Biomedical subjects
Publications and source records attributed to A Buchberger.
The Hsp70 co-chaperone CHIP has recently gained attention as a regulator of protein turnover. CHIP has now been reported to be a component of the ubiquitination cascade, specifically an E3 ligase. CHIP appears to be part of a system that diverts incorrectly folded proteins from chaperones to the proteasome.
The UBX domain is an 80 amino acid residue module that is present typically at the carboxyl terminus of a variety of eukaryotic proteins. In an effort to elucidate the function of UBX domains, we solved the three-dimensional structure of the UBX domain of human Fas-associated factor-1 (FAF1) by NMR spectroscopy. The structure has a beta-Grasp fold characterised by a beta-beta-alpha-beta-beta-alpha-beta secondary-structure organisation. The five beta strands are arranged into a mixed sheet in the order 21534. The longer first helix packs across the first three strands of the sheet, and a second shorter 3(10) helix is located in an extended loop connecting strands 4 and 5. In the absence of significant sequence similarity, the UBX domain can be superimposed with ubiquitin with an r.m.s.d. of 1.9 A, suggesting that the two structures share the same superfold, and an evolutionary relationship. However, the absence of a carboxyl-terminal extension containing a double glycine motif and of suitably positioned lysine side-chains makes it highly unlikely that UBX domains are either conjugated to other proteins or part of mixed UBX-ubiquitin chains. Database searches revealed that most UBX domain-containing proteins belong to one of four evolutionarily conserved families represented by the human FAF1, p47, Y33K, and Rep8 proteins. A role of the UBX domain in ubiquitin-related processes is suggested.
Pinealectomy enhances tumor growth and metastatic spread in experimental animals. This effect is only in part due to melatonin since melatonin-free pineal extracts containing yet unidentified pineal substances have also shown tumor inhibiting activity. Despite numerous reports suggesting melatonin as a potential anti-cancer agent there have not been sufficient clinical trials to define the actual therapeutic potential of melatonin for the treatment of human cancers. To help fill this gap, we used a chemosensitivity assay designed to test the sensitivity of tumors from individual patients towards chemotherapeutic drugs for assessing the effect of melatonin and pineal extracts on primary human tumor cells. Primary cell cultures from seven ovarian and six mammary tumors were incubated with melatonin, the pineal extract YC05R (containing substances between 500 and 1000 daltons) and chemotherapeutic drugs. The pineal extract YC05R inhibited growth of all tumors in a dose-dependent manner. Physiological concentrations of melatonin (10(-8)-10(-10) M) inhibited the growth of one out of six mammary carcinomas in a dose-dependent manner. Primary cell cultures from three ovarian tumors were affected by melatonin in different ways, i.e., two were inhibited and one was slightly stimulated. There was no correlation between sensitivity towards melatonin and sex steroid receptor status, stage or grade of the tumor. It is concluded that, 1), melatonin may be an inhibitor of human mammary and ovarian carcinoma in individual cases and, 2), the pineal gland contains very active anti-tumor substances inhibiting both, the mammary and ovarian tumors, tested. These substances require chemical and biological identification.
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Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.
Comparative genome analysis may provide novel insights into gene evolution and function. To investigate the von Hippel-Lindau (VHL) disease tumor suppressor gene, we sequenced the VHL gene in seven primate species. Comparative analysis was performed for human, primate, and rodent VHL genes and for a putative Caenorhabditis elegans VHL homologue identified by database analysis. The VHL gene has two translation initiation sites (at codons 1 and 54); however, the relative importance of the full-length translation product (pVHL30) and that translated from the second internal translation initiation site (pVHL19) is unclear. The N-terminal sequence of pVHL30 contains eight copies of a GXEEX acidic repeat motif in human and higher primates, but only three copies were present in the marmoset, and only one copy was present in rodent VHL genes. Evolutionary analysis suggested that the N-terminal repetitive sequence in pVHL30 was of less functional importance than those regions present in both pVHL30 and pVHL19. The VHL gene product is reported to form complexes with various proteins including elongin B, elongin C, VBP-1, fibronectin, Spl, CUL2, and HIF-1. Although most of the regions in pVHL that had been implicated in binding specific proteins demonstrated evolutionary conservation, the carboxy-terminal putative VBP-1 binding site was less well conserved, suggesting that VBP-1 binding may have less functional significance. Although an amino acid substitution (K171T) close to the pVHL elongin binding region was found in baboon, analysis of the structure of human pVHL suggested that this substitution would not interfere with pVHL/elongin C interaction. In general, there was a good correlation between the pVHL domains that demonstrated most evolutionary conservation and those that were most frequently mutated in tumors. Analysis of human/C. elegans conservation and human germline and somatic mutation patterns identified a highly conserved mutation cluster region between codons 74 and 90. However, this region is likely to be important for the structural integrity of pVHL rather than representing an additional protein binding domain.
