Cyclic AMP pathway activation and extracellular zinc induce rapid intracellular zinc mobilization in Candida albicans

LK was supported by Innovation Fund Denmark, DK (4019-00019B). Pcovery ApS received funding from Wellcome Trust, Research Councils, UK (100480/Z/12), Novo Seeds, DK and Boehringer Ingelheim Venture Fund, D. DW is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (102549/Z/13/Z), the Medical Research Council and University of Aberdeen (MR/N006364/1) and received support from a Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology (097377/Z/11/Z). The funders had no part in study design, data collection and interpretation, or the decision to submit the work for publication.Peer reviewedPublisher PD

Data_Sheet_1.docx

<p>Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen.</p

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL’s α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL’s catalytic site, including β2, β3–α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3–α3 and progress to β5 and β4–α4, ultimately leading to the irreversible unfolding of regions that form LPL’s catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL’s α/β-hydrolase domain (T(m) of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (T(m) of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism

Femme des environs de Moscou : [estampe] / [Jean-Baptiste Le Prince]

[Femme des environs de Moscou]Référence bibliographique : IFF18 LE PRINCE (Jean-Baptiste), 109Référence bibliographique : Hédou, Le Prince, 9

Electrostatic sheathing of lipoprotein lipase is essential for its movement across capillary endothelial cells.

GPIHBP1, an endothelial cell (EC) protein, captures lipoprotein lipase (LPL) within the interstitial spaces (where it is secreted by myocytes and adipocytes) and transports it across ECs to its site of action in the capillary lumen. GPIHBP1's 3-fingered LU domain is required for LPL binding, but the function of its acidic domain (AD) has remained unclear. We created mutant mice lacking the AD and found severe hypertriglyceridemia. As expected, the mutant GPIHBP1 retained the capacity to bind LPL. Unexpectedly, however, most of the GPIHBP1 and LPL in the mutant mice was located on the abluminal surface of ECs (explaining the hypertriglyceridemia). The GPIHBP1-bound LPL was trapped on the abluminal surface of ECs by electrostatic interactions between the large basic patch on the surface of LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the surface of ECs. GPIHBP1 trafficking across ECs in the mutant mice was normalized by disrupting LPL-HSPG electrostatic interactions with either heparin or an AD peptide. Thus, GPIHBP1's AD plays a crucial function in plasma triglyceride metabolism; it sheathes LPL's basic patch on the abluminal surface of ECs, thereby preventing LPL-HSPG interactions and freeing GPIHBP1-LPL complexes to move across ECs to the capillary lumen

ATP hydrolysis and growth inhibition by library hit compounds.

<p>MIC is defined as 50% growth inhibition, <i>n</i> = 3.</p

IC₅₀ values of compounds 4–12 against ATP hydrolysis activity of fungal and mammalian P-type ATPases.

<p><i>n</i> = 3.</p

Chemical structure of the tetrahydrocarbazole scaffold (top), three initial hits from the library screening (1–3) and nine rationally designed tetrahydrocarbazole analogues used in this study (4–12).

<p>The chiral center is indicated by an asterisk.</p