Biomedical subjects

Udo Oppermann

Publications and source records attributed to Udo Oppermann.

At least 19 recordsLinked to original sources

PubMed2007

Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity.

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17beta-HSD (17beta-hydroxysteroid dehydrogenase) activity. 17beta-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3beta,17beta-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.

17-Hydroxysteroid Dehydrogenases↗

PubMed2007

Carbonyl reductases: the complex relationships of mammalian carbonyl- and quinone-reducing enzymes and their role in physiology.

Carbonyl groups are frequently found in endogenous or xenobiotic compounds. Reactive carbonyls, formed during lipid peroxidation or food processing, and xenobiotic quinones are able to covalently modify DNA or amino acids. They can also promote oxidative stress, the products of which are thought to be an important initiating factor in degenerative diseases or cancer. Carbonyl groups are reduced by an array of distinct NADPH-dependent enzymes, belonging to several oxidoreductase families. These reductases often show broad and overlapping substrate specificities and some well-characterized members, e.g., carbonyl reductase (CBR1) or NADPH-quinone reductase (NQO1) have protective roles toward xenobiotic carbonyls and quinones because metabolic reduction leads to less toxic products, which can be further metabolized and excreted. This review summarizes the current knowledge on structure and function relationships of the major human and mammalian carbonyl reductases identified.

Alcohol Oxidoreductases↗

PubMed2006

Reversible sequestration of active site cysteines in a 2Fe-2S-bridged dimer provides a mechanism for glutaredoxin 2 regulation in human mitochondria.

Human mitochondrial glutaredoxin 2 (GLRX2), which controls intracellular redox balance and apoptosis, exists in a dynamic equilibrium of enzymatically active monomers and quiescent dimers. Crystal structures of both monomeric and dimeric forms of human GLRX2 reveal a distinct glutathione binding mode and show a 2Fe-2S-bridged dimer. The iron-sulfur cluster is coordinated through the N-terminal active site cysteine, Cys-37, and reduced glutathione. The structures indicate that the enzyme can be inhibited by a high GSH/GSSG ratio either by forming a 2Fe-2S-bridged dimer that locks away the N-terminal active site cysteine or by binding non-covalently and blocking the active site as seen in the monomer. The properties that permit GLRX2, and not other glutaredoxins, to form an iron-sulfur-containing dimer are likely due to the proline-to-serine substitution in the active site motif, allowing the main chain more flexibility in this area and providing polar interaction with the stabilizing glutathione. This appears to be a novel use of an iron-sulfur cluster in which binding of the cluster inactivates the protein by sequestering active site residues and where loss of the cluster through changes in subcellular redox status creates a catalytically active protein. Under oxidizing conditions, the dimers would readily separate into iron-free active monomers, providing a structural explanation for glutaredoxin activation under oxidative stress.

Amino Acid Sequence↗

PubMed2006

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding.

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.

Crystallization↗

PubMed2006

The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs.

Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N-BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x-ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme-inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs.

Animals↗

PubMed2006

Active site variability of type 1 11beta-hydroxysteroid dehydrogenase revealed by selective inhibitors and cross-species comparisons.

The NADPH-dependent enzyme type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) activates in a tissue-specific manner circulating pro-glucocorticoid hormones (cortisone in humans) to the 11beta-OH ligand (cortisol in humans), which is able to bind to its cognate receptor and regulate gene transcription. Modulation of this pre-receptor activation mechanism by selective enzyme inhibitors is a desirable goal in the treatment of insulin resistance and related metabolic disorders. Like most other hydroxysteroid dehydrogenases 11beta-HSD1 belongs to the evolutionarily conserved enzyme superfamily of short-chain dehydrogenases/reductases (SDR). The enzyme is anchored within the endoplasmic reticulum through an N-terminal transmembrane domain. In this study we aimed to characterize the active site of mammalian 11beta-HSD1 by determining primary structures from several mammalian lines (cat, hamster, cynomolgus, chimpanzee, dog) thus increasing substantially available sequence information, and allowing us to determine highly variable and constant parts within the primary structure. These regions were mapped to the recently determined three-dimensional structure and are mostly found around the substrate binding site. Furthermore we performed inhibition studies by using different series of inhibitors, comprising 11beta-HSD1 selective arylsulfonamidothiazoles and the unselective steroid-based compound carbenoxolone. The different arylsulfonamidothiazoles display distinct inhibition profiles versus the mammalian species tested, with several tight binding inhibitors for the human enzyme (Ki approximately 50 nM), intermediate for mouse, and weak or not binding inhibitors for rat and guinea pig (Ki>3 microM). Analysis of the inhibition mode reveals that the tight binding inhibitor BVT.528 is a competitive inhibitor for the human form, whereas the related compound BVT.2733 displays a mixed-type inhibition pattern versus the mouse enzyme. Taken together, this structure-activity study provides increased insight into active site complexity and catalytic mechanism of 11beta-HSD1, useful for further inhibitor design.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2006

