM1 are largely defective in degrading misfolded proteins in the mitochondrial matrix, are respiratory deficient, and lose integrity of mitochondrial DNA. In order to analyze whether E. coli Lon protease is functionally equivalent to mitochondrial PIM1 protease, yeast cells lacking the PIM1 gene were transformed with a construct consisting of a mitochondrial targeting sequence fused onto the Lon protease. In these cells, the fusion protein was expressed and imported into mitochondria, and the targeting sequence was removed. In the absence of PIM1 protease, the E. coli Lon protease mediated the degradation of misfolded proteins in the matrix space in cooperation with the mitochondrial hsp70 system. These cells maintained the integrity of the mitochondrial genome and the respiratory function at 30 degrees C but not at 37 degrees C. Stabilization of mitochondrial DNA in Deltapim1 cells depended on protein degradation by the E. coli Lon protease, as a proteolytically inactive Lon variant was not capable of substituting for a loss of PIM1 protease. These results demonstrate functional conservation of Lon-like proteases from prokaryotes to eukaryotes and shed new light on the role of Lon-like proteases in mitochondrial biogenesis.

ATP-Dependent Proteases↗
PubMed1996

Role of the mitochondrial DnaJ homologue, Mdj1p, in the prevention of heat-induced protein aggregation.

The role of the mitochondrial Hsp70 system in the prevention of heat-induced protein aggregation was studied in isolated mitochondria from Saccharomyces cerevisiae. Firefly luciferase was employed as a thermolabile tester protein. After shift to 40 degrees Celsius transient increase of mt-Hsp70/luciferase complex was observed, which required functional Mdj1p and Mge1p, the mitochondrial homologues of DnaJ and GrpE. The kinetics of luciferase aggregation, however, were not influenced by mutations in either mt-Hsp70 or Mge1p. Only the absence of Mdj1p led to enhanced protein aggregation. Thus, a central role in the transient protection against heat stress is attributed to this mitochondrial DnaJ homologue.

Bacterial Proteins↗
PubMed1996

Molecular structure and dynamics of some potent 5-HT3 receptor antagonists. Insight into the interaction with the receptor.

The molecular structure and the dynamic behaviour of some potent 5-HT3 antagonists structurally related to quipazine have been investigated by NMR spectroscopy and by computational methods in order to gain insight into the structure-activity relationships at a molecular level. The role of the different dynamic behaviour of these compounds in the binding to 5-HT3 receptors is discussed. A model of ligand-receptor interaction has been developed on the basis of molecular orbital calculations and on the reference ligands quipazine, ondansetron and LY278584. The interaction model proposed herein rationalizes the observed agonist-antagonist shift between quipazine and investigated compounds with the assumption of different but overlapping binding domains for antagonists and agonists at the 5-HT3 receptor.

Affinity Labels↗
PubMed1996

Quantitative EEG in long-term survivors of acute lymphoblastic leukemia.

Conventional and quantitative aspects of electroencephalographic recordings obtained during a follow-up surveillance study in long-term survivors of acute lymphoblastic leukemia in childhood were investigated with respect to differences in central nervous system prophylaxis given during antileukemic therapy and compared with data derived from healthy controls. Central nervous system prophylaxis consisted either of cranial irradiation (18 Gray, group A, n = 8) or intermediate high-dose methotrexate (2000 mg/m2; group B, n = 5), each combined with intrathecal methotrexate. Conventional electroencephalographic analysis revealed comparable results in all three study groups. However, quantitative electroencephalography showed significantly increased absolute power scores for all frequency bands in both long-term survivor groups. Relative power estimates revealed a significant increase in delta/tau activities in both prophylaxis groups compared to healthy controls, which were countered by decreased percentage power scores in the alpha-range. Quantitative electroencephalographic comparisons between both central nervous system prophylaxis groups revealed only small differences in quantity, not quality, of the observed power disturbances with slightly higher deviations in irradiated long-term survivors than in nonirradiated ones. Topographical distributions of spectral band power were comparable between all three study groups without evidence for therapy-related topographical differences.

Adolescent↗
PubMed1996

A competitive enzyme immunoassay for the pyrrolizidine alkaloids of the senecionine type.

We developed an immunoassay with antibodies against retrorsine to detect the closely related senecionine, the main alkaloid in various Asteraceae. Sensitivity is about 23 pg (68 fmol) for senecionine. Cross reactivity of monocrotalin, retrorsine N-oxide, senkirkine (< 0.1%), and seneciphylline (3.6-34.5%) was resolved. The alkaloid content in leaves of Petasites hybridus (Asteraceae) was determined to be 3.86 ppm, calculated as senecionine, which was far less than in rhizomes (104.8 ppm). Additionally, we proved specificity by taking immunograms from different parts of Petasites hybridus. The test is a suitable tool for assessing the toxic potential of medicinal plants containing senecionine.

Animals↗
PubMed1996

Visual-evoked potentials in long-term survivors of acute lymphoblastic leukemia in childhood. The German Late Effects Working Group.

