Conformationally restricted calpain inhibitors.

The cysteine protease calpain-I is linked to several diseases and is therefore a valuable target for inhibition. Selective inhibition of calpain-I has proved difficult as most compounds target the active site and inhibit a broad spectrum of cysteine proteases as well as other calpain isoforms. Selective inhibitors might not only be potential drugs but should act as tools to explore the physiological and pathophysiological roles of calpain-I. α-Mercaptoacrylic acid based calpain inhibitors are potent, cell permeable and selective inhibitors of calpain-I and calpain-II. These inhibitors target the calcium binding domain PEF(S) of calpain-I and -II. Here X-ray diffraction analysis of co-crystals of PEF(S) revealed that the disulfide form of an α-mercaptoacrylic acid bound within a hydrophobic groove that is also targeted by a calpastatin inhibitory region and made a greater number of favourable interactions with the protein than the reduced sulfhydryl form. Measurement of the inhibitory potency of the α-mercaptoacrylic acids and X-ray crystallography revealed that the IC50 values decreased significantly on oxidation as a consequence of the stereo-electronic properties of disulfide bonds that restrict rotation around the S-S bond. Consequently, thioether analogues inhibited calpain-I with potencies similar to those of the free sulfhydryl forms of α-mercaptoacrylic acids.

[New concepts for pressure-controlled glaucoma implants]. / Neue Konzepte für druckgesteuerte Glaukomimplantate.

In industrialized countries glaucoma is one of the most common causes that leads to blindness. It is also the most common cause of irreversible blindness worldwide. In addition to local treatment of intraocular pressure and filtering glaucoma surgery, alloplastic implants are increasingly being used in glaucoma therapy. As long-term results published in the literature of commonly used implants are unsatisfactory, it seems useful to search for new concepts. In order to avoid the well-known short-term and long-term postoperative complications a pressure-controlled microstent with antiproliferative surface modifications was developed. Additionally, the functionality of such a microstent should be investigated using an animal glaucoma model. This paper describes the concept of a microstent which drains aquous humour from the anterior chamber into the suprachoroidal space. In addition, the glaucoma models described in the literature are discussed. Unfortunately, none of the methods could be reproduced permanently. First results show a correct implantation of a coated microstent with valve where the anti-proliferative effect could be demonstrated histologically. The promising results should lead to further investigations and the final goal will be the testing of the stent in the human eye.

[Concept of a pressure-controlled microstent for glaucoma therapy]. / Konzept eines druckgesteuerten Mikrostents für die Glaukomtherapie.

A pressure-controlled microstent could permanently normalise the intraocular pressure (IOP) for open-angle glaucoma therapy by drainage into the suprachoroidal space. The complex requirements demand new technical solutions as well as an improved understanding of specific cell biological processes at the implant's surface to develop effective local drug delivery (LDD) concepts and surface modifications. Fluid mechanical requirements were derived from physiological data and the analysis of commercial glaucoma implants. The technological basics for the production of suitable structures are refined ultra-short pulse laser technology and 2-photon polymerisation (2PP). All known glaucoma implants induce unwanted cell proliferation resulting in a loss of function. It is assumed that the activity of fibroblasts is low in the suprachoroidal space. However, it was seen that LDD concepts are required to control cell proliferation. Fibroblasts from sclera and choroidea were isolated und cultured as the most relevant cell types for in vitro investigation. Potential materials and drugs were investigated by cell viability tests for biocompatibility or suppression of cell viability. The fluid mechanical analysis leads to smallest stent lumina (ID = 50 µm) at anatomically suitable implant lengths (7 - 10 mm). Only pressure control can manage the individual conditions with changing IOP. Finite element analysis of valves showed the need for highly flexible structures. This can be achieved by combining basic structures with micromechanically active valves added by 2PP. The potential materials show perfect in vitro and in vivo biocompatibility. Ormocers which are best suited for 2PP are also highly biocompatible. The selected drugs paclitaxel and triamcinolon acetonide open a wide therapeutic window to impair fibroblast growth. The surgical procedure was established by implantation of prototypes in rabbit eyes, connecting the anterior chamber with the suprachoroidal space. Highly flexible implants are required for correct placement within the eye. The new concept of the microstent combines biomechanical approaches, technologies for microfabrication and current LDD concepts and opens new perspectives for glaucoma therapy.

