The chemokine receptor Cxcr3 is not essential for acute cardiac allograft rejection in mice and rats.

Chemokine receptors have gained attention as potential targets for novel therapeutic strategies. We investigated the mechanisms of allograft rejection in chemokine receptor Cxcr3-deficient mice using a model of acute heart allograft rejection in the strain combination BALB/c to C57BL/6. Allograft survival was minimally prolonged in Cxcr3-deficient mice compared to wild-type (wt) animals (8 vs. 7 days) and treatment with a subtherapeutic dose of cyclosporine A (CsA) led to similar survival in Cxcr3-deficient and wt recipients (13 vs. 12 days). At rejection grafts were histologically indistinguishable. Microarray analysis revealed that besides Cxcr3 only few genes were differentially expressed in grafts or in spleens from transplanted or untransplanted animals. Transcript analysis by quantitative RT-PCR of selected cytokines, chemokines, or chemokine receptors or serum levels of selected cytokines and chemokines showed similar levels between the two groups. Furthermore, in a rat heart allograft transplantation model treatment with a small molecule CXCR3 antagonist did not prolong survival despite full blockade of Cxcr3 in vivo. In summary, Cxcr3 deficiency or pharmacologic blockade does not diminish graft infiltration, tempo and severity of rejection. Thus, Cxcr3 does not appear to play a pivotal role in the allograft rejection models described here.

Distribution of calcineurin A isoenzyme mRNAs in rat thymus and kidney.

The distribution of the mRNAs encoding the different isoforms of the catalytic subunit (A subunit) of calcineurin has been investigated in rat thymus and kidney using in situ hybridization histochemistry with specific antisense oligonucleotide probes. In the thymus, the mRNAs of the A beta isoforms were the predominant transcripts and showed very intense hybridization signals in the cortical areas. The A alpha mRNAs were expressed at low levels. A beta 2 mRNA was expressed at higher levels than A beta 3 mRNA, but no difference could be detected between the expression levels of A alpha 1 and A alpha 2. In the kidney, highest calcineurin A mRNA hybridization signals were found in the medulla. Signal intensities of A alpha mRNAs were comparable to those of A beta mRNAs. A alpha 1 mRNA level was extremely weak, and A beta 2 mRNA expression was slightly higher than A beta 3 mRNA expression. A tissue-specific distribution pattern of the alternatively spliced isoforms of calcineurin A, as suggested by these preliminary data from thymus and kidney, may be critical in understanding the toxic side-effects associated with the use of the immunosuppressive, calcineurin-inhibiting compounds cyclosporin A and FK506.

Solution structure of a cysteine rich domain of rat protein kinase C.

Intracellular protein phosphorylation by protein kinase C (PKC) plays a major role in the translation of extracellular signals into cellular events. Speculations on the structural basis for PKC activation are based on sequence homology between their cysteine-rich domains (CRD) and the DNA-binding 'zinc-fingers'. We produced a fragment comprising the second CRD (CRD2) of rat PKC-alpha and determined its three-dimensional structure in solution by NMR spectroscopy. This revealed that CRD2 adopts a globular fold allowing two non-consecutive sets of zinc-binding residues to form two separate metal-binding sites. The fold is different to those previously proposed and allows insight into the molecular topology of a family of homologous proteins.

Mapping of the immunophilin-immunosuppressant site of interaction on calcineurin.

The interaction of the immunosuppressive complexes cyclosporin A-cyclophilin A and FK506 binding protein-FK506 with the Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin has been investigated by means of photoaffinity labeling and chemical cross-linking. Photolabeling of purified bovine brain calcineurin with the affinity label [O-[4-[4-(1-diazo-2,2,2-trifluoroethyl)benzoyl]aminobutanoyl]-D- serine8]cyclosporin in the presence of cyclophilin A results, in addition to the labeling of cyclophilin itself, in the transfer of some of the chemical probe to both the catalytic subunit A and the regulatory subunit B of calcineurin. Chemical cross-linking studies with disuccinimidyl suberate in the presence of either cyclophilin A, B, or C in complex with cyclosporin A or FK506 binding protein-FK506 result on the other hand in the apparently exclusive and strictly immunosuppressant-dependent formation of covalent immunophilin-calcineurin B subunit products. Cross-linking of immunophilins to calcineurin B subunit requires the presence of subunit A. In the present study, using a set of recombinant maltose-binding protein fusion products representing different stretches of the catalytic subunit A, we were able to map the minimal calcineurin A sequence necessary for immunophilin-ligand-calcineurin B interaction to occur.

Differential distribution of calcineurin A alpha isoenzyme mRNA's in rat brain.