Visual-evoked potentials (VEPs) were studied in 92 asymptomatic long-term survivors of acute lymphoblastic leukemia (ALL) in first complete remission 6-7 years after cessation of therapy in order to detect therapy-related disturbances in conduction velocity capacities within central nerve fibers. Subjects were grouped with respect to CNS prophylaxis: a) ith. MTX and 18 GY cranial irradiation (group A, n = 58), b) ith. MTX and iv. MHD-MTX, no cranial irradiation (group B, n = 34). At follow-up VEPs were abnormal in 10 subjects (10.9%) with comparable rates in both CNS prophylaxis groups (A: 7/58 [12.1%], B: 3/34 [8.8%]; p = 0.56). Participants with radiological signs of leukodystrophy (n = 14), all within the irradiated CNS prophylaxis group showed significantly prolonged P100 latencies and had a 6.1-fold increased relative risk to develop VEP disturbances. No correlation could be established between VEP outcome and illness- or treatment-related parameters. VEP outcome was not correlated with age at diagnosis or gender. Thus, VEP recordings showed a close relationship with radiation-induced CNS white-matter disturbances. However, their value for clinical practice, routinely performed follow-up evaluations or standardized posttreatment surveillance studies is negligible.

Age of Onset↗
PubMed1996

The molecular chaperone Hsp78 confers compartment-specific thermotolerance to mitochondria.

Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.

Cell Compartmentation↗
PubMed1995

Hsp78, a Clp homologue within mitochondria, can substitute for chaperone functions of mt-hsp70.

Hsp78 is a Clp homologue within mitochondria of Saccharomyces cerevisiae. Deletion of HSP78 does not cause any detectable changes in wild type cells, but results in a petite phenotype in the ssc1-3 mutant strain carrying a temperature-sensitive allele of mt-hsp70. When overexpressed in the ssc1-3 mutant strain, hsp78 suppresses the defect in mitochondrial protein import under permissive conditions in vitro and interacts directly with newly imported polypeptide chains. As a molecular chaperone, hsp78 prevents the aggregation of misfolded proteins in the matrix of mitochondria under conditions of impaired mt-hsp70 function. However, unlike misfolded proteins associated with mt-hsp70, hsp78-bound polypeptides are not efficiently degraded by the ATP-dependent PIM1 protease. Thus, hsp78 can partially substitute for mt-hsp70 functions in the assembly of mitochondria and may be part of a salvage pathway if mt-hsp70 is limiting.

Amino Acid Sequence↗
PubMed1995

Novel, potent, and selective 5-HT3 receptor antagonists based on the arylpiperazine skeleton: synthesis, structure, biological activity, and comparative molecular field analysis studies.

Synthesis and pharmacological evaluation of a series of condensed quinoline derivatives bearing a basic nitrogen on piperazine or [(dimethylamino)ethyl]thio moieties attached at the 2-position of the quinoline nucleus are described. 5-HT receptor binding studies revealed, for most of the compounds studied, nanomolar affinity for the 5-HT3 receptor subtype. The most active compound, benzopyrano[3,4-c]quinoline derivative 5f, displayed a Ki value very similar to that reported for quipazine along with an improved selectivity. Functional and in vivo testing carried out on three selected compounds showed that 5f,j,n are potent 5-HT3 receptor antagonists with potencies in the same range as the best known 5-HT3 receptor antagonists ondansetron, tropisetron, and zacopride. The crystal and molecular structures of compounds 5f,j,n were determined by single-crystal X-ray diffraction and used as starting structures for molecular modeling studies. Comparative molecular field analysis (CoMFA) was applied to binding constants of compounds 5a-p and 6a-h. The cross-validated r2, derived from partial least-squares calculations, indicated a good predictive capacity for affinity values in the series of compounds investigated. Evidence for the prediction capacity is provided in the form of plots of actual vs predicted pKi values. The steric and electrostatic features of the CoMFA-derived model are presented as standard coefficient contour maps of steric and electrostatic fields.

Animals↗
PubMed1995

Computer-aided molecular modeling, synthesis, and biological evaluation of 8-(benzyloxy)-2-phenylpyrazolo[4,3-c]quinoline as a novel benzodiazepine receptor agonist ligand.

Using computer-aided conformational analysis, based on molecular dynamics simulation, cluster analysis, and Monte Carlo techniques, we have designed and synthesized compounds in which a benzyloxy substituent has been incorporated into a series of pyrazoloquinoline benzodiazepine receptor (BZR) ligands. Earlier studies had shown that the benzyloxy group could act as part of the agonist pharmacophoric determinant in the beta-carboline ring system. Furthermore, the agonist beta-carboline had been correlated with a binding site orientation and volume fit for an agonist 6-phenylimidazobenzodiazepine carboxylate. The present study was undertaken to determine whether the benzyloxy substituent could be used as an agonist pharmacophoric descriptor for the phenylpyrazolo[4,3-c]quinolin-3-one BZR ligands. The results of a determination of GABA shift ratios for the synthetic ligands indicate that 8-(benzyloxy)-2-phenylpyrazolo[4,3-c]quinolin-3-one can be predicted to be an agonist at the BZR.

Animals↗
PubMed1994

Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.

ATP-Dependent Proteases↗
PubMed1994

The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE.

Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.

Adenosine Triphosphatases↗