Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role

Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.     

Inhibition of the activity of SRC and Abl tyrosine protein kinases by the binding of the Wiskott-Aldrich syndrome protein

The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible.     

Catalytic and thermodynamic properties of tetrahydromethanopterin-dependent serine hydroxymethyltransferase from Methanococcus jannaschii

The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of Cbeta of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C1) units for several essential biosynthetic processes. In many Archaea, C1 units are carried by modified pterin-containing compounds, which, although structurally related to tetrahydropteroylglutamate, play a distinct functional role. Tetrahydromethanopterin, and a few variants of this compound, are the modified folates of methanogenic and sulfate-reducing Archaea. Little information on SHMT from Archaea is available, and the metabolic role of the enzyme in these organisms is not clear. This contribution reports on the purification and characterization of recombinant SHMT from the hyperthermophilic methanogen Methanococcus jannaschii. The enzyme was characterized with respect to its catalytic, spectroscopic, and thermodynamic properties. Tetrahydromethanopterin was found to be the preferential pteridine substrate. Tetrahydropteroylglutamate could also take part in the hydroxymethyltransferase reaction, although with a much lower efficiency. The catalytic features of the enzyme with substrate analogues and in the absence of a pteridine substrate were also very similar to those of SHMT isolated from Eucarya or Eubacteria. On the other hand, the M. jannaschii enzyme showed increased thermoactivity and resistance to denaturating agents with respect to the enzyme purified from mesophilic sources. The results reported suggest that the active site structure and the mechanism of SHMT are conserved in the enzyme from M. jannaschii, which appear to differ only in its ability to bind and use a modified folate as substrate and increased thermal stability.     

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells

The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21(Cip1/Waf1) and p27(Kip1) is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27(Kip1) and p21(Cip1) synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27(Kip1), p21(Cip1) double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27(Kip1) single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21(Cip1)-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27(Kip1), p21(Cip1) double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27(Kip1), p21(Cip1) double-null granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8-10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27(Kip1) and p21(Cip1) is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.     

Functional plasticity of dendritic cell subsets as mediated by CD40 versus B7 activation

Murine dendritic cells (DCs) can present Ag in an immunogenic or tolerogenic fashion, the distinction depending on either the occurrence of specialized DC subsets or the maturation or activation state of the DC. Although DC subsets may be programmed to direct either tolerance or immunity, it is not known whether appropriate environmental stimulation can result in complete flexibility of a basic program. Using splenic CD8(-) and CD8(+) DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we show that both the in vivo and in vitro activities of either subset can be altered by ligation of specific surface receptors. Otherwise immunogenic CD8(-) DCs become tolerogenic upon B7 ligation by soluble CTLA-4, a maneuver that initiates immunosuppressive tryptophan catabolism. In contrast, CD40 ligation on tolerogenic CD8(+) DCs makes these cells capable of immunogenic presentation. Thus, environmental conditioning by T cell ligands may alter the default function of DC subsets to meet the needs of flexibility and redundancy.     

Experimental manipulation of egg carotenoids affects immunity of barn swallow nestlings

The yolk of bird eggs contains maternal carotenoids that may act as antioxidants thus influencing offspring performance and survival. However, to our knowledge, this hypothesis has not been subjected to experimental tests and the function of transmission of carotenoids to the egg is largely unknown. We directly manipulated the concentration of the main carotenoid (lutein) in the eggs of barn swallows (Hirundo rustica) and analysed the effect of experimental manipulation on growth of nestlings and two fundamental components of their acquired immunity. Nestlings hatched from lutein-inoculated eggs had larger T-cell-mediated immune response compared with those of two control groups. T-cell-mediated immune response predicted nestling survival until fledging. However, lutein inoculation did not affect antibody response to an immunogen, body mass, tarsus length or plumage development. Nestling body mass and plumage development declined with egg laying order, but the effects of lutein inoculation were independent of egg laying order for all traits. Our results show that maternal yolk carotenoids can have a major effect in promoting a fundamental component of immunity that predicts offspring survival and suggests that adaptive early maternal effects can be mediated by transmission of antioxidants to eggs.     

Cardiovascular hybrid drugs: new benzazepinone derivatives as bradycardic agents endowed with selective beta1-non-competitive antagonism

The synthesis and pharmacological profile of some hybrid compounds bearing both the benzazepinone moiety present in Zatebradine and typical beta-blocker aryloxypropanolamine groups are described. The new compounds proved to be endowed with negative chronotropic and inotropic activity and are weak vasorelaxant agents. The cardiodepressant action is probably due to selective beta(1)-noncompetitive reversible antagonism. Both enantiomers of the most active compound 5c were synthesized and they showed a different cardiovascular profile, that is (+)-(R)-enantiomer displays affinity for cardiac beta(1)-adrenoceptors, while (-)-(S)-enantiomer shows specificity for vessel smooth muscle.     

Synthesis and pharmacological characterisation of 2,4-dicarboxy-pyrroles as selective non-competitive mGluR1 antagonists

Metabotropic glutamate receptors (mGluRs) are an unusual family of G-protein coupled receptor (GPCR), and are characterised by a large extracellular N-terminal domain that contains the glutamate binding site. We have identified a new class of non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonists, 2,4-dicarboxy-pyrroles which are endowed with nanomolar potency. They interact within the 7 transmembrane (7TM) domain of the receptor and show antinociceptive properties when tested in a number of different animal models.