Dystrophin in adult zebrafish muscle.

Mutations in the human dystrophin gene are implicated in the fatal muscle wasting disease Duchenne Muscular Dystrophy (DMD). This gene expresses a sarcolemmal-associated protein that is evolutionarily conserved, underpinning its important role in the architecture of muscle. In terms of DMD modelling, the mouse has served as a suitable vertebrate species but the pathophysiology of the disease in the mouse does not entirely mimic human DMD. We have examined the zebrafish in order to expand the repertoire of vertebrate species for muscle disease modelling, and to dissect further the functional interactions of dystrophin. We report here the identification of an apparent zebrafish orthologue of the human dystrophin gene that expresses a 400-kDa protein that is localised to the muscle membrane surface. These data suggest that the zebrafish may prove to be a beneficial vertebrate model to examine the role and functional interactions of dystrophin in disease and development.

The structures of caspases-1, -3, -7 and -8 reveal the basis for substrate and inhibitor selectivity.

BACKGROUND: Peptide inhibitors of caspases have helped define the role of these cysteine proteases in biology. Structural and biochemical characterization of the caspase enzymes may contribute to the development of new drugs for the treatment of caspase-mediated inflammation and apoptosis. RESULTS: The crystal structure of the previously unpublished caspase-7 (Csp7; 2.35 A) bound to the reversible tetrapeptide aldehyde inhibitor acetyl-Asp-Glu-Val-Asp-CHO is compared with crystal structures of caspases-1 (2.3 A), -3 (2.2 A), and -8 (2.65 A) bound to the same inhibitor. Csp7 is a close homolog of caspase-3 (Csp3), and these two caspases possess some quarternary structural characteristics that support their unique role among the caspase family. However, although Csp3 and Csp7 are quite similar overall, they were found to have a significantly different substitution pattern of amino acids in and around the S4-binding site. CONCLUSIONS: These structures span all three caspase subgroups, and provide a basis for inferring substrate and inhibitor binding, as well as selectivity for the entire caspase family. This information will influence the design of selective caspase inhibitors to further elucidate the role of caspases in biology and hopefully lead to the design of therapeutic agents to treat caspase-mediated diseases, such as rheumatoid arthritis, certain neurogenerative diseases and stroke.

Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.

Expression and purification of human interleukin-1beta converting enzyme from Trichoplusia ni insect cells using a baculovirus expression system.

Employing a baculovirus expression system, human interleukin-1beta converting enzyme (ICE) has been expressed in Trichoplusia ni High-Five insect cells and purified. ICE was expressed with an N-terminal T7 epitope, thus allowing its purification using immobilized anti-T7 antibodies. The recombinant ICE was purified to >95% homogeneity, the one minor contaminant being the baculovirus anti-apoptotic protein (P35) which was copurified. The purified recombinant ICE was biologically active, cleaving both pIL-1beta and poly(ADP-ribose) polymerase substrates. The kinetic properties of the purified recombinant ICE compare favorably with native ICE purified from a THP-1 monocytic line.

Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae.

cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1-181 of NS3; NS3 (181)) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae. NS3 (181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography. The purified NS3(181), protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.

Crystal structure of p38 mitogen-activated protein kinase.

p38 mitogen-activated protein kinase is activated by environmental stress and cytokines and plays a role in transcriptional regulation and inflammatory responses. The crystal structure of the apo, unphosphorylated form of p38 kinase has been solved at 2.3 A resolution. The fold and topology of p38 is similar to ERK2 (Zhang, F., Strand, A., Robbins, D., Cobb, M. H., and Goldsmith, E. J. (1994) Nature 367, 704-711). The relative orientation of the two domains of p38 kinase is different from that observed in the active form of cAMP-dependent protein kinase. The twist results in a misalignment of the active site of p38, suggesting that the orientation of the domains would have to change before catalysis could proceed. The residues that are phosphorylated upon activation of p38 are located on a surface loop that occupies the peptide binding channel. Occlusion of the active site by the loop, and misalignment of catalytic residues, may account for the low enzymatic activity of unphosphorylated p38 kinase.

Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide.

An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.

Structure and mechanism of inosine monophosphate dehydrogenase in complex with the immunosuppressant mycophenolic acid.

