Biomedical subjects

Martin Haslbeck

Publications and source records attributed to Martin Haslbeck.

At least 19 recordsLinked to original sources

PubMed2006

Factors masking HMGB1 in human serum and plasma.

High mobility group box 1 protein (HMGB1) is a ubiquitously expressed architectural chromosomal protein. Recently, it has become obvious that HMGB1 can also act as a proinflammatory mediator when actively secreted during cell activation or passively released from necrotic cells. HMGB1 appears to play an important role in the pathogenesis of diseases, including sepsis and rheumatoid arthritis. However, easy, sensitive, and reliable detection systems are required to investigate the clinical significance of HMGB1 in clinical samples for diagnosis and prognosis of diseases. Here, we describe sensitive ELISAs for the detection of HMGB1 in cell culture medium and cell lysates. However, these assays failed to reliably quantitate HMGB1 in serum and plasma when compared with immunoblot analysis. We found that serum/plasma components bind to HMGB1 and interfere with its detection by ELISA systems. In most serum/plasma samples investigated, including those from healthy individuals, we detected IgG antibodies binding to HMGB1. The titers of these antibodies correlated with the capacity of sera to interfere with the detection of recombinant HMGB1 by ELISA. Furthermore, HMGB1 coimmunoprecipitated with several proteins including IgG1, as identified by mass spectrometry. These HMGB1 interacting proteins are currently characterized and may contribute to complex formation, masking, and possibly, modulation of cytokine activity of HMGB1.

Biomarkers↗

PubMed2006

Identification and characterization of a stress-inducible and a constitutive small heat-shock protein targeted to the matrix of plant peroxisomes.

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL>) and a functional PTS2 (RLx5HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one- and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions.

Amino Acid Sequence↗

PubMed2006

Recombinant expression and in vitro refolding of the yeast small heat shock protein Hsp42.

Small Hsps represent a variation on the theme of protection of proteins from irreversible aggregation by reversible interaction with chaperone proteins. While different sHsps are highly heterogeneous in sequence and size, the common trait is the presence of a conserved alpha-crystallin domain. In addition sHsps assemble into large oligomeric complexes where dimers represent the basic building blocks. Hsp42, a member of the sHsp family in the cytosol of S. cerevisiae, forms ordered oligomers with a barrel-like structure. Here, we present the recombinant expression and purification of Hsp42. We demonstrate, that Hsp42 is expressed in inclusion bodies and can be resolubilized and folded to correct, active oligomers. This indicates that in contrast to thermal unfolding, the chemical disassembly and unfolding of Hsp42 is fully reversible. In comparison to the purification of mature Hsp42 from yeast, its recombinant expression leads to a substantial increase in the yield of the protein and to a reduction of contamination caused by aggregation prone proteins complexed by Hsp42. In addition, the recombinant Hsp42 is fully active as a chaperone in an energy independent manner.

Animals↗

PubMed2006

Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26.

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.

Binding Sites↗

PubMed2006

MotD of Sinorhizobium meliloti and related alpha-proteobacteria is the flagellar-hook-length regulator and therefore reassigned as FliK.

The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates.

Amino Acid Sequence↗

PubMed2005

Disassembling protein aggregates in the yeast cytosol. The cooperation of Hsp26 with Ssa1 and Hsp104.

In all organisms studied, elevated temperatures induce the expression of a variety of stress proteins, among them small Hsps (sHsp). sHsps are chaperones that prevent the unspecific aggregation of proteins by forming stable complexes with unfolded polypeptides. Reactivation of captured proteins requires the assistance of other ATP-dependent chaperones. How sHsps and ATP-dependent chaperones work together is poorly understood. Here, we analyzed the interplay of chaperones present in the cytosol of Saccharomyces cerevisiae. Specifically, we characterized the influence of Hsp104 and Ssa1 on the disassembly of Hsp26 x substrate complexes in vitro and in vivo. We show that recovery of proteins from aggregates in the cell requires the chaperones to work together with defined but overlapping functions. During reactivation, proteins are transferred from a stable complex with Hsp26 to Hsp104 and Hsp70. The need for ATP-dependent chaperones depends on the type of sHsp x substrate complex. Although Ssa1 is able to release substrate proteins from soluble Hsp26 x substrate complexes, Hsp104 is essential to dissociate substrate proteins from aggregates with incorporated sHsps. Our results are consistent with a model of several interrelated defense lines against protein aggregation.

Adenosine Triphosphatases↗

PubMed2005

Some like it hot: the structure and function of small heat-shock proteins.

Small heat-shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. Recent evidence suggests that they maintain protein homeostasis by binding proteins in non-native conformations, thereby preventing substrate aggregation. Some members of the sHsp family are inactive or only partially active under physiological conditions, and transition toward the active state is induced by specific triggers, such as elevated temperature. Release of substrate proteins bound to sHsps requires cooperation with ATP-dependent chaperones, suggesting that sHsps create a reservoir of non-native proteins for subsequent refolding.

