Phosphodiesterase type IV inhibitors in the treatment of multiple sclerosis.

Multiple sclerosis is an autoimmune disease with inflammatory lesions localized to the white matter of the central nervous system. Early on, the disease is characterized by episodes of exacerbations and remissions. During exacerbations there is an acute inflammatory infiltrate characterized by the presence of mononuclear cells, monocytes, and T lymphocytes. These cells produce proinflammatory cytokines that have been implicated in the amplification of the inflammatory response as well as in the damage of oligodendrocytes. The inflammation ultimately results in loss of myelin and oligodendrocyte cell death (demyelination). Thus therapies aimed at preventing the inflammatory response may have a beneficial effect on the course of the disease. One such therapy is treatment with inhibitors of phosphodiesterase type IV. These drugs have proven to be extremely effective in the prevention and treatment of experimental allergic encephalomyelitis, the animal model for multiple sclerosis. These experiments, as well as other data discussed here, provide a rationale for the treatment of multiple sclerosis with inhibitors of phosphodiesterase type IV.

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases.

The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate diacarboxylic amino acids with similar rate constants. However, eTATase exhibits approximately 10(2)-10(4)-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to alpha-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L-Asp and L-Glu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidino group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acid.

The type IV phosphodiesterase specific inhibitor mesopram inhibits experimental autoimmune encephalomyelitis in rodents.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological features reminiscent of those seen in multiple sclerosis and thus serves as an animal model for this disease. Inhibition of type IV phosphodiesterase (PDE IV) in animals with this disease has been shown to result in amelioration of disease symptoms. Here we describe the immunomodulatory activity of the novel potent and selective PDE IV inhibitor mesopram. In vitro, mesopram selectively inhibits the activity of type 1 helper T (Th1) cells without affecting cytokine production or proliferation of type 2 helper T (Th2) cells. Administration of mesopram to rodents inhibits EAE in various models. Clinically, EAE is completely suppressed by mesopram in Lewis rats. This is accompanied by a reduction of inflammatory lesions in spinal cord and brain. RT-PCR analysis revealed a marked reduction in the expression of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the brains of these animals. Furthermore, the ex vivo production of Th1 cytokines by activated spleen cells derived from mesopram-treated animals is significantly reduced compared to vehicle-treated controls. Amelioration of the clinical symptoms is also observed during chronic EAE in mesopram-treated SJL mice as well as in relapsing-remitting EAE in SWXJ mice using a therapeutic treatment regimen. These data demonstrate the anti-inflammatory activity of mesopram and provide a rationale for its clinical development.

Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change.

The strictly conserved active site residue, Asp222, which forms a hydrogen-bonded salt bridge with the pyridine nitrogen atom of the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase (AATase), was replaced with alanine (D222A) in the Escherichia coli enzyme. The D222A mutant exhibits non-hyberbolic saturation behavior with amino acid substrates which appear as apparent negative cooperativity in steady-state kinetic analyses. Single turnover progress curves for D222A are well described by the sum of two exponentials, contrasting with the monophasic kinetics of the wild-type enzyme. An active/inactive heterodimer containing the D222A mutation retains this biphasic kinetic response, proving that the observed cooperativity is not the result of induced allostery. The anomalous behavior is explained by a hysteretic kinetic model involving two slowly interconverting enzyme forms, only one of which is catalytically competent. The slow functional transition between the two forms has a half-life of approximately 10 mins. Preincubation of the mutant with the dicarboxylic inhibitor maleate shifts the equilibrium population of the enzyme towards the catalytically active form, suggesting that the slow transition is related to the domain closure known to occur upon association of this inhibitor with the wild-type enzyme. The importance of Asp222 in the chemical steps of transamination is confirmed by the approximately 10(5)-fold decrease in catalytic competence in the D222A mutant, and by the large primary C alpha-deuterium kinetic isotope effect (6.7 versus 2.2 for the wild-type). The transamination activity of the D222A mutant is enhanced 4- to 20-fold by reconstitution with the co-factor analog N-methylpyridoxal-5'-phosphate (N-MePLP), and the C alpha-proton abstraction step is less rate determining, as evidenced by the decrease in the primary kinetic isotope effect from 6.7 to 2.3. These results suggest that the conserved interaction between the protonated pyridine nitrogen of PLP and the negatively charged carboxylate of Asp222 is important not only for efficient C alpha-proton abstraction, but also for conformational transitions concomitant with the transamination process.

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.

Alternating arginine-modulated substrate specificity in an engineered tyrosine aminotransferase.

Mutation of six residues of Escherichia coli aspartate aminotransferase results in substantial acquisition of the transamination properties of tyrosine amino-transferase without loss of aspartate transaminase activity. X-ray crystallographic analysis of key inhibitor complexes of the hexamutant reveals the structural basis for this substrate selectivity. It appears that tyrosine aminotransferase achieves nearly equal affinities for a wide range of amino acids by an unusual conformational switch. An active-site arginine residue either shifts its position to electrostatically interact with charged substrates or moves aside to allow access of aromatic ligands.

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase.

To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.

Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli.

