Biomedical subjects

A Buchberger

Publications and source records attributed to A Buchberger.

At least 37 records · Page 2Linked to original sources

PubMed1997

GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism.

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.

Adenosine Diphosphate↗

PubMed1997

Chicken NKx2-8, a novel homeobox gene expressed during early heart and foregut development.

cNkx2-8 represents a novel member of the NK2-family transcription factors. The gene contains three highly conserved regions, the TN-, NK2-, and homeodomains which are diagnostic for this group of proteins. cNkx2-8 is expressed during chick embryogenesis in ventral foregut endoderm, myocardial mesoderm, epithelium of the branchial arches and the dorsal mesocardium. While cNkx2-8 expression partially overlaps with other NK genes, such as Nkx2-5 and Nkx2-3, its onset and aspects of its expression domains are specific. Thus, structural data and the expression profile suggest that cNkx2-8 constitutes a new homeobox protein which may cooperate with its known relatives in defining an antero-ventral field including the developing heart and pharyngeal endoderm.

Animals↗

PubMed1997

Interaction of Hsp70 chaperones with substrates.

Determination of the structure of the substrate binding domain of the Escherichia coli Hsp70 chaperone, DnaK, and the biochemical characterisation of the motif it recognizes within substrates provide insights into the principles governing Hsp70 interaction with polypeptide chains. DnaK recognizes extended peptide strands composed of up to five consecutive hydrophobic residues within and positively charged residues outside the substrate binding cavity.

Binding Sites↗

PubMed1996

Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding.

GroEL and DnaK with their cofactors constitute the major chaperone systems promoting protein folding in the Escherichia coli cytosol. The ability of GroEL to bind and promote folding of a substrate released from DnaK led to the proposal that the DnaK and GroEL systems act successively along a protein folding pathway. Here we have investigated the role of both systems in preventing aggregation and assisting refolding of firefly luciferase denatured by guanidinium chloride and heat. We find that DnaK and GroEL compete with each other for binding to non-native luciferase. Addition of ATP and co-operating proteins results in release of luciferase from either chaperone in a non-native conformation. Only a small fraction of luciferase molecules released from GroEL can reach the native state. Instead, the released luciferase must bind repeatedly to the DnaK system, and only then is it able to fold to the native state. Thus, during a folding reaction, DnaK and GroEL do not obligatorily act in succession by promoting earlier and later protein folding steps, respectively. Rather, the two chaperone systems and perhaps others can form a lateral network of co-operating proteins. This chaperone network is proposed to be of particular importance for the assisted refolding of proteins that are unfolded by stress treatment such as heat shock and whose size is too large to allow folding inside the substrate binding cavity of the GroEL ring underneath GroES.

Bacterial Proteins↗

PubMed1996

Chick NKx-2.3 represents a novel family member of vertebrate homologues to the Drosophila homeobox gene tinman: differential expression of cNKx-2.3 and cNKx-2.5 during heart and gut development.

NKx homeodomain proteins are members of a growing family of vertebrate transcription factors with strong homology to the NK genes in Drosophila. Here, we describe the cloning of cNKx-2.3 and cNKx-2.5 cDNAs and their expression during chick development. Both genes are expressed in the developing heart with distinct but overlapping spatio-temporal patterns. While cNKx-2.5 is activated in early precardiac mesoderm and continues to be uniformly expressed throughout the mature heart, expression of NKx-2.3 starts later in differentiated myocardial cells with regional differences compared to NKx-2.5. Additionally, both genes are expressed in adjacent domains of the developing mid- and hindgut mesoderm as well as in branchial arches. The highly conserved structure of cNKx-2.5 and its similar expression to mouse and Xenopus NKx-2.5 genes and to the Drosophila gene tinman argue that it constitutes the chick homologue of these genes. Different temporal and spatial activity of cNKx-2.3 in heart and gut as well as in a regionally restricted expression domain in the neural tube suggest that cNKx-2.3 is a member of the NK-2 gene family which may be involved in specifying mesodermally and ectodermally derived cell types in the embryo.

Animals↗

PubMed1996

Expression of nerve growth factor and neurotrophin receptors in testicular cells suggest novel roles for neurotrophins outside the nervous system.