The structure of inosine-5'-monophosphate dehydrogenase (IMPDH) in complex with IMP and mycophenolic acid (MPA) has been determined by X-ray diffraction. IMPDH plays a central role in B and T lymphocyte replication. MPA is a potent IMPDH inhibitor and the active metabolite of an immunosuppressive drug recently approved for the treatment of allograft rejection. IMPDH comprises two domains: a core domain, which is an alpha/beta barrel and contains the active site, and a flanking domain. The complex, in combination with mutagenesis and kinetic data, provides a structural basis for understanding the mechanism of IMPDH activity and indicates that MPA inhibits IMPDH by acting as a replacement for the nicotinamide portion of the nicotinamide adenine dinucleotide cofactor and a catalytic water molecule.

Inhibition of IMPDH by mycophenolic acid: dissection of forward and reverse pathways using capillary electrophoresis.

The objective of this work was to contribute to the understanding of mechanisms for IMPDH inhibition. We over-expressed hamster type II IMPDH in Escherichia coli, purified the protein to apparent homogeneity, and used capillary electrophoresis to quantify enzyme turnover events accompanying inhibition by mycophenolic acid (MPA). We dissected two convergent pathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates and linked to enzyme catalysis, and a slower "reverse" pathway apparently not involving catalysis. MPA-inhibition occurred rapidly in the forward direction by interrupting the enzyme turnover cycle, after IMP and NAD+ binding, after hydride transfer, and after NADH release. Slow inhibition, without substrate turnover, was achieved by incubating free enzyme with excess XMP and MPA. We propose that mycophenolic acid inhibits IMPDH by trapping a transient covalent product of the hydride transfer reaction (IMPDH approximately XMP*) before a final hydrolysis step that precedes XMP and enzyme release in the forward reaction pathway. Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, chemically defined complexes for structural studies. IMPDH samples inhibited by MPA in the forward and reverse pathways yielded similar, high-quality crystals that are currently undergoing X-ray diffraction analyses.

Structure and mechanism of interleukin-1 beta converting enzyme.

Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

Enthalpy of hydrogen bond formation in a protein-ligand binding reaction.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort to probe the energetic contributions of single protein-ligand hydrogen bonds formed in the binding reactions. Changing tyrosine-82 to phenylalanine in FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12 complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic stabilization of binding in both H2O and D2O. High-resolution crystallographic analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl group in unliganded FKBP-12 are displaced upon formation of the protein-ligand complexes. A thermodynamic analysis is presented that suggests that the removal of polar atoms from water contributes a highly unfavorable enthalpy change to the formation of C=O...HO hydrogen bonds as they occur in the processes of protein folding and ligand binding. Despite the less favorable enthalpy change, the entropic advantage of displacing two water molecules upon binding leads to a slightly more favorable free-energy change of binding in the reactions with wild-type FKBP-12.

15N NMR relaxation studies of the FK506 binding protein: backbone dynamics of the uncomplexed receptor.

Backbone dynamics of the major tacrolimus (FK506) binding protein (FKBP-12, 107 amino acids) have been studied using 15N relaxation data derived from proton-detected two-dimensional 1H-15N NMR spectroscopy. 15N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and heteronuclear NOEs were determined for over 85% of the backbone amide 15N nuclei. A model free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559; Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4559-4570] was used to derive values for the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and the chemical exchange line width (Rex) for each N-H bond vector. The final optimized overall correlation time (tau m) was 9.2 ns. The average order parameter (S2) describing the amplitude of motions on the picosecond time scale was found to be 0.88 +/- 0.06. Motions on the picosecond time scale are restricted at the N and C termini, consistent with previous NMR structural studies indicating well-defined beta-strands in these regions. With the exception of the flap region from resides 82 to 87, no regions appear to be significantly disordered on the picosecond time scale. Residues in several regions of the protein exhibit high Rex terms, indicating possible motions on the millisecond to microsecond time scale due to chemical exchange and/or conformational averaging effects. Possible effects of tacrolimus (FK506) binding on FKBP-12 dynamics are discussed in the context of previously determined solution structures for FKBP-12 in the uncomplexed [Michnick et al. (1991) Science 252, 836-839; Moore et al. (1991) Nature 351, 248-250] and complexed [Meadows et al. (1993) Biochemistry 32, 754-765] states.

