Biomedical subjects

T Hesterkamp

Publications and source records attributed to T Hesterkamp.

12 recordsLinked to original sources

PubMed2001

Binding specificity of Escherichia coli trigger factor.

The ribosome-associated chaperone trigger factor (TF) assists the folding of newly synthesized cytosolic proteins in Escherichia coli. Here, we determined the substrate specificity of TF by examining its binding to 2842 membrane-coupled 13meric peptides. The binding motif of TF was identified as a stretch of eight amino acids, enriched in basic and aromatic residues and with a positive net charge. Fluorescence spectroscopy verified that TF exhibited a comparable substrate specificity for peptides in solution. The affinity to peptides in solution was low, indicating that TF requires ribosome association to create high local concentrations of nascent polypeptide substrates for productive interaction in vivo. Binding to membrane-coupled peptides occurred through the central peptidyl-prolyl-cis/trans isomerase (PPIase) domain of TF, however, independently of prolyl residues. Crosslinking experiments showed that a TF fragment containing the PPIase domain linked to the ribosome via the N-terminal domain is sufficient for interaction with nascent polypeptide substrates. Homology modeling of the PPIase domain revealed a conserved FKBP(FK506-binding protein)-like binding pocket composed of exposed aromatic residues embedded in a groove with negative surface charge. The features of this groove complement well the determined substrate specificity of TF. Moreover, a mutation (E178V) in this putative substrate binding groove known to enhance PPIase activity also enhanced TF's association with a prolyl-free model peptide in solution and with nascent polypeptides. This result suggests that both prolyl-independent binding of peptide substrates and peptidyl-prolyl isomerization involve the same binding site.

Amino Acid Sequence↗

PubMed1999

A reporter gene assay for inhibitors of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) plays a key role in sugar uptake and metabolic regulation in bacteria. PTS proteins form a divergent phosphorylation cascade. Enzyme I (EI) is at the top of the cascade and mediates phosphoryl-transfer from phosphoenolpyruvate to the phosphoryl-carrier protein HPr, which then distributes the phosphoryl-groups to the different carbohydrate transporters. In addition, some PTS proteins have a regulatory function in catabolite repression, inducer exclusion and chemotaxis which is modulated by their degree of phosphorylation in response to the availability of substrates. Using as a reporter the IacZ gene under control of the bgl t2 transcriptional terminator and as an effector the transcriptional antiterminator LicT from B. subtilis, a two-plasmid reporter gene system was constructed in order to monitor PTS activity. LicT, when present at low concentration in E. coli, is inactivated by EI/HPr-dependent phosphorylation and conversely is active in a ptsl- mutant lacking El. Active LicT allows for transcriptional readthrough at bgl t2, resulting in a full-length lacZ transcript. Beta-galactosidase activities are increased 4-8-fold in a ptsl+ strain growing on PTS substrates relative to growth on non-PTS substrates and approximately 30-fold in a ptsl- mutant. This gain-of-function in response to dephosphorylation of El or lack of active El can be used to monitor changes of El activity caused by mutations and environmental factors and for screening and validation of inhibitors of the PTS as potentially novel antibacterial compounds.

Bacterial Proteins↗

PubMed1998

Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli.

Folding of newly synthesized cytosolic proteins has been proposed to require assistance by Hsp70 chaperones. We investigated whether two Hsp70 homologs of Escherichia coli, DnaK and HscA, have this role in vivo. Double mutants lacking dnaK and hscA were viable and lacked defects in protein folding at intermediate temperature. After heat shock, a subpopulation of pre-existing proteins slowly aggregated in mutants lacking DnaK, but not HscA, whereas the bulk of newly synthesized proteins displayed wild-type solubility. For thermolabile firefly luciferase, DnaK was dispensable for de novo folding at 30 degrees C, but essential for aggregation prevention during heat shock and subsequent refolding. DnaK and HscA are thus not strictly essential for folding of newly synthesized proteins. DnaK instead has functions in refolding of misfolded proteins that are essential under stress.

Bacterial Proteins↗

PubMed1997

The amino-terminal 118 amino acids of Escherichia coli trigger factor constitute a domain that is necessary and sufficient for binding to ribosomes.

Escherichia coli trigger factor has prolyl-isomerase and chaperone activities and associates with nascent polypeptide chains. Trigger factor has a binding site on ribosomes, which is a prerequisite for its efficient association with nascent chains and its proposed function as a cotranslational folding catalyst. We set out to identify the domain of trigger factor that mediates ribosome binding. Of a series of recombinant fragments, the amino-terminal fragments, TF (1-144) and TF (1-247), cofractionated with ribosomes from cell extracts and rebound to isolated ribosomes in vitro. They competed efficiently with full-length trigger factor for stoichiometric binding to a single site on the large ribosomal subunit. However, TF (1-144) and TF (1-247) differed from full-length trigger factor in that their association with ribosomes was not strengthened by the presence of nascent chains, indicating a role for carboxyl-terminal trigger factor segment in sensing the translational status. The domain responsible for ribosome binding was further investigated by limited proteolysis of recombinant fragments. A stable domain comprising the amino-terminal 118 residues was identified that was still capable of ribosome binding and thus represents a novel structural and functional element of trigger factor.

Amino Acid Isomerases↗

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

The Escherichia coli trigger factor.

