Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase.
; ; et al
in Nature (2011), 473(7346), 234-8
Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle ... [more ▼]
Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses. [less ▲]Detailed reference viewed: 163 (18 ULg)
Heart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3).
; Toussaint, Louise ; et al
in Biochemical Journal (2010), 431(2), 267-75
On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation ... [more ▼]
On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [gamma-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBbeta-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBalpha/beta siRNA or PKBalpha siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBalpha. [less ▲]Detailed reference viewed: 25 (2 ULg)
Effects of marginal iron overload on iron homeostasis and immune function in alveolar macrophages isolated from pregnant and normal rats.
; ; et al
in Biometals (2009), 22(2), 211-23
The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx ... [more ▼]
The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy +/- a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFkappaB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms. [less ▲]Detailed reference viewed: 12 (0 ULg)
Comparative Fe and Zn K-edge X-ray absorption spectroscopic study of the ferroxidase centres of human H-chain ferritin and bacterioferritin from Desulfovibrio desulfuricans.
in Journal of Biological Inorganic Chemistry : A Publication of the Society of Biological Inorganic Chemistry (2009), 14(1), 35-49
Iron uptake by the ubiquitous iron-storage protein ferritin involves the oxidation of two Fe(II) ions located at the highly conserved dinuclear ‘‘ferroxidase centre’’ in individual subunits. We have ... [more ▼]
Iron uptake by the ubiquitous iron-storage protein ferritin involves the oxidation of two Fe(II) ions located at the highly conserved dinuclear ‘‘ferroxidase centre’’ in individual subunits. We have measured X-ray absorption spectra of four mutants (K86Q, K86Q/E27D, K86Q/E107D, and K86Q/E27D/E107D, involving variations of Glu to Asp on either or both sides of the dinuclear ferroxidase site) of recombinant human H-chain ferritin (rHuHF) in their complexes with reactive Fe(II) and redoxinactive Zn(II). The results for Fe–rHuHf are compared with those for recombinant Desulfovibrio desulfuricans bacterioferritin (DdBfr) in three states: oxidised, reduced, and oxidised/Chelex -treated. The X-ray absorption nearedge region of the spectrum allows the oxidation state of the iron ions to be assessed. Extended X-ray absorption fine structure simulations have yielded accurate geometric information that represents an important refinement of the crystal structure of DdBfr; most metal–ligand bonds are shortened and there is a decrease in ionic radius going from the Fe(II) to the Fe(III) state. The Chelex -treated sample is found to be partly mineralised, giving an indication of the state of iron in the cycled-oxidised (reduced, then oxidised) form of DdBfr, where the crystal structure shows the dinuclear site to be only half occupied. In the case of rHuHF the complexes with Zn(II) reveal a surprising similarity between the variants, indicating that the rHuHf dinuclear site is rigid. In spite of this, the rHuHf complexes with Fe(II) show a variation in reactivity that is reflected in the iron oxidation states and coordination geometries. [less ▲]Detailed reference viewed: 16 (1 ULg)
High-resolution X-ray structures of human apoferritin H-chain mutants correlated with their activity and metal-binding sites.
in Journal of Molecular Biology (2007), 365(2), 440-52
Ferritins are a family of proteins distributed widely in nature. In bacterial, plant, and animal cells, ferritin appears to serve as a soluble, bioavailable, and non-toxic form of iron provider. Ferritins ... [more ▼]
Ferritins are a family of proteins distributed widely in nature. In bacterial, plant, and animal cells, ferritin appears to serve as a soluble, bioavailable, and non-toxic form of iron provider. Ferritins from animal sources are heteropolymers composed of two types of subunit, H and L, which differ mainly by the presence (H) or absence (L) of active ferroxidase centres. We report the crystallographic structures of four human H apoferritin variants at a resolution of up to 1.5 Angstrom. Crystal derivatives using Zn(II) as redox-stable alternative for Fe(II), allows us to characterize the different metal-binding sites. The ferroxidase centre, which is composed of sites A and B, binds metal with a preference for the A site. In addition, distinct Zn(II)-binding sites were found in the 3-fold axes, 4-fold axes and on the cavity surface near the ferroxidase centre. To study the importance of the distance of the two metal atoms in the ferroxidase centre, single and double replacement of glutamate 27 (site A) and glutamate 107 (site B), the two axial ligands, by aspartate residues have been carried out. The consequences for metal binding and the correlation with Fe(II) oxidation rates are discussed. [less ▲]Detailed reference viewed: 2 (0 ULg)