myo-Inositol monophosphatase: a challenging target for mood stabilising drugs.

myo-Inositol monophosphatase has long been a target for drug discovery. Recent work has given detailed insight into its mechanism and dynamics plus new activities and some novel series of inhibitors. The goal of a bio-available inhibitor for this enzyme, however, remains a major challenge for medicinal chemistry.

Design, synthesis and characterisation of a peptide with oxaloacetate decarboxylase activity.

The synthesis of Oxaldie-3, a synthetic 31-residue peptide with oxaloacetate decarboxylase activity, is described. Biophysical characterisation by gel filtration, CD and NMR spectroscopy indicated that the peptide adopted a folded structure in solution. Oxaldie-3 was an efficient catalyst at concentrations as low as 2 microM, 100-fold lower than the previously described Oxaldie-2, which relied on aggregating alpha-helices for activity. Oxaldie-3 speeded decarboxylation by more than three orders of magnitude relative to simple amines.

Thermodynamics of DNA binding of MM17, a 'single chain dimer' of transcription factor MASH-1.

MASH-1, a member of the basic helix-loop-helix (bHLH) family of transcriptional regulators, is a central factor for the regulation of the differentiation of committed neuronal precursor cells of the peripheral nervous system. We have previously produced MM17, a single chain version of this dimeric protein, by linking the C-terminal end of the first subunit to the N-terminal residue of the second subunit through a flexible peptide linker. We have now determined by isothermal titration calorimetry the thermodynamic parameters characterising the DNA binding reactions of MM17. The DNA binding specificity was relatively low and comparable to that observed for wild-type MASH bHLH. At 32 degrees C and pH 7, the concentration of MM17 at which 50% DNA binding occurred was determined as 22.8 and 152 nM for binding to MCK-S and the heterologous SP-1, respectively. Similarly to MASH bHLH the free energy of the association was only slightly temperature dependent, while both the entropy and the enthalpy change were strong functions of temperature. The free energy of DNA binding was independent of the pH for the pH range between 6 and 8. Dissection of the entropy change of the association reaction suggested that the two basic domains and the linker region between the subunits underwent a folding transition from a mainly unfolded to a predominantly ordered conformation. Therefore, like wild-type MASH bHLH, the DNA binding reaction of MM17 follows an induced fit mechanism.

Acquisition of myogenic specificity through replacement of one amino acid of MASH-1 and introduction of an additional alpha-helical turn.

The homologous transcription factors Myf-5, MyoD, myogenin, MRF-4, and MASH-1 bind with high affinity and modest sequence specificity to DNA containing an E-box (CANNTG). This similarity of the in vitro DNA binding specificity is in sharp contrast to the high physiological specificity displayed by these proteins. Myf-5, MyoD, myogenin, and MRF-4 induce cells to differentiate along a myogenic pathway, while MASH-1 promotes the differentiation of neuronal precursor cells. We show here that MASH-1 can be converted into a protein capable of inducing myogenesis in fibroblasts by replacing leucine (130) of MASH-1 with lysine and introducing an additional turn into its basic recognition helix. These changes do not significantly alter the DNA binding properties of the proteins in cell free conditions. Crystallographic data for the DNA complexes of MyoD and E12 suggest that Leu (130) points away from the DNA into the solvent. We postulate that the identity of the amino acid in position 130 is important for protein-protein interactions that might affect the DNA binding specificities displayed by BHLH-proteins in vivo and form the molecular basis of the different physiological properties of the myogenic and neurogenic BHLH-proteins.