E. coli trigger factor is an abundant cytosolic protein originally identified by its ability to maintain the precursor of a secretory protein in a translocation competent form. Recent studies shed new light on the function of this protein. Trigger factor was found to be a peptidyl-prolyl-cisltrans-isomerase capable of catalysing protein folding in vitro, to associate with nascent cytosolic and secretory polypeptide chains, and to cooperate with the GroEL chaperone in promoting proteolysis of an unstable polypeptide in vivo. These findings suggest roles for trigger factor in various folding processes of secretory as well as cytosolic proteins.

Amino Acid Isomerases↗

PubMed1996

Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains.

Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.

Amino Acid Isomerases↗

PubMed1996

Identification of the prolyl isomerase domain of Escherichia coli trigger factor.

E. coli trigger factor is a protein of 48 kDa which was recently identified as a ribosome-bound peptidyl-prolyl-cis/transisomerase (PPIase) capable of catalysing protein folding in vitro. We found trigger factor in association with nascent polypeptide chains, suggesting a function in the co-translational folding of proteins. Sequence comparisons revealed a homology of a segment of trigger factor with PPIases of the FK506 binding protein (FKBP) family. Here, we report on the purification of trigger factor and a domain assignment of its polypeptide chain by microsequencing and mass spectroscopy of proteolytic fragments. Two proteases generated fragments of 12-13 kDa molecular weight that encompass the predicted FKBP domain and possess PPIase activity in vitro. Sequence alignment of the known trigger factor proteins demonstrates a high degree of conservation within this central functional domain of the protein.

Amino Acid Isomerases↗

PubMed1996

Growing up in a dangerous environment: a network of multiple targeting and folding pathways for nascent polypeptides in the cytosol.

The first events in the lives of proteins are the most hazardous. Starting at the ribosome, nascent polypeptides undergo complex folding processes endangered by aggregation reactions. Proteins with organellar destinations require correct targeting to the translocation machineries and prevention from premature folding. The high precision and speed of these processes is ensured by a cystosolic system consisting of molecular chaperones, folding catalysts and targeting factors. This review focuses on the interactions of this system with nascent polypeptides and discusses new concepts for protein folding in the cytosol. It is proposed that folding and targeting are promoted by a flexible network of multiple unassisted and assisted pathways.

Journal Article↗

PubMed1993

The actin monomers in the ternary gelsolin: 2 actin complex are in an antiparallel orientation.

Gelsolin forms ternary complexes with two actin monomers in the presence of Ca2+, which nucleate actin polymerization and cap the barbed ends of filaments. It has therefore been assumed that the two actins are oriented in a similar manner to the terminal subunits in the genetic helix of F-actin. We have tested this using chemical cross-linking with N,N'-1,4-phenylenedimaleimide. For all conditions tested, we identified as the only cross-linked dimeric species an actin dimer indistinguishable from the lower actin dimer of 86 kDa. This lower dimer was previously identified in the initial phase of actin polymerization and also when actin paracrystals are chemically cross-linked [Millonig, R., Salvo, H. & Aebi, U. (1988) J. Cell Biol. 106, 785-796]. It probably defines a contact between adjacent monomers oriented in an antiparallel orientation. In contrast, when F-actin is cross-linked by the same reagent, an upper dimer of apparent molecular mass 115 kDa is formed, which corresponds to adjacent monomers in the genetic helix. The formation of this upper dimer was specifically inhibited by addition of gelsolin to F-actin. Evidence is presented for a Cys374-Cys374 cross-link in the lower dimer. Isolated lower dimer binds to gelsolin in a 1:1 stoichiometry, but it inhibits nucleation of polymerization by gelsolin. Other gelsolin constructs that bind two actin subunits (e.g. the N-terminal half of the molecule, which has severing and capping but no nucleating activity) also form only lower dimer when cross-linked with N,N'-1,4-phenylenedimaleimide. Only segment 2-6 (gelsolin fragment devoid of the N-terminal segment 1) induces an upper dimer orientation of the two actins under nucleating conditions. Our evidence suggests that the two actins associated with gelsolin are not fixed in the orientation of adjacent subunits in F-actin; instead they have a flexible orientation with respect to each other, which permits cross-linking into a stable antiparallel form that does not correspond to the presumed nucleating conformation.

Actins↗

PubMed1993

[Magnetic resonance spectroscopy of breast cancer].

Conservative therapeutic concepts with initial chemotherapy for patients with breast cancer represent a challenge to non-invasive techniques for monitoring response to therapy. Experimental magnetic resonance spectroscopy studies have been able to show exemplary applications for therapy monitoring of breast cancer patients. The characteristic phosphomonoester resonances and their changes during therapy are possible clinical parameters. The additional information which can be obtained from proton and carbon spectroscopy increases the amount of detectable metabolites. On-going studies are investigating clinical applications of multinuclear spectroscopic studies in patients with breast cancer.

Breast Neoplasms↗

PubMed1992

Functional characterisation of serum DNase I in MRL-lpr/lpr mice.

The autosomal defect in Fas antigen leads CD4-CD8-T-cells to accumulate in lymph nodes and spleen of MRL-lpr/lpr mice. MRL-lpr/lpr mice present increased levels of DNase I as compared to the control strain MRL-+/+. This DNase I, which most probably originates from the accumulated CD4-CD8-T-cells, cleaves nuclear DNA with a strong preference for internucleosomal sites yielding, in the presence of both Ca2+ and Mg2+, a pattern of fragments typical for apoptosis. Furthermore, we show that this "apoptosis-ladder" can be obtained with purified DNase I in presence of normal serum.

Animals↗