Specific antisense oligonucleotide probes for the alpha isoforms of the catalytic subunit (A-subunit) of calcineurin were prepared and the distribution of A alpha 1 and A alpha 2 mRNA's has been studied in rat brain using in situ hybridization histochemistry. Clear regional differences have been observed for the A alpha 1 and A alpha 2 isoforms. The predominant form, A alpha 1, was found to be preferentially expressed in the caudate putamen, the pyramidal cell layer of the hippocampus, specific cortical cell layers, the cerebellar granular cell layer and some other brain areas. On the other hand, the A alpha 2 isoform, although being generally less abundant than A alpha 1, gave an intense autoradiography signal in the dentate gyrus of the hippocampus and was the major transcript in the amygdala, the superior and the inferior colliculus, the central gray matter and the reticular formation. These regional differences might reflect specific functions exerted by the two alternatively spliced isoenzymes in the CNS and opens the perspective of interfering with defined calcineurin-dependent signal transduction pathways using isoform-specific compounds.

Cyclosporins. Structure-activity relationships.

Cyclosporin A (Sandimmun) achieves immunosuppressive activity by complex formation with cyclophilin and subsequent binding of the binary complex to and inhibiting protein phosphatase 2B (calcineurin). Complexes of nonimmunosuppressive cyclophilin binding cyclosporin analogues do not inhibit protein phosphatase 2B, suggesting a crucial role for this enzyme in T cell activation. Binding of cyclosporin A to cyclophilins A, B, and C, respectively, results in complexes of significantly different inhibitory potency. The cyclosporin molecule thus has two functional domains, one mediating cyclophilin binding and a second one endowing affinity of the complex to calcineurin, thereby inhibiting its enzyme activity. Structure-activity studies and x-ray crystallography of cyclosporin-cyclophilin complexes indicate a crucial role of leucine side chains in positions 4 and 6 of the cyclosporin macrocycle for the calcineurin interaction.

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase.

Lactate dehydrogenases are of considerable interest as stereospecific catalysts in the chemical preparation of enantiomerically pure alpha-hydroxyacid synthons. For such applications in synthetic organic chemistry it would be desirable to have enzymes which tolerate elevated temperatures for prolonged reaction times, to increase productivity and to extend their applicability to poor substrates. Here, two examples are reported of significant thermostabilizations, induced by site-directed mutagenesis, of an already thermostable protein, the L-lactate dehydrogenase (EC 1.1.1.27, 35 kDa per monomer subunit) from Bacillus stearothermophilus. Thermal inactivation of this enzyme is accompanied by irreversible unfolding of the native protein structure. The replacement of Arg171 by Tyr stabilizes the enzyme against thermal inactivation and unfolding. This stabilizing effect appears to be based on improved interactions between the subunits in the core of the active dimeric or tetrameric forms of the enzyme. The thermal stability of L-lactate dehydrogenase variants with an active site Arg residue, either in the 171 (wild-type) or in the 102 position, is further increased by sulfate ions. The two stabilizing effects are additive, as found for the Arg171Tyr/Gln102Arg double mutant, for which the stability of the protein in 100 mM sulfate solution reaches that of L-lactate dehydrogenases from extreme thermophiles. All mutant proteins retain significant catalytic activity, both in the presence and absence of stabilizing salts, and are viable catalysts in preparative scale reactions.

On the effects of replacing the carboxylate-binding arginine-171 by hydrophobic tyrosine or tryptophan residues in the L-lactate dehydrogenase from Bacillus stearothermophilus.

For L-lactate dehydrogenases (LDH's), the interaction of the guanidinium group of their Arg 171 residue with the carboxylate group of an alpha-keto acid is of primary importance in orienting the substrate productively at the active site. LDH's such as that of Bacillus stearothermophilus (BSLDH) are of practical importance for the preparation of chiral 2-hydroxy acids used as synthons in asymmetric synthesis but would even be more valuable in this regard if their specificities were broader. With a view to tailoring the specificity of BSLDH toward carbonyl substrates that lack an alpha-carboxyl group such as ketones, site-directed mutagenesis has been applied to replace Arg 171 by the approximately isosteric, but hydrophobic, amino acids Tyr and Trp. The mutant enzymes exhibit remarkably good catalytic activities toward representative alpha-keto acids RCOCOOH, where R = Me, Et, n-Pr, n-Bu, and CH2OH, although for the mutant enzymes the kcat/KM's are lower by approximately 10(3)-10(4)-fold than those for native BSLDH. Surprisingly, the 171----Tyr/Trp enzymes are significantly more active than 171----Lys (Hart et al., 1987a), for which an interaction of a positively charged side chain with substrate COO- is retained. Preparative-scale 171----Trp catalyzed reduction of pyruvate gave optically pure L-lactate, showing that L stereospecificity of such LDH enzymes was unaffected by the loss of Arg 171. The retention of L stereospecificity is attributed to secondary polar or hydrogen-bonding associations of Arg 109 and Thr246, respectively, with the substrate COO-function that are of sufficient magnitude to maintain "normal" substrate orientation.(ABSTRACT TRUNCATED AT 250 WORDS)

On the effect on specificity of Thr246----Gly mutation in L-lactate dehydrogenase of Bacillus sterothermophilus.

The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.