The present study was designed to clarify the non-neurotrophic role for neurotrophins in mouse testis. By means of SI nuclease protection assay we could demonstrate that the gene coding for the low-affinity nerve growth factor (NGF) receptor p75NGFR is transiently expressed during germ cell development. Gene expression for p75NGFR was detected in late-meiotic spermatocytes and early spermatids and was found to be co-expressed with trkB and trkC, two tyrosine kinase receptors, commonly regarded as the high-affinity receptors for brain-derived neurotrophic factor and neurotrophin-3. Gene transcripts for the high-affinity NGF receptor trkA were found exclusively in non-germ cells. Isolated Leydig cells, peritubular myoid cells and Sertoli cells, but not germ cells, could be identified as potential testicular NGF sources. Non-germ cells respond after incubation for several days with a sharp induction in NGF synthesis, which is accompanied by a loss of phenotypic expression patterns. The fact that p75NGFR mRNA expression was induced in cultured Sertoli cells and peritubular myoid cells suggests an autocrine mode of NGF action in these cells. Induction of NGF synthesis in cultured Leydig cells could be prevented by the glucocorticoid dexamethasone. Results indicate different roles for the individual neurotrophins in distinct testicular compartments and suggest that these neurotrophins might support testicular functions by signalling between individual cell types in an autocrine and paracrine manner.

Animals↗

PubMed1995

Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication.

Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.

Adenosine Diphosphate↗

PubMed1995

The role of ATP in the functional cycle of the DnaK chaperone system.

Hsp70 chaperons interact with protein substrates in an ATP-dependent manner to prevent aggregation and promote protein folding. For the Escherichia coli homolog DnaK, we have characterized the ATP hydrolysis cycle as well as the effects of the DnaJ and GrpE cofactors on substrate interaction to reach conclusions on the functional cycle. DnaK ATPase was stimulated by substrates (ninefold) and DnaJ (13-fold) through stimulation of the rate limiting step, gamma-phosphate cleavage (approximately tenfold slower than ADP release). Substrates stimulate ATPase after binding with high affinity (KA < 10 microM) to preformed DnaK-ATP complexes. The rapid binding kinetics lead to the conclusion that ATP-bound DnaK is the primary form initiating interaction with substrates for chaperone activity. The resulting DnaK-ATP-substrate complexes, however, are also characterized by rapid dissociation of bound substrate, but can be stabilized by hydrolysis of ATP (stimulated either by the substrate itself or DnaJ through their effects on the rate-limiting step). Stimulation of the gamma-phosphate cleavage reaction by DnaJ is much more efficient (complete conversion of bound ATP to ADP within five seconds) than that by substrates, indicating the special and important role for DnaJ in stabilization of DnaK-substrate interactions.

Adenosine Triphosphate↗

PubMed1994

The myogenin gene is activated during myocyte differentiation by pre-existing, not newly synthesized transcription factor MEF-2.

The myogenin gene, a member of the gene family encoding muscle-specific basic-helix-loop-helix transcription factors, is activated in myoblasts at the onset of differentiation and can be induced in fibroblasts by forced expression of MyoD or its relatives. Here, we report that a small proximal promoter region of the Myf-4 gene, the human myogenin homolog, suffices to direct muscle-specific expression and up-regulation by MyoD. The minimal promoter contains an E-box and a MEF-2 consensus element. Paradoxically, we find that the MEF-2 binding site but not the E-box is necessary for cell type-specific expression and activation by MyoD in tissue culture cells. This suggests an activating mechanism which is independent of direct protein interactions at the E-box. MEF-2 binding complexes were detected in myoblasts and myotubes, as well as in fibroblasts with no strict correlation to myogenin expression. Moreover, transcription of myogenin could be induced in the presence of potent inhibitors of protein synthesis. From these results we conclude that myogenin expression is not mediated primarily through de novo synthesis of MEF-2 but rather involves a post-translational mode of activation.

Animals↗

PubMed1994

The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171.

Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP-dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP-dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK-dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate-171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.

Adenosine Triphosphatases↗

PubMed1994

A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE.

The activity of DnaK (Hsp70) chaperones in assisting protein folding relies on DnaK binding and ATP-controlled release of protein substrates. The ATPase activity of DnaK is tightly controlled by the nucleotide exchange factor GrpE. We find that GrpE interacts stably with the amino-terminal ATPase domain of DnaK. Analysis of the mutant DnaK756 protein, which has a lower affinity for GrpE, reveals a role for residue Gly 32 in GrpE binding. Gly 32 is located in an exposed loop near the nucleotide binding site of DnaK. Deletion of this loop prevents stable GrpE binding, ATPase stimulation by GrpE, and DnaK chaperone activity. Conservation of this loop within the Hsp70 family suggests that cooperation between Hsp70 and GrpE-like proteins may be a general feature of this class of chaperone.

Adenosine Triphosphatases↗

PubMed1992

Regulation of myogenin expression in normal and transformed myogenic cell lines.