Structure and function of human 17beta-hydroxysteroid dehydrogenases.

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the NAD(P)(H) dependent oxidoreduction at C17 oxo/beta-hydroxyl groups of androgen and estrogen hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands, since the conversion "switches" between the 17beta-OH receptor ligands and their inactive 17-oxo metabolites. At present, 14 mammalian 17beta-HSDs are described, of which at least 11 exist within the human genome, encoded by different genes. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of sex steroid hormone levels. Broad and overlapping substrate specificities with enzymes involved in lipid metabolism suggest interactions of several 17beta-HSDs with other metabolic pathways. Several 17beta-HSDs enzymes constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and possibly immunity.

17-Hydroxysteroid Dehydrogenases↗

PubMed2006

Type 1 11beta-hydroxysteroid dehydrogenase as universal drug target in metabolic diseases?

Glucocorticoid hormones play essential roles in adaptation to stress, regulation of metabolism and inflammatory responses. Their effects primarily depend on their binding to intracellular receptors leading to altered target gene transcription as well as on cell-type specific biotransformation between 11beta-hydroxy glucocorticoids and their 11-oxo metabolites. The latter effect is accomplished by two different 11beta-hydroxysteroid dehydrogenase isozymes, constituting a shuttle system between the receptor ligand cortisol and its non-binding precursor cortisone. Whereas the type 1 enzyme (11beta-HSD1) is in vitro a NADP(H)- dependent bidirectional enzyme, it reduces in most instances in vivo cortisone to active cortisol. The type 2 enzyme is an exclusive NAD+ dependent dehydrogenase of glucocorticoids, thus "protecting" the mineralocorticoid receptor against illicit occupation by cortisol. Inhibition of tissue-specific glucocorticoid activation by 11beta-HSD1 constitutes a promising target in the treatment of metabolic and cardiovascular diseases. Pharmacological inhibition leads in animal models to lowered hepatic glucose production and increased insulin sensitivity, the primary goals in therapy of diabetes mellitus. Importantly, 11beta-HSD1 activity appears to be intrinsically linked to all features of the metabolic syndrome, which could at least in animal experiments be modulated by use of synthetic selective inhibitors. Importantly, these features include not only insulin resistance but also dyslipidemia, obesity and arterial hypertension. Animal studies and pharmacological experiments suggest further unrelated target areas, for example improvement of cognitive function and treatment of glaucoma, due to the role of glucocorticoids and cellular activation by 11beta-HSD1 in these pathologies. The recent development of specific 11beta-HSD1 inhibitors coupled with advances on structural knowledge and regulation of the 11beta-HSD1 target has undoubtedly promoted the understanding of glucocorticoid control of metabolic regulation. Taken together, it appears that inhibitors against 11beta-HSD1 constitute a promising avenue for novel treatment strategies against the underlying causes of cardiovascular and other metabolic diseases.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed2005

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

Amino Acid Motifs↗

PubMed2005

Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease.

Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.

Aspartic Acid↗

PubMed2004

The crystal structure of guinea pig 11beta-hydroxysteroid dehydrogenase type 1 provides a model for enzyme-lipid bilayer interactions.