The N-terminal methionine is a major determinant of the DNA binding specificity of MEF-2C.

Members of the MEF-2 family of transcriptional regulators positively modulate the activity of basic helix-loop-helix proteins in both myogenic and neurogenic cell lineages. Previous work had shown that MEF-2C(2-117), a protein fragment comprising the dimerization and DNA-binding domains of MEF-2C but lacking the N-terminal methionine, bound to AT-rich DNA sequences with high affinity. MEF-2C(2-117) did not discriminate between different AT-rich sequences. We now report the in vitro DNA binding properties of a MEF-2C fragment containing the N-terminal methionine. Measurements of the apparent dissociation constants of the complexes of GG-MEF-2C(1-117) revealed that different AT-rich sequences are bound with different affinities; in particular MEF site containing DNA (CTATAAATAG) is bound preferentially to DNA containing a SRF site (CATAAATG). Strikingly, when the shorter AT run consisted of six alternating thymines and adenines, almost wild-type affinity was observed. Irrespective of the particular DNA sequence, all circular dichroism spectra of the DNA complexes of GG-MEF-2C(1-117) were superimposable and characterized by an identical maximal ellipticity at 269.5 nm, suggesting similar DNA conformations. Bending analysis by circular permutation assay revealed that on complex formation MEF-2C(2-117) induced cognate DNA to bend by 49 degrees, while heterologous DNA remained unbent. In the presence of the N-terminal methionine, however, all DNA sequences were bent by 70 degrees. The above results suggest an important function for the N-terminal methionine in properly orientating MEF-2C on the DNA.

Arginine (348) is a major determinant of the DNA binding specificity of transcription factor E12.

The basic helix-loop-helix proteins (BHLH) E12 and E47 bind to DNA in a cell-type specific fashion as heterodimers with transcription factors such as MyoD, Myf-5, MRF-4, myogenin, and MASH-1 and -2 which are critical regulators of cellular differentiation. We have measured the apparent dissociation constants (KD) of the complexes of E12 and several E12 mutants with various oligonucleotides. Glutamate (345) of E12, which is hydrogen bonded to a CpA dinucleotide, and arginine (348), a residue that does not directly interact with the nucleobases, are major determinants of the DNA binding specificity of E12. R(348) is in direct contact with both the phosphate backbone and the carboxylate of E(345), thereby locking the side chain conformation of E(345). In its locked conformation the glutamate residue interacts favourably only with E-box containing DNA, the natural target of BHLH-proteins, while repulsive interactions destabilise the complexes with all other DNA sequences.

Thermodynamics of the DNA binding reaction of transcription factor MASH-1.

MASH-1, a member of the basic helix-loop-helix (BHLH) family of transcription factors, promotes the differentiation of committed neuronal precursor cells. We have determined the thermodynamic parameters of the DNA binding reaction of the BHLH domain of MASH-1 (MASH-BHLH) by isothermal titration calorimetry and found that the specificity of the binding reaction was rather low. At 27 degrees C, the association constant for binding was 5.13 (+/-0.51) x 10(8) M-1 for an E-box containing oligonucleotide, while for a heterologous DNA sequence it was 5.14 (+/-1.93) x 10(7) M-1. The reaction enthalpy and the reaction entropy were strongly dependent on the temperature, but the reaction free energy was almost independent of temperature. The association reaction was enthalpically driven throughout the physiological temperature range and characterized by a large negative heat capacity change. No change in the protonation state of the protein and/or the DNA was observed at pH 6. Within experimental error, the reaction was independent of pH between pH 6 and 8. Dissection of the entropy change of the binding reaction indicated that binding was coupled to local protein folding of approximately 25 amino acids per protein subunit. The circular dichroism spectra of free and DNA-bound MASH-BHLH revealed the formation of additional alpha-helical structure comprising approximately 25 amino acids upon complex formation. Therefore, while the basic region was in an alpha-helical conformation in the DNA complex, in free MASH-BHLH it was substantially unfolded even at concentrations where the protein is mainly dimeric. The association between MASH-1 and DNA is therefore an example of "induced fit".