The control of myogenin (Myf-4), one of the muscle-specific regulatory proteins, is particularly interesting since its expression appears obligatory in myoblasts at the onset of differentiation. We isolated the human Myf-4 (myogenin) gene and determined promoter elements which direct cell type-specific expression and are subject to transactivation by the muscle transcription factors Myf-5 and MyoD1 in fibroblasts. Extrinsic signals such as serum components and purified growth factors or potential intracellular signals such as cAMP down-regulate transcription of the myogenin gene. Constitutive expression of the catalytic subunit of PKA completely suppresses transactivation of the myogenin promoter by Myf-5 or MyoD1 suggesting that cAMP may act via phosphorylation by PKA. In contrast to normal myogenic cell lines in which differentiation and myogenin expression can be induced by the removal of serum components, retinoic acid (RA) is required for differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C. This model system was utilized to investigate factors which influence the balance between the transformed state and differentiation. Administration of retinoic acid to BA-Han-1C cells leads to the accumulation of myogenin mRNA approximately 48 h after the addition of RA. This late induction requires ongoing protein- and DNA-synthesis suggesting that trans- and cis-acting factors may be involved in the control. The critical involvement of myogenin in the process of terminal muscle differentiation was also demonstrated in the rat L6 muscle cell line which has been blocked for differentiation by the transforming protein E1a of Ad5 adenovirus. In cells which stably express E1a, myogenin expression is completely suppressed while Myf-5 continues to be synthesized normally. However, E1a inhibits the transactivator function of Myf-5, as demonstrated on GAL4-Myf5 chimeric proteins. A possible interpretation of this result is that Myf-5 or factors activated by Myf-5 are required for the expression of myogenin and myogenin itself is necessary for the terminal differentiation of myoblasts.

Blood↗

PubMed1991

Transcription of the muscle regulatory gene Myf4 is regulated by serum components, peptide growth factors and signaling pathways involving G proteins.

The muscle regulatory protein myogenin accumulates in differentiating muscle cells when the culture medium is depleted for serum. To investigate the regulation of myogenin gene expression, we have isolated and characterized the Myf4 gene which encodes the human homologue of murine myogenin. Serum components, basic FGF (b-FGF), transforming growth factor beta (TGF-beta), and EGF, agents which suppress differentiation of muscle cells in vitro, down-regulate the activity of the Myf4 gene, suggesting that it constitutes a nuclear target for the negative control exerted by these factors. The 5' upstream region containing the Myf4 promoter confers activity to a CAT reporter plasmid in C2C12 myotubes but not in fibroblasts and undifferentiated myoblasts. Unidirectional 5' deletions of the promoter sequence reveal that integral of 200 nucleotides upstream of the transcriptional start site are sufficient for cell type-specific expression. The forced expression of the muscle determining factors, MyoD1, Myf5, and Myf6 and to a lesser degree Myf4, results in the transactivation of the Myf4 promoter in C3H mouse 10T1/2 fibroblasts. Pathways potentially involved in conveying signals from the cell-surface receptors to the Myf4 gene were probed with pertussis- and cholera toxin, forskolin, and cAMP. Dibutyryl-cAMP and compounds that stimulate adenylate cyclase inhibit the endogenous Myf4 gene and the Myf4 promoter in CAT and LacZ reporter constructs. Conversely, pertussis toxin which modifies Gi protein stimulates Myf4 gene expression. In summary, our data provide evidence that the muscle-specific expression of the Myf4 gene is subject to negative control by serum components, growth factors and a cAMP-dependent intracellular mechanism. Positive control is exerted by a pertussis toxin-sensitive pathway that presumably involves G proteins.

Adenylate Cyclase Toxin↗

PubMed1989

Effect of photoperiod and acclimation temperature on nonshivering thermogenesis and GDP-binding of brown fat mitochondria in the Djungarian hamster Phodopus s. sungorus.

Acclimation to short photoperiod at 23 degrees C constant Ta caused P. sungorus to improve their NST capacity from 752 to 1,082 mW. Chronic cold exposure in short photoperiod further enhanced the NST capacity, reaching a maximum level of 1,573 mW at -5 degrees C acclimation temperature. Improvements in NST capacity were always accompanied by an increase in brown fat mitochondrial mass and GDP-binding of brown fat mitochondria, in proportion with the cold load applied during temperature acclimation (23 degrees, 15 degrees, 5 degrees, -5 degrees C). Brown fat mitochondrial protein increased from 7.41 mg (23 degrees C Ta, long photoperiod) through 21.6 mg (23 degrees C Ta, short photoperiod) and 81.6 mg (-5 degrees C Ta, short photoperiod). This approximately 10-fold increase was accompanied by a approximately 35-fold increase in GDP-binding (2.0, 7.3 and 71.6 nmol GDP bound, respectively), demonstrating that the increase in capacity for uncoupled respiration in brown fat is of primary significance for thermogenic acclimation to cold as well as to short photoperiod.