The metabolic reduction of 11-keto groups in glucocorticoid steroids such as cortisone leads to the nuclear receptor ligand cortisol. This conversion is an example of pre-receptor regulation and constitutes a novel pharmacological target for the treatment of metabolic disorders such as insulin resistance and possibly other derangements observed in the metabolic syndrome, such as hyperlipidemia, hypertension, and lowered insulin secretion. This reaction is carried out by the NADPH-dependent type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), an enzyme attached through an integral N-terminal transmembrane helix to the lipid bilayer and located with its active site within the lumen of the endoplasmic reticulum. Here we report the crystal structure of recombinant guinea pig 11beta-HSD1. This variant was determined in complex with NADP at 2.5 A resolution and crystallized in the presence of detergent and guanidinium hydrochloride. The overall structure of guinea pig 11beta-HSD1 shows a clear relationship to other members of the superfamily of short-chain dehydrogenases/reductases but harbors a unique C-terminal helical segment that fulfills three essential functions and accordingly is involved in subunit interactions, contributes to active site architecture, and is necessary for lipid-membrane interactions. The structure provides a model for enzyme-lipid bilayer interactions and suggests a funneling of lipophilic substrates such as steroid hormones from the hydrophobic membrane environment to the enzyme active site.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2004

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2004

Tic32, an essential component in chloroplast biogenesis.

Chloroplast protein import across the inner envelope is facilitated by the translocon of the inner envelope of chloroplasts (Tic). Here we have identified Tic32 as a novel subunit of the Tic complex. Tic32 can be purified from solubilized inner envelope membranes by chromatography on Tic110 containing affinity matrix. Co-immunoprecipitation experiments using either Tic32 or Tic110 antisera indicated a tight association between these polypeptides as well as with other Tic subunits, e.g. Tic40, Tic22, or Tic62, whereas the outer envelope protein Toc75 was not found in this complex. Chemical cross-linking suggests that Tic32 is involved late in the overall translocation process, because both the precursor form as well as the mature form of Rubisco small subunit can be detected. We were unable to isolate Arabidopsis null mutants of the attic32 gene, indicating that Tic32 is essential for viability. Deletion of the attic32 gene resulted in early seed abortion because the embryo was unable to differentiate from the heart stage to the torpedo stage. The homology of Tic32 to short-chain dehydrogenases suggests a dual role of Tic32 in import, one as a regulatory component and one as an important subunit in the assembly of the entire complex.

Amino Acid Sequence↗

PubMed2004

Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance.

Expression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.

Archaeal Proteins↗

PubMed2003

Expanded substrate screenings of human and Drosophila type 10 17beta-hydroxysteroid dehydrogenases (HSDs) reveal multiple specificities in bile acid and steroid hormone metabolism: characterization of multifunctional 3alpha/7alpha/7beta/17beta/20beta/21-HSD.

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the conversion of 17beta-OH (-hydroxy)/17-oxo groups of steroids, and are essential in mammalian hormone physiology. At present, eleven 17beta-HSD isoforms have been defined in mammals, with different tissue-expression and substrate-conversion patterns. We analysed 17beta-HSD type 10 (17beta-HSD10) from humans and Drosophila, the latter known to be essential in development. In addition to the known hydroxyacyl-CoA dehydrogenase, and 3alpha-OH and 17beta-OH activities with sex steroids, we here demonstrate novel activities of 17beta-HSD10. Both species variants oxidize the 20beta-OH and 21-OH groups in C21 steroids, and act as 7beta-OH dehydrogenases of ursodeoxycholic or isoursodeoxycholic acid (also known as 7beta-hydroxylithocholic acid or 7beta-hydroxyisolithocholic acid respectively). Additionally, the human orthologue oxidizes the 7alpha-OH of chenodeoxycholic acid (5beta-cholanic acid, 3alpha,7alpha-diol) and cholic acid (5beta-cholanic acid). These novel substrate specificities are explained by homology models based on the orthologous rat crystal structure, showing a wide hydrophobic cleft, capable of accommodating steroids in different orientations. These properties suggest that the human enzyme is involved in glucocorticoid and gestagen catabolism, and participates in bile acid isomerization. Confocal microscopy and electron microscopy studies reveal that the human form is localized to mitochondria, whereas Drosophila 17beta-HSD10 shows a cytosolic localization pattern, possibly due to an N-terminal sequence difference that in human 17beta-HSD10 constitutes a mitochondrial targeting signal, extending into the Rossmann-fold motif.