We have identified a novel chicken gene, cMespo, which encodes a basic-helix-loop-helix (bHLH) protein with sequence homology to a subgroup of bHLH transcription factors that have been implicated in somitogenesis. cMespo transcripts are first found in the primitive streak of gastrulating chick embryos (HH stage 4) and continue to accumulate in presomitic mesoderm (PSM) until somite formation has been concluded. cMespo, however, is not expressed within somites or in tailbud mesoderm. The expression domain of cMespo in PSM largely overlaps with delta-1 but spares a region of several prospective somites at the rostral end of PSM in which c-Meso and Cek-8 are expressed.
The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant, dnaK756, which rendered the cells resistant to lytic infection with bacteriophage lambda. The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivo and in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.
The basic helix-loop-helix (bHLH) transcription factor myogenin plays a crucial role in terminal differentiation of committed myoblasts into mature myocytes. Transcriptional activation of the myogenin gene requires coordinate action of myocyte enhancer factor 2 (MEF2) proteins and the myogenic bHLH regulators, MyoD or Myf5. Here we show that transcription of the myogenin gene in differentiated cells correlates with MEF2 and NF1 binding to their cognate sites in the proximal myogenin promoter but not with binding of Myf5 or MyoD to the E-box. The importance of MEF2 activity was further demonstrated by expression of antisense MEF2 RNA which repressed MEF2 and Myf5-mediated MEF2 site-dependent reporter gene activation and the synergistic transactivation of a myogenin CAT reporter by Myf5 and MEF2. Adenovirus E1A which has previously been shown to specifically interfere with myogenin gene transcription also inhibited the cooperative transactivation by Myf5/MEF2 and MEF2. Consistently, coimmunoprecipitation studies revealed impaired MEF2/Myf5 protein-protein interactions. These results support a model of transcriptional activation and stabilization of myogenin expression in which DNA-bound MEF2 recruits myogenic bHLH factors into an active but E1A-sensitive transcription factor complex.
Delusion as a phenomenon was always in the focus of psychiatric interest. Explanations for its origin reach from disturbed perception or affect to deficits in cognition. In our study we investigated 20 deluded, 20 depressive and 20 healthy subjects in order to find out differences in decision making, while a neutral test situation. Our hypothesis was that deluded subjects need less information for decision making and tend less to change their decision, made before, than both control groups will do this. For examination our hypothesis a modified version of "Probabilistic Inference Task" by Philips and Edwards was performed. In summary we found that deluded subjects need less information for decisions making than the control groups. Furthermore, decision making of deluded subjects seems more impulsive and less referring to formal logical criteria than it was found in depressed and healthy volunteers.
Muscle enhancer factor 2 (MEF2) proteins are important transcription factors for muscle-specific gene activation. Four family members are known in mammals, referred to as MEF2A, MEF2B, MEF2C, and MEF2D. Here we report the isolation and expression pattern of the chick Mef2a gene (cMef2a). cMef2a expression starts in precardiac mesoderm of HH stage 8 embryos. During further embryonic development expression continues in the heart tube and later in atrium and ventricle. A second cMef2a expression domain appears in somites of stage 13 embryos. Somitic cMef2a expression is limited to the myotome and is not found in newly formed somites until the muscle-specific transcription factors MyoD and myogenin are present. This suggests that activation of the cMef2a gene in skeletal muscle is dependent on these basic helix-loop-helix transcription factors. cMef2a expression in heart and skeletal muscle continues into adulthood when it is also seen in intestinal mesenchyme and in brain.
Previous studies on human breast cancer patients showed a decline in circulating melatonin levels corresponding to primary tumor growth and an increase when relapse occurred. The aim of the current investigation was to study in an experimental model possible mechanisms involved. Inbred female F344 Fischer rats were used for serial passages derived from a chemically induced mammary adenocarcinoma. Animals with slow-growing carcinosarcomas at passage 2 showed a significant elevation of nocturnal urinary melatonin (23. 00-07.00 h; +50%, p < 0.05) and a nominal increase in plasma melatonin (+41%; 02.00-03.00 h). By contrast, these parameters were significantly depressed in animals with fast-growing sarcomas (urinary melatonin: -22%, p < 0.025; plasma melatonin: -56%, p < 0. 01). At passage 2 nocturnal pineal N-acetylserotonin (02.00-03.00 h) was significantly enhanced (+62%, p < 0.05) probably due to an increased activity of serotonin-N-acetyltransferase (SNAT, +45%), the rate-limiting step of pineal melatonin biosynthesis converting serotonin to N-acetylserotonin. The activation of SNAT may be due to a stimulation of the sympathetic nervous system (urinary noradrenaline; NA: +243%, p < 0.005) when the cellular immune system responded towards tumor growth (urinary biopterin, +214%, p < 0.005). At passage 12 SNAT and N-acetylserotonin were unaffected but a depletion of plasma tryptophan (-34%, p < 0.0001), the precursor amino acid of melatonin, was found. The marginal decline in pineal serotonin (-18%, p < 0.05) disputes that the drastic depletion in circulating melatonin (-56%, p < 0.01) can be exclusively explained by a reduced availability of tryptophan. Therefore, the involvement of an additional mechanism has to be postulated, such as a degradation of melatonin via indoleamine 2,3-dioxygenase, an extrahepatic enzyme which has been detected in tumor tissue and is related to tryptophan 2,3-dioxygenase (TDO). TDO occurs only in the liver, is highly specific for L-tryptophan and is induced by glucocorticoids which would account for the observed depletion of plasma tryptophan resulting from a tumor-associated activation of the hypothalamo-pituitary-adrenal axis (urinary corticosterone +208%, p < 0.01). These findings present first explanations for the previously observed modulation of melatonin levels in cancer patients but also illustrate the high degree of complexity of mechanisms involved in the interactions between tumor growth and the immunoneuroendocrine system.