Heat-Shock Proteins, Small↗

PubMed2004

Oncogenic mutations reduce the stability of SRC kinase.

The oncogenic potential of the viral tyrosine kinase v-Src is due to its constitutive activity. Unlike the highly homologous cellular c-Src kinase, a C-terminal deletion of the regulatory tail and numerous point mutations make the viral kinase uncontrollable. To determine the basis of these differences, we analysed the structure and stability of v-Src and c-Src in vitro. We show that the stability of v-Src against unfolding and irreversible aggregation is significantly lower than that of c-Src. Furthermore, in v-Src hydrophobic residues are more exposed already in the native state. In consequence, v-Src was found to be inactive close to physiological temperatures. We thus suggest that the ensemble of mutations that transform c-Src into the oncogenic variant cause a concomitant destabilisation of the kinase.

Amino Acid Sequence↗

PubMed2004

A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization.

Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.

Amino Acid Sequence↗

PubMed2004

IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis.

The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.

Catalysis↗

PubMed2004

A rapid method for analyzing recombinant protein inclusion bodies by mass spectrometry.

The identification and characterization of a protein overexpressed in insoluble inclusion bodies in Escherichia coli are the first crucial and time-limiting steps in recombinant protein expression. Here, a straightforward approach to the analysis of recombinant proteins in inclusion bodies is presented. Inclusion bodies were dissolved in 8M urea and analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry without prior desalting. Mass determination was achieved by direct spotting of the samples onto the MALDI target and serial dilution in the matrix. The masses of four different proteins, expressed in inclusion bodies, were determined with a mass accuracy better than 0.1%. Furthermore, protein modifications, such as N-terminal processing of single amino acids or artificial cyanylation caused by incubation of the inclusion bodies with urea at elevated temperatures, could be detected. Similarly, tryptic digests were directly analyzed in 2M urea to obtain peptide mass fingerprints for identification and more detailed information on the primary protein structure and secondary modifications. Due to the presence of ammonia in the urea-containing buffers, no Na(+) adducts were observed in the peptide mass fingerprint analysis. Taken together, the rapid and robust procedures presented here greatly facilitate the analysis of recombinant proteins.

Antibodies, Monoclonal↗

PubMed2004

RNA stem-loop enhanced expression of previously non-expressible genes.

The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem-loop (stem length, 7 bp; DeltaG(0) = -9.9 kcal/mol) in front of various gene sequences. In each case, the stem-loop was inserted 15 nt downstream from the start codon. Insertion of the stem-loop allowed in vitro expression of five previously non-expressible genes and enhanced the expression of all other genes investigated. Analysis of the RNA structure proved that the stem-loop was formed in vitro, and demonstrated that stabilization of the ribosome binding site is due to stem-loop introduction. By theoretical RNA structure analysis we showed that the inserted RNA stem-loop suppresses long-range interactions between the translation initiation domain and gene-specific mRNA sequences. Thus the inserted RNA stem-loop supports the formation of a separate translational initiation domain, which is more accessible to ribosome binding.

Base Sequence↗

PubMed2004

Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the unspecific aggregation of proteins. So far, Hsp26 was the only unambiguously identified member of the sHsp family in Saccharomyces cerevisiae. We show here that the sHsp system in the cytosol of S. cerevisiae consists of two proteins, Hsp26 and Hsp42. Hsp42 forms large dynamic oligomers with a barrel-like structure. In contrast to Hsp26, which functions predominantly at heat shock temperatures, Hsp42 is active as a chaperone under all conditions tested in vivo and in vitro. Under heat shock conditions, both Hsp42 and Hsp26 suppress the aggregation of one-third of the cytosolic proteins. This subset is about 90% overlapping for Hsp42 and Hsp26. The sHsp substrates belong to different biochemical pathways. This indicates a general protective function of sHsps for proteome stability in S. cerevisiae. Consistent with this observation, sHsp knockout strains show phenotypical defects. Taken together, our results define Hsp42 as an important player for protein homeostasis at physiological and under stress conditions.

Cytosol↗

PubMed2004

Analysis of the regulation of the molecular chaperone Hsp26 by temperature-induced dissociation: the N-terminal domail is important for oligomer assembly and the binding of unfolding proteins.

Small heat shock proteins (sHsps) are molecular chaperones that efficiently bind non-native proteins. All members of this family investigated so far are oligomeric complexes. For Hsp26, an sHsp from the cytosol of Saccharomyces cerevisiae, it has been shown that at elevated temperatures the 24-subunit complex dissociates into dimers. This dissociation seems to be required for the efficient interaction with unfolding proteins that results in the formation of large, regular complexes comprising Hsp26 and the non-native proteins. To gain insight into the molecular mechanism of this chaperone, we analyzed the dynamics and stability of the two oligomeric forms of Hsp 26 (i.e. the 24-mer and the dimer) in comparison to a construct lacking the N-terminal domain (Hsp26DeltaN). Furthermore, we determined the stabilities of complexes between Hsp26 and non-native proteins. We show that the temperature-induced dissociation of Hsp26 into dimers is a completely reversible process that involves only a small change in energy. The unfolding of the dissociated Hsp26 dimer or Hsp26DeltaN, which is a dimer, requires a much higher energy. Because Hsp26DeltaN was inactive as a chaperone, these results imply that the N-terminal domain is of critical importance for both the association of Hsp26 with non-native proteins and the formation of large oligomeric complexes. Interestingly, complexes of Hsp26 with non-native proteins are significantly stabilized against dissociation compared with Hsp26 complexes. Taken together, our findings suggest that the quaternary structure of Hsp26 is determined by two elements, (i) weak, regulatory interactions required to form the shell of 24 subunits and (ii) a strong and stable dimerization of the C-terminal domain.