17-Hydroxysteroid Dehydrogenases↗

PubMed2003

Molecular mechanisms underlying WOX1 activation during apoptotic and stress responses.

Human WWOX gene encodes a putative tumor suppressor WW domain-containing oxidoreductase WOX1 (also known as WWOX or FOR). A high frequency of loss of heterozygosity (LOH) of this gene has been shown in prostate, lung, breast and other cancers. In addition, numerous aberrant WWOX mRNA transcripts have been found in cancer cells. WOX1 is a proapoptotic protein. In response to stress or apoptotic stimuli, WOX1 became phosphorylated at Tyr33, which enabled its complex formation with activated p53 and JNK1. The p53/WOX1 complex translocated to the mitochondria and further to the nuclei to mediate apoptosis. WOX1 mutants, which were inactivated for nuclear translocation or Tyr33 phosphorylation, failed to induce apoptosis, indicating that activation of WOX1 via Tyr33 phosphorylation, followed by nuclear translocation, is essential for inducing cell death. WOX1 induced apoptosis synergistically with p53. In contrast, transiently activated JNK1 induced anti-apoptotic response, and this protective activity inhibited WOX1-induced apoptosis. Taken together, WOX1 is involved in stress and apoptotic responses, and is likely to regulate the activation of both p53 and JNK1.

Apoptosis↗

PubMed2003

High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system.

In this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag could not be cleaved, even under stringent conditions, possibly due to steric hindrance, thus preventing further purification. In contrast, the GST fusion system could increase protein expression level and the corresponding fusion protein could be easily cleaved by thrombin. After binding to glutathione sepharose, the fusion protein was cleaved on column, and a roughly purified protein fraction was eluted. This fraction was purified by heating at 80 degrees C for 10 min, followed by centrifugation. The correct total mass and N-terminal primary structure were confirmed by mass spectrometry and Edman degradation. Both constructs were used for in vitro expression, and similar results were obtained, indicating higher expression levels of the GST tag vs. intein/chitin tag. Taken together, our results suggest that the GST fusion system can be used as a considerable alternative to synthetic genes for the expression of rare codon genes. The affinity chromatography purification followed by a heating step is an efficient and convenient method for thermostable protein purification.

Archaea↗

PubMed2003

Hot spots in Tcf4 for the interaction with beta-catenin.

The interaction of beta-catenin with T-cell factor (Tcf) 4 plays a central role in the Wnt signaling pathway and has been discussed as a possible site of intervention for the development of anti-cancer drugs. In this study, we performed Ala-scanning mutagenesis of all Tcf4 residues in the Tcf-beta-catenin interface and studied the binding energetics of these mutants using isothermal titration calorimetry. Binding of Tcf4 was found to be highly cooperative. Single site mutations of most Tcf4 residues resulted in a significant reduction in binding enthalpies but in similar binding constants as compared with wild type Tcf4. Interestingly, this was also true for residues that are disordered in the reported crystal structures. The mutation D16A caused the largest reduction in binding constant (50-fold) accompanied by a large unfavorable enthalpy change (DeltaDeltaHobs) of +8 kcal/mol at 25 degrees C. In contrast, the mutation of the Tcf residues Glu24 and Glu28, which have been proposed as an interaction hot spot due to their location in a field of strong positive electrostatic potential on the beta-catenin surface (charge button), resulted only in a significant reduction of binding enthalpies, which were largely compensated for by unfavorable entropic contributions to the binding. Other mutations that significantly reduced Tcf binding constants were D11A and alanine mutations of the hydrophobic residues Leu41, Val44, and Leu48. The measured thermodynamic data are discussed with the available structural information of Tcf-beta-catenin crystal structures and allow us to propose possible sites for development of Tcf antagonists.

Amino Acid Sequence↗