Stimulation of oxygen consumption at the cytochrome A3 level inhibits aldosterone biosynthesis from 18-hydroxycorticosterone

A mitochondrial preparation from duck adrenal gland was used, under aerobic conditions, to show that the oxygen requirement for the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) is at the cytochrome P-450 level only. Vitamin C and tetramethyl-p-phenylene-diamine (TMPD) were used to increase oxygen consumption at the cytochrome a3 level, thereby decreasing its availability to cytochrome P-450. The vitamin C plus TMPD system acts as an 'oxygen trap'. Results show that despite reducing equivalents provided by L-malate, vitamin C plus TMPD strongly inhibits aldosterone biosynthesis from 18-hydroxycorticosterone (89%). Moreover, we used KCN in order to block oxygen consumption, even in the presence of vitamin C plus TMPD. Under these conditions, the inhibition of aldosterone biosynthesis from 18-hydroxycorticosterone is reduced by 51%. The reversal of this inhibition by KCN was evident but only partial. According to polarographic and electron microscopy studies, the reversal of inhibition can only be explained by an increased availability of oxygen at the cytochrome P-450 level. Experiments performed under aerobic conditions, without a nitrogen atmosphere, show that oxygen is required in the transformation of 18-hydroxycorticosterone into aldosterone, at the cytochrome P-450 level. This suggests that a classical hydroxylating mechanism is involved.     

Action of different nucleotides on the last step of aldosterone synthesis

The effect of various nucleotides on the last step of aldosterone biosynthesis, the so-called "18 oxidation" (transformation of 18-hydroxycorticosterone to aldosterone), was studied by incubation of tritiated 18-hydroxycorticosterone with untreated duck adrenal mitochondria in vitro. The study was carried out in the absence or in the presence of antimycin A which blocks the respiratory chain. Results show that, when oxidative phosphorylation chain functions normally, GTP and CTP had no effect, UTP stimulated this reaction but ADP and ATP inhibited the transformation of 18-hydroxycorticosterone into aldosterone to the same extent. For this reason ATP is included in all controls for experiments studying the effect of ATP when "18 oxidation" is inhibited by antimycin A. When oxidative phosphorylation chain is inhibited by antimycin A, ATP is able to reverse the inhibition of "18 oxidation" induced by antimycin A, in the presence of succinate. Under these conditions UTP is not able to reverse the inhibition induced by antimycin A; GTP and CTP had no effect. Effects of ATP and UTP on the last step of aldosterone biosynthesis are related to different mechanisms. ATP clearly acts as an energy source for "18 oxidation" in the presence of succinate. The role of UTP must still be determined.     

Unexpected dimerization of a tripeptide comprising a β,γ‐diamino acid

We have previously reported the synthesis of enantiopure β,γ‐diamino acids and relevant short α/γ‐peptide containing such building blocks. Complete nuclear magnetic resonance (NMR) studies, together with molecular modeling, highlighted the ability of a β,γ‐diamino acid to induce various intramolecular turns. In this paper, we describe for the first time the formation of a dimeric structure constituted by α/γ/α‐tripeptide and stabilized by intermolecular interactions. A structural model is proposed based on extensive NMR measurements.     

High affinity Grb2-SH3 domain ligand incorporating Cbeta-substituted prolines in a Sos-derived decapeptide

Peptide ligands that disrupt MAPK pathways are of great interest for a better understanding of these signalling cascades and represent therefore an attractive target to control cell degenerative processes. In that context, selective disruption of the upstream Grb2/Sos complex in the Ras/MAPK cascade has focused extensive work. The Sos PPII decapeptide, which interacts with the Grb2-SH3 domains, has been modified in various positions and the best inhibitors designed so far are either dimeric ligands or peptoid analogues of the VPPPVPPRRR sequence. We report the synthesis of new Grb2 ligands in which the key Val5 residue has been replaced by a cis C(beta)-substituted proline. Both fluorescence and ITC assays have been employed to measure the affinity of these substituted peptides for a recombinant Grb2 protein. Whereas proline in position 5 completely abolished the binding potency, a cis C(beta)-methyl-L-proline restored the affinity. Other cis C(beta)-proline substituents led to a complete loss of binding potency. Combining the best modifications: a cis C(beta)-methylproline 5, N-acetylation, C-carboxamide and dimerization yielded a 560-fold affinity enhancement compared to the wild-type VPPPVPPRRR sequence. This study shows that C(beta)-substituted prolines may constitute a new alternative for PPII ligands, combining entropy and enthalpy beneficial effects.     

Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis

Tissue mechanics have been shown to play a key role in the regulation of morphogenesis in animals [1-4] and may have an equally important role in plants [5-9]. The aerial organs of plants are formed at the shoot apical meristem following a specific phyllotactic pattern [10]. The initiation of an organ from the meristem requires a highly localized irreversible surface deformation, which depends on the demethylesterification of cell wall pectins [11]. Here, we used atomic force microscopy (AFM) to investigate whether these chemical changes lead to changes in tissue mechanics. By mapping the viscoelasticity and elasticity in living meristems, we observed increases in tissue elasticity, correlated with pectin demethylesterification, in primordia and at the site of incipient organs. Measurements of tissue elasticity at various depths showed that, at the site of incipient primordia, the first increases occurred in subepidermal tissues. The results support the following causal sequence of events: (1) demethylesterification of pectin is triggered in subepidermal tissue layers, (2) this contributes to an increase in elasticity of these layers-the first observable mechanical event in organ initiation, and (3) the process propagates to the epidermis during the outgrowth of the organ.     

A low-temperature heteronuclear NMR study of two exchanging conformations of metal-bound pyoverdin PaA from Pseudomonas aeruginosa

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdin PaA, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, D-Ser-L-Arg-D-Ser-L-FoOHOrn-(L-Lys-L-FoOHOrn-L-Thr-L-Thr) (FoOHOrn: delta N-formyl-delta N-hydroxyornithine), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the L-Lys side chain. Ferric iron is chelated by the catechol group on the chromophore and the two hydroxyornithine side chains. In aqueous solution, the (1)H-NMR spectrum of pyoverdin PaA-Ga(III), in which Ga(III) is used instead of Fe(III) for spectroscopic purposes, showed clear evidence of exchange broadening, preventing further structural characterization. The use of cryo-solvents allowed measurements to be made at temperatures as low as 253 K where two distinct conformations with roughly equivalent populations could be observed. (13)C and (15)N labeling of pyoverdin PaA enabled complete assignment of both forms of pyoverdin PaA-Ga(III) at 253 and 267 K, using triple-resonance multidimensional NMR experiments commonly applied to doubly labeled proteins.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.