The effects of four single amino acid replacements on the stability and folding of the alpha subunit of tryptophan synthase from Escherichia coli have been investigated by ultraviolet differences spectroscopy. In previous studies [Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586], it had been shown that the urea-induced unfolding at pH 7.8, 25 degrees C, proceeds by the initial unfolding of the less stable carboxyl domain (residues 189-268) followed by the unfolding of the more stable amino domain (residues 1-188). The effects of the Phe-22----Leu, Glu-49----Met, Gly-234----Asp, and Gly-234----Lys mutants on the equilibrium unfolding process can all be understood in terms of the domain unfolding model. With the exception of the Glu-49----Met replacement, the effects on stability are small. In contrast, the effects of three of the four mutations on the kinetics of interconversion of the native form and one of the stable partially folded intermediates are dramatic. The results for the Phe-22----Leu and Gly-234----Asp mutations indicate that these residues play a key role in the rate-limiting step. The Glu-49----Met mutation increases the stability of the native form with respect to that of the intermediate but does not affect the rate-limiting step. The Gly-234----Lys mutation does not affect either the stability or the kinetics of folding for the transition between native and intermediate forms. The changes in stability calculated from the unfolding and refolding rate constants agree quantitatively with those obtained from the equilibrium data. When considered with the results from a previous study on the Gly-211----Glu replacement [Matthews, C. R., Crisanti, M. M., Manz, J. T., & Gepner G. L. (1983) Biochemistry 22, 1445], it can be concluded that the rate-limiting step in the conversion of the intermediate to the native conformation involves either domain association or some other type of molecule-wide phenomenon.

Folding of homologous proteins: conservation of the folding mechanism of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids.

The equilibrium and kinetic properties for the urea-induced unfolding of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids were compared to determine the role of protein folding in evolution. The parent proteins differ at 40 positions in the sequence of 268 amino acids, and the hybrids differ by up to 15 amino acids from the Escherichia coli alpha subunit. The results show that all the proteins follow the same folding mechanism and are consistent with a previously proposed hypothesis [Hollecker, M., & Creighton, T. E. (1983) J. Mol. Biol. 168, 409; Krebs, H., Schmid, F. X., & Jaenicke, R. (1983) J. Mol. Biol. 169, 619] that the folding mechanisms are conserved in homologous proteins. Analysis of the kinetic data suggests that the 15 positions at which the parent proteins differ in the amino folding unit, residues 1-188, do not play a role in a rate-limiting step in folding that has been previously identified as the association of the amino and carboxyl folding units [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T. S., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965]. One or more of the 25 positions at which the parent proteins differ in the carboxyl folding unit, residues 189-268, do appear to play a role in this same rate-limiting step.

Myomegalin is a novel protein of the golgi/centrosome that interacts with a cyclic nucleotide phosphodiesterase.

Subcellular targeting of the components of the cAMP-dependent pathway is thought to be essential for intracellular signaling. Here we have identified a novel protein, named myomegalin, that interacts with the cyclic nucleotide phosphodiesterase PDE4D, thereby targeting it to particulate structures. Myomegalin is a large 2,324-amino acid protein mostly composed of alpha-helical and coiled-coil structures, with domains shared with microtubule-associated proteins, and a leucine zipper identical to that found in the Drosophila centrosomin. Transcripts of 7.5-8 kilobases were present in most tissues, whereas a short mRNA of 2.4 kilobases was detected only in rat testis. A third splicing variant was expressed predominantly in rat heart. Antibodies against the deduced sequence recognized particulate myomegalin proteins of 62 kDa in testis and 230-250 kDa in heart and skeletal muscle. Immunocytochemistry and transfection studies demonstrate colocalization of PDE4D and myomegalin in the Golgi/centrosomal area of cultured cells, and in sarcomeric structures of skeletal muscle. Myomegalin expressed in COS-7 cells coimmunoprecipitated with PDE4D3 and sequestered it to particulate structures. These findings indicate that myomegalin is a novel protein that functions as an anchor to localize components of the cAMP-dependent pathway to the Golgi/centrosomal region of the cell.

Effect of single amino acid replacements on the folding and stability of dihydrofolate reductase from Escherichia coli.

The role of the secondary structure in the folding mechanism of dihydrofolate reductase from Escherichia coli was probed by studying the effects of amino acid replacements in two alpha helices and two strands of the central beta sheet on the folding and stability. The effects on stability could be qualitatively understood in terms of the X-ray structure for the wild-type protein by invoking electrostatic, hydrophobic, or hydrogen-bonding interactions. Kinetic studies focused on the two slow reactions that are thought to reflect the unfolding/refolding of two stable native conformers to/from their respective folding intermediates [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445-5452]. Replacements at three different positions in helix alpha B selectively alter the relaxation time for unfolding while a single replacement in helix alpha C selectively alters the relaxation time for refolding. This behavior is characteristic of mutations that change the stability of the protein but do not affect the rate-limiting step. In striking contrast, replacements in strands beta F and beta G can affect both unfolding and refolding relaxation times. This behavior shows that these mutations alter the rate-limiting step in these native-to-intermediate folding reactions. It is proposed that the intermediates have an incorrectly formed beta sheet whose maturation to the structure found in the native conformation is one of the slow steps in folding.