Single chain dimers of MASH-1 bind DNA with enhanced affinity.

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.

DNA recognition and bending.

DNA-binding proteins recognize their DNA targets not only through the formation of specific contacts with the nucleotide bases but also through inherent properties of the DNA sequence, including increased bendability and rigidity. Consideration of the properties of both the protein and the DNA is required before the sequence specificity and the observed DNA bend in DNA-protein complexes can be understood.

Covalently linking BHLH subunits of MASH-1 increases specificity of DNA binding.

MASH-1, a member of the basic-helix-loop-helix (BHLH) family of transcription factors, promotes the differentiation of committed neuronal precursor cells. In vitro, MASH-1 displays only marginal DNA sequence specificity. We have produced a MASH-1 variant, MASH-GGC, by introducing the tripeptide Gly-Gly-Cys at the C-terminal end of the BHLH domain. Under reducing conditions the properties of MASH-GGC and of the BHLH domain of MASH-1 were very similar. Like MASH-1, reduced MASH-GGC showed little specificity of DNA binding. CD spectroscopy revealed that both proteins underwent a conformational change from a largely unfolded to a mainly alpha-helical conformation upon binding to DNA. When the subunits of MASH-GGC were linked through a disulfide bond, the folded conformation was stable over a wide concentration range (2.5 nM to 2 microM) even in the absence of DNA. Oxidized MASH-GGC bound to E-box-containing sequences half-maximally at 148 nM, compared to 458 nM for the reduced form. Therefore, even when the change from a monomeric to a dimeric species was taken into account, the affinity for E-box-containing DNA sequences was increased. Surprisingly, the apparent dissociation constant for the complex with DNA not containing E-box sequences was increased upon oxidation. Therefore, despite the large distance between the disulfide bridge and the protein-DNA interface, covalently linking the subunits of MASH-1 increased the specificity of DNA binding significantly. In vivo, such an increase of the intrinsic DNA binding specificity might be achieved through interactions with other proteins of the transcriptional machinery.

High affinity binding of MEF-2C correlates with DNA bending.

To regulate lineage-specific gene expression in many cell types, members of the myocyte enhancer factor-2 (MEF-2) family of transcription factors cooperate with basic helix-loop-helix (bHLH) proteins, which show only limited intrinsic DNA binding specificity. We investigated the DNA binding properties of MEF-2C in vitro and show that the inherent bendability of the MEF site is one of the principal structural characteristics recognized by MEF-2C. Measurements of the apparent dissociation constants of MEF-2C complexes with several DNA sequences revealed that MEF-2C bound with high affinity to DNA sequences containing a MEF site. Mutations in the MEF site which did not affect the bendability of the DNA changed the free energy of binding only marginally. However, reducing the intrinsic bendability of the DNA binding site through an AA-->GC substitution increased the half-maximal binding concentration of MEF-2C by almost one order of magnitude. Electrophoretic mobility shift assays revealed markedly reduced MEF-2C binding to DNA containing 2,6-diaminopurine. On binding to MEF-2C the maximum ellipticity at 275 nm in the CD spectrum of DNA containing a MEF site was red shifted by 4 nm and its intensity reduced significantly, while a slight blue shift of <1 nm was observed for a mutant DNA sequence with reduced bendability (AA-->GC). Bending analysis by circular permutation assay revealed that the DNA in the cognate complex was bent by 49 degrees , while the DNA in the complex with the mutant oligonucleotide was largely unbent.

High level expression in soluble form, one step purification, and characterization of the DNA binding domain of MEF-2C.