Base Sequence↗

PubMed2004

MotE serves as a new chaperone specific for the periplasmic motility protein, MotC, in Sinorhizobium meliloti.

The flagella of Sinorhizobium meliloti rotate solely clockwise and vary their rotary speed to provoke changes in the swimming path. This mode of motility control has its molecular corollary in two novel motility proteins, MotC and MotD, present in addition to the ubiquitous MotA/MotB energizing proton channel. MotC binds to the periplasmic portion of MotB, whereas MotD interacts with FliM at the cytoplasmic face of the rotor. We report here the assignment and analysis of a fifth motility protein, MotE. Deletion of motE resulted in aggregation and decay of the periplasmic MotC protein and, as a consequence, in paralysis of the cell. The 179-residue MotE protein bears an N-terminal signal peptide and is rapidly secreted to the periplasm, where it forms stable dimers that are linked by a disulphide bridge between the cysteine 53 residues. Both, the monomeric and the dimeric MotE bind to MotC, and dimerization is essential for MotE stability in the periplasm. We conclude that MotE is a periplasmic chaperone specific for MotC being responsible for its proper folding and stability. We also propose that the MotE dimer serves as a shuttle to target MotC to its binding site at MotB.

Amino Acid Sequence↗

PubMed2003

Sti1 is a novel activator of the Ssa proteins.

The molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop. In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction. With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins. Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro. The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90. Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system. Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1. Thus, Sti1 is a potent novel effector for the Hsp70 ATPase.

Adenosine Triphosphatases↗

PubMed2003

GroE-dependent expression and purification of pig heart mitochondrial citrate synthase in Escherichia coli.

Citrate synthase (CS) is a dimeric, mitochondrial protein, composed of two identical subunits (M(r) 48969 each). The nuclear-encoded alpha-helical protein is imported into mitochondria post-translationally where it catalyses the first step of the citric cycle. Furthermore, the pathway of thermal unfolding as well as the folding pathway was studied extensively, making CS a well-suited substrate protein for studying chaperone function. In chaperone research the quality of the substrate proteins is essential to guaranty the reproducibility of the results. In this context, we here describe the GroE-enhanced recombinant expression and purification of CS. CS was expressed in E. coli by using an arabinose regulated T7 promotor. Under standard expression conditions only insoluble, inactive CS was detected. Interestingly, the expression of soluble and active CS was possible when GroEL/GroES was co-expressed. Furthermore, a shift to lower expression temperatures increased the amount of soluble, active CS. We describe for the first time, the purification of CS in soluble and active form by following a CiPP strategy (capture, intermediate purification, polishing). After the initial capturing step on DEAE-Sephacel the protein was further purified on a Q-Sepharose column. After these two steps of anion-exchange chromatography a final size-exclusion chromatography step on a Superdex 75-pg column yields CS with a purity over 99%. Using this expression and purification strategy 1 mg CS per g E. coli wet weight were purified.

Animals↗

PubMed2003

Recombinant expression and purification of Ssa1p (Hsp70) from Saccharomyces cerevisiae using Pichia pastoris.

Heat shock proteins with a molecular mass of 70000 (Hsp70s) are a ubiquitous class of ATP-dependent molecular chaperones involved in the folding of cellular proteins. Sequencing the entire genome of Saccharomyces cerevisiae revealed 14 different genes for Hsp70 proteins in different cellular compartments. Among these 14 Hsp70s, the subclass of Ssa (Ssa1p-Ssa4p) is abundant and essential in the cytosol. Since high yield expression of cytoplasmic Ssa1p is inefficient in Saccharomyces cerevisiae and recombinant expression in E. coli yields low protein levels, we chose Pichia pastoris as the recombinant expression system. In Pichia pastoris, expression levels of Ssa1p are high and Ssa1p is soluble and correctly folded. Also, we present a new protocol for purification of Ssa1p. Previously described purifications include ATP-agarose chromatography leading to Ssa1p partially complexed with ATP. Our optimized purification protocol follows the CiPP strategy (capture, intermediate purification, polishing) avoiding ATP-agarose chromatography, which allows detailed studies on the ATP-dependent Hsp70 functions. We obtained Ssa1p in high purity and 400 times higher quantity compared to previous studies.

Chromatography, Ion Exchange↗