Physical characterisation and over-expression of the Bacillus caldotenax superoxide dismutase gene.

The gene (sod) encoding Bacillus caldotenax (BC) Mn-superoxide dismutase (MnSOD) has been cloned in Escherichia coli and its entire nucleotide sequence determined. Within the coding region of the gene there were 21 nucleotide differences to the previously sequenced sod of Bacillus stearothermophilus (BS). The predicted amino acid sequence of BCMnSOD had two amino acid dissimilarities to the BSMnSOD, containing Asp and Val at positions 13 and 188, respectively, compared to Glu and Ile at the respective equivalent positions of BSMnSOD. Recombinant BCMnSOD was shown to be functionally active in E. coli, both in vitro and in vivo, and was produced at levels representing over 40% of the cells' soluble protein by coupling sod transcription to the E. coli trp promoter. The sequenced region of DNA was also found to encompass part of a second open-reading frame, of unknown function, previously noted 3' to the B. stearothermophilus gene.

Molecular cloning and nucleotide sequence determination of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Escherichia coli.

The gene (sod) encoding Bacillus stearothermophilus Mn-superoxide dismutase (MnSOD) has been cloned in Escherichia coli and its entire nucleotide sequence determined. With the exception of the post-translationally cleaved N-terminal methionine residue, the predicted amino acid sequence exhibits complete identity to the previously determined amino acid sequence. The recombinant MnSOD was shown to be functionally active in E. coli both in vitro and in vivo, and was expressed to 49% of the soluble cell protein by coupling its transcription to the E. coli trp promoter. The sequenced region of DNA was also found to encompass a second open reading frame. The putative encoded polypeptide exhibited no significant primary sequence homology to any currently characterised protein.

The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing.

A series of nic- cloning vectors have been constructed analogous to the pUC plasmids but which are smaller in size and carry more extensive polylinker regions within the lacZ' gene. The vectors pMTL20 and pMTL21 carry six additional sites (AatII, MluI, NcoI, BglII, XhoI and StuI) to those present in pUC18 and pUC19, while pMTL22 and -23 possess eleven new cloning sites (ActII, MluI, NcoI, BglII, XhoI, StuI, NaeI, EcoRV, ClaI, NdeI and NruI). More importantly, the relative order of the restriction sites within the polylinker of these latter vectors has been totally rearranged, relative to pUC18 and pUC19, to facilitate the conversion of DNA fragments with incompatible ends to fragments with compatible termini. The availability of such DNA fragments is a crucial requirement when M13 templates are generated for dideoxy sequencing by the sonication procedure. Derivatives of these vectors have also been constructed which demonstrate improved segregational stability by incorporation of the pSC101 par locus. During the construction of these new vectors data were obtained which demonstrated that the pUC and pMTL plasmids contain a previously unreported single base pair difference within the RNA I/RNA II region (compared to pBR322) responsible for a three-fold increase in plasmid copy number. The pUC and pMTL plasmids were also shown to be functionally nic-, thus affording the lowest categorisation in genetic manipulation experiments.

Measurement of thymus weight, lumbar node weight and progesterone levels in syngeneically pregnant, allogeneically pregnant, and pseudopregnant mice.

Female CBA mice were mated to fertile CBA males, to vasectomized CBA males, to fertile C57BL males or to vasectomized C57BL males. After allogeneic or syngeneic mating the extent of thymic involution on the 10th day of pregnancy and pseudopregnancy was similar. Lumbar lymph node weight was not affected by pseudopregnancy but increased similarly in allogeneic and syngeneic pregnancies. Serum progesterone levels on the 10th day of pseudopregnancy were similar to those of non-pregnant females, and significantly lower than those of pregnant females. On the 4th to 7th days progesterone levels in pseudopregnant animals were equal to those in pregnant animals. Progesterone levels and thymic involution were similar in syngeneically and allogeneically pregnant females. Progesterone levels were negatively correlated with thymus weight but reached significance only when the mating was allogeneic. It is suggested that there is an interaction between progesterone concentrations and the degree of thymic involution during pregnancy.