Hsp70 chaperones assist protein folding by ATP-controlled cycles of substrate binding and release. ATP hydrolysis is the rate-limiting step of the ATPase cycle that causes locking in of substrates into the substrate-binding cavity of Hsp70. This key step is strongly stimulated by DnaJ cochaperones. We show for the Escherichia coli Hsp70 homolog, DnaK, that stimulation by DnaJ requires the linked ATPase and substrate-binding domains of DnaK. Functional interaction with DnaJ is affected by mutations in an exposed channel located in the ATPase domain of DnaK. It is proposed that binding to this channel, possibly involving the J-domain, allows DnaJ to couple substrate binding with ATP hydrolysis by DnaK. Evolutionary conservation of the channel and the J-domain suggests conservation of the mechanism of action of DnaJ proteins.
The segmentation of somites from the paraxial mesoderm is a crucial event in vertebrate embryonic development; however, the mechanisms underlying this process are not well understood. In a yeast two-hybrid screen we have identified the novel basic-helix-loop-helix (bHLH) protein cMeso-1 which is expressed in the presomitic mesoderm of early chicken embryos. Initially the gene is activated in the epiblast and transcripts concentrate later in and around the primitive streak. When the segmental plate is laid down the cMeso-1 expression domain successively retracts toward the caudal end but a second domain appears in bilateral stripes in the anterior paraxial mesoderm. This highly dynamic domain of cMeso-1 transcripts demarcates the area immediately posterior to the next prospective pair of somites in cyclic waves which apparently correspond to the formation of new somites. Loss of cMeso-1 function by antisense RNA or oligonucleotides results in severe attenuation of somitogenesis suggesting that it plays an important role in setting up the segmentation process. The dynamic and periodically reiterated expression of cMeso-1 along the anteroposterior axis is not dependent on anterior structures or the propagation of a signal along the anteroposterior axis but seems to follow an intrinsic patterning program which is already set up in the segmental plate.
The cDNA cFKH-1 encodes a chicken winged helix/forkhead domain transcription factor that presents a dynamic expression pattern during chicken embryogenesis. Transcripts accumulate predominantly in early paraxial mesoderm, developing somites, and within mesenchymal precursors of skeletal structures. cFKH-1 RNA is first detected in the developing mesoderm of HH stage 6 embryos. During subsequent development cFKH-1 RNA accumulates in a dorsal domain of the anterior presomitic mesoderm and later in all cells of the epithelial somites before it becomes limited to the sclerotome when somites compartmentalise. cFKH-1 expression persists in the sclerotome, forming the vertebrae and in mesenchymal condensations in limb buds that will give rise later to the appendicular bones. In differentiated chondrocytes and definitive bone structures, however, cFKH-1 expression is down-regulated. Additional expression domains are found in mesenchyme of branchial arches and the head, in the dorsal aorta, and weakly in the endocardium. Based on its expression pattern and the structure of the forkhead DNA-binding domain cFKH-1 constitutes a chicken relative to the murine family of fkh-1/MF1 and MFH-1 factors. The embryonic expression of the cFKH-1 gene defines distinct mesodermal domains and suggests that it may regulate gene expression in mesenchymal cell lineages that will form cartilage in trunk and limb buds.
We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their common low-affinity receptor p75(neurotrophin receptor) (p75NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite-promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell-derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF-mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75NTR-overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor-overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high-affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low-affinity receptor in an autocrine fashion.
The differential diagnosis of psychogenic vs. organic amnestic syndromes may cause difficulty in certain cases. Here, we report a case of psychogenic amnesia which occurred after alcohol intoxication and mild head trauma. The initial memory deficit was very severe consisting of near-complete retrograde amnesia and anterograde amnesia covering 12 hours. The deficits resolved within a 4-week period of time. Brain CT and MRI scans revealed two circumscribed lesions of the right temporal lobe which were interpreted as old posttraumatic lesions. To ascertain the diagnosis, diffusion-weighted MR imaging (DWI) and brain perfusion SPECT were performed. The basal temporal lobes neither showed focal changes of perfusion, nor enhanced signal intensity on DWI as has been recently reported in patients with transient global amnesia. Later, the dissociative nature of the disorder could be confirmed by the exploration of recent psychological conflicts and the delayed type of recovery. We regard diffusion-weighted MRI as a powerful means to differentiate acute amnestic syndromes.
The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8-[4-[(N'-methylanthraniloyl)amino]butyl]-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes. The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation. The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.