7 research outputs found
Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2
AbstractBackground: Phosphatidylinositol transfer protein (PI-TP), which has the ability to transfer phosphatidylinositol (PI) from one membrane compartment to another, is required in the inositol lipid signalling pathway through phospholipase CâÎČ (PLCâÎČ) that is regulated by GTP-binding protein(s) in response to extracellular signals. Here, we test the hypothesis that the principal role of PI-TP is to couple sites of lipid hydrolysis to sites of synthesis, and so to replenish depleted substrate for PLCâÎČ.Results We have designed an experimental protocol that takes advantage of the different rates of release of endogenous PI-TP and PLC-ÎČ from HL60 cells permeabilized with streptolysin O. We have examined the kinetics of stimulated inositol lipid hydrolysis in cells depleted of PI-TP, but not of endogenous PLC-ÎČ, in the presence and absence of exogenous PI-TP. Linear time-courses were observed in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5âČ[Îł-thio]-triphosphate (GTPÎłS) or the receptor-directed agonist fMetLeuPhe as activators. In addition, depletion from the cells of both PI-TP and PLC-ÎČ isoforms by extended permeabilization (40 minutes) allowed us to control the levels of PLCâÎČ present in the cells. Once again, PI-TP increased the rates of reactions. To identify whether the role of PI-TP was to make available the substrate phosphatidylinositol bisphosphate (PIP2) for the PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP. We found that PI-TP was essential for the synthesis of PIP2.Conclusion The predicted function of PI-TP in inositol lipid signalling is the provision of substrate for PLCâÎČ from intracellular sites where PI is synthesized. We propose that PI-TP is in fact a co-factor in inositol lipid signalling and acts by interacting with the inositol lipid kinases. We hypothesize that the preferred substrate for PLCâÎČ is not the lipid that is resident in the membrane but that provided through PI-TP
Crystallization and Preliminary X-ray Diffraction Studies on ADP-ribosylation Factor 1
ADP-ribosylation factor 1 (ARF-1) is a member of a family of small G-proteins that regulate both intracellular vesicle transport and phospholipase D activity. Crystals of ARF-1 suitable for X-ray diffraction analysis have been grown in the presence of GDP by the hanging drop vapour diffusion method. Crystal grows in space C2 with cell dimensions a = 122.36 Ă
, b = 45.01 Ă
, c = 91.96 Ă
and ÎČ = 133.62° and diffract to at least 2.3 Ă
resolution. A second crystal form has been characterized (space group C2, a = 69.70 Ă
, b = 45.25 Ă
, c = 60.45 Ă
, ÎČ = 109.6°) but does not grow reproducibly
Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells
Developmentally acquired PKA localisation in mouse oocytes and embryos
<p>Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development.</p>
<p>Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.</p>
Phosphatidylinositol Transfer Proteins: One Big Happy Family or Strangers with the Same Name?
Search for intermediate-mass black hole binaries in the third observing run of Advanced LIGO and Advanced Virgo
International audienceIntermediate-mass black holes (IMBHs) span the approximate mass range 100â105âMâ, between black holes (BHs) that formed by stellar collapse and the supermassive BHs at the centers of galaxies. Mergers of IMBH binaries are the most energetic gravitational-wave sources accessible by the terrestrial detector network. Searches of the first two observing runs of Advanced LIGO and Advanced Virgo did not yield any significant IMBH binary signals. In the third observing run (O3), the increased network sensitivity enabled the detection of GW190521, a signal consistent with a binary merger of mass âŒ150âMâ providing direct evidence of IMBH formation. Here, we report on a dedicated search of O3 data for further IMBH binary mergers, combining both modeled (matched filter) and model-independent search methods. We find some marginal candidates, but none are sufficiently significant to indicate detection of further IMBH mergers. We quantify the sensitivity of the individual search methods and of the combined search using a suite of IMBH binary signals obtained via numerical relativity, including the effects of spins misaligned with the binary orbital axis, and present the resulting upper limits on astrophysical merger rates. Our most stringent limit is for equal mass and aligned spin BH binary of total mass 200âMâ and effective aligned spin 0.8 at 0.056 Gpcâ3 yrâ1 (90% confidence), a factor of 3.5 more constraining than previous LIGO-Virgo limits. We also update the estimated rate of mergers similar to GW190521 to 0.08 Gpcâ3 yrâ1.Key words: gravitational waves / stars: black holes / black hole physicsCorresponding author: W. Del Pozzo, e-mail: [email protected]â Deceased, August 2020