Acclimatization↗

PubMed1988

Cytochrome oxidase activity in brown fat varies with reproductive response and use of torpor in deer mice.

Reproductive responses and thermogenic properties of brown adipose tissue (BAT) were evaluated in individuals from an outbred population of deer mice (Peromyscus maniculatus nebrascensis) after 10 weeks exposure to short photoperiod (8:16 light:dark) and cold ambient temperature (2 degrees C). Deer mice populations are composed of phenotypes that differ in their reproductive response to environmental cues. These phenotypes also differ in body temperature regulation as indicated by their use of daily torpor. By comparing BAT responses among individuals of different phenotypes, we were able to assess the association between environmentally induced changes in reproduction and metabolism. Short/cold days caused increased proliferation of BAT and higher cytochrome oxidase activity. However, the magnitude of these changes varied with reproductive phenotype and use of daily torpor. BAT weight in short/cold day exposed males with normal sized testes more than doubled while total cytochrome oxidase activity increased by 30% as compared to controls. In contrast, short/cold day exposed deer mice with atrophic testes that employed daily torpor exhibited a 64% increase in BAT weight and 100% increase in total cytochrome oxidase activity, compared to control mice. Cytochrome oxidase activity in nontorpid deer mice with atrophic testes was intermediate to these two groups. Our results demonstrate a response of BAT to short/cold days that varies with individual reproductive response. This finding suggests that there exists a common integrative mechanism for temperature and photoperiod to regulate both seasonal reproductive and metabolic adjustments.

Adipose Tissue, Brown↗

PubMed1987

Circadian rhythms of pineal N-acetyltransferase activity in the Djungarian hamster, Phodopus sungorus, in response to seasonal changes of natural photoperiod.

The aim of this study was to describe the regular annual pattern of the daily melatonin synthesis in Djungarian hamsters, Phodopus sungorus sungorus. The hamsters were maintained from birth in natural photoperiodic conditions and in bimonthly intervals the day/night rhythms of pineal N-acetyltransferase (NAT) were measured. Analysis of the circadian profiles of NAT activity showed that the duration of elevated melatonin synthesis closely reflects the duration of the scotophase throughout the seasons. Thus the duration of elevated melatonin seems to represent a direct humoral signal transmitting the photoperiodic message. The duration of the nightly melatonin pulse appears to be influenced mainly by the time of dawn rather than by the time of dusk. Additional information about the time of year might be encoded in the total amount of melatonin synthesized per day, whereas the amplitude of the nightly melatonin peak seems to be of minor importance.

Acetyltransferases↗

PubMed1985

Sources of heat during nonshivering thermogenesis in Djungarian hamsters: a dominant role of brown adipose tissue during cold adaptation.

To assess the thermogenic importance of BAT in Djungarian hamsters we removed about 40% of their BAT and compared their thermogenic abilities before and after the operation. BAT was weighed and assayed for its respiratory properties (Cox, mitochondria). Following removal of BAT we observed considerable reductions of NST. The comparison of NST with BAT weight and with respiratory properties of BAT following partial removal of BAT revealed that at least three different pathways for heat production were involved in NST. In cold-adapted hamsters (values for warm-adapted hamsters in parentheses) we estimated that 66.2% (37.0%) of all NST was produced by mitochondrial respiration in BAT; 16.3% (38.4%) was produced in other organ sites but required the presence of BAT, i.e. there was a mediatory action of BAT on thermogenesis in other organ sites. A further 11.5% (23%) of NST occurred outside of and independent of BAT. Mitochondrial respiration in BAT was the only compartment of NST which increased its contribution during cold adaptation (238 mW to 1,062 mW), whereas the other sources of heat remained largely unchanged.

Acclimatization↗

PubMed1983

Photoperiod and temperature effects on adrenal tyrosine hydroxylase and its relation to nonshivering thermogenesis.

Exposure to short photoperiod increased the activity of adrenal tyrosine hydroxylase (TOH). During summer, the shortening of photoperiod at constant Ta of 23 degrees C stimulated TOH from 7.28 to 10.94 nmoles L-Dopa (h . pair of adrenals), which was as effective as a chronic exposure to 15 degrees C Ta, elevating TOH to 9.1 nmoles L-Dopa/(h . pair of adrenals). The stimulation of TOH by photoperiod or cold exposure in Djungarian hamsters was well correlated with an increase in their ability for nonshivering thermogenesis.

Adrenal Glands↗