Members of the MEF-2 family of transcription factors act as coregulators of basic helix-loop-helix (BHLH) proteins in the control of lineage specific gene expression in many cell types through direct interaction between the respective DNA binding domains. To make possible a thorough biochemical, biophysical, and structural characterization of the properties of myocyte enhancer factor (MEF) proteins and of their interactions with BHLH-proteins, a simple system for high level expression and rapid purification of myocyte enhancer factor-2C (MEF-2C) was developed. A T7 expression system was used to produce in high yield in Escherichia coli an N-terminal fragment of MEF-2C comprising both the MADS box and the MEF domain. Purification by a single round of cation-exchange chromatography on a Resource-S HPLC column at elevated pH afforded an essentially pure protein. Recombinant MEF-2C (1-117) bound with high affinity to the MEF consensus DNA binding site (CTATAAATAG). Mutations in this sequence that replaced adenines with thymine or vice versa did not significantly alter the affinity for MEF-2C(1-117). The introduction of G-C pairs into the core of the MEF-site, however, dramatically increased the concentration of MEF-2C(1-117) needed for half maximal DNA binding. We propose an explanation of the DNA binding specificity of MEF-2C based on the intrinsic bending properties of the unbound DNA.

Basic helix-loop-helix protein MyoD displays modest DNA binding specificity.

The expression of MyoD can activate muscle specific genes and myogenic differentiation in many cell types. The hypothesis that the DNA binding specificity of MyoD is responsible for its biological specificity was tested. Homodimers of MyoD bind to E-box containing DNA with high affinity, but do not form stable and well defined complexes with heterologous DNA sequences. The physiologically active heterodimer of MyoD and E12 binds an oligonucleotide containing an E-box sequence with an affinity only two orders of magnitude higher than a completely unrelated DNA sequence, stressing the importance of cooperative interactions with other proteins of the transcriptional machinery for specific gene activation.

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

Synthesis, structure and activity of artificial, rationally designed catalytic polypeptides.

Biological macromolecules with catalytic activity can be created artificially using two approaches. The first exploits a system that selects a few catalytically active biomolecules from a large pool of randomly generated (and largely inactive) molecules. Catalytic antibodies and many catalytic RNA molecules are obtained in this way. The second involves rational design of a biomolecule that folds in solution to present to the substrate an array of catalytic functional groups. Here we report the synthesis of rationally designed polypeptides that catalyse the decarboxylation of oxaloacetate via an imine intermediate. We determine the secondary structures of the polypeptides by two-dimensional NMR spectroscopy. We are able to trap and identify intermediates in the catalytic cycle, and to explore the kinetics in detail. The formation of the imine by our artificial oxaloacetate decarboxylases is three to four orders of magnitude faster than can be achieved with simple amine catalysts: this performance rivals that of typical catalytic antibodies.

Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression.

Murine Hox genes are organized into four clusters that share many features with the homeotic clusters of Drosophila. This evolutionary conservation and the clear relationships between the position of a gene within a cluster and its expression pattern have led to the suggestion that the structure of the cluster is essential for proper regulation. Using a Hox-2.6-lacZ reporter gene in transgenic mice we have shown that the overall expression pattern of the endogenous Hox-2.6 gene can be reconstructed when it is isolated from the complex. The transgene was expressed in the proper tissues, with the correct spatial distribution and temporal pattern. Furthermore, direct comparison by in situ hybridization revealed that the levels of transgene expression are similar to those of the endogenous gene. This has allowed us to define three elements that regulate particular aspects of the Hox-2.6 pattern, two of which act as spatially specific enhancers. One enhancer, region A, directed expression only in the neural tube, whereas the other, region C, specified the majority of the Hox-2.6 pattern. Both were also capable of imposing the correct boundaries of expression on heterologous promoters. The definition of such elements will allow the characterization of the trans-acting factors that mediate spatial regulation in the mammalian embryo.

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3'----5')-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable 'module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.