Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol-sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol-sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.Fungal infections have recently become a growing threat to human health, especially in persons whose immune systems are compromised (for example, by human immunodeficiency virus and cancer chemotherapy). Only a few effective antifungal agents are currently in use; these include the polyenes, the fluorocytes, and the azole derivatives. One important problem is the increase of drug resistance, particularly against azole antimyotics and fluorocytosine (1). Resistance against polyene antibiotics is still a rare event, which makes these antibiotics particularly interesting as antifungal agents. The polyene antibiotics have a ring structure in which a conjugated double bond system is located opposite to a number of hydroxyl functions. Often a mycosamine group is present in combination with a carboxyl moiety, rendering the molecule amphoteric (Fig. 1). In the past convincing evidence has been presented that several members of this class of antibiotics target sterols and in particular ergosterol, the abundant and main sterol of fungal membranes (2, 3). Different types of polyene antibiotics were shown to have different modes of action despite that they share a common target. The larger polyenes like amphotericin B and nystatin form pores together with ergosterol in the plasma membrane that collapse vital ion gradients, thereby killing the cells. The smaller uncharged filipin also destroys the membrane barrier, but by a completely different mechanism. Filipin forms large complexes with sterols between the leaflets of the lipid bilayer, resulting in loss of the barrier function (2). Natamycin (also called pimaricin) is a very effective member of the polyene antibiotic family with a large standing record of applications. It is produced by Streptomyces natalensis and used against fungal infections, but it is also widely utiliz...

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The proline-, glutamic acid-, serine- and threonine-rich (PEST) family of protein tyrosine phosphatases (PTPs) includes proline-enriched phosphatase (PEP)/lymphoid tyrosine phosphatase (LYP), PTP-PEST, and PTP-hematopoietic stem cell fraction (HSCF). PEP/LYP is a potent inhibitor of T-cell activation, principally by suppressing the activity of Src family protein tyrosine kinases (PTKs). This function seems to be dependent, at least in part, on the ability of PEP to bind C-terminal Src kinase (Csk), a PTK also involved in inactivating Src kinases. Interestingly, a polymorphism of LYP in humans (R620W) is a significant risk factor for autoimmune diseases including type 1 diabetes, rheumatoid arthritis, and lupus. The R620W mutation may be a 'gain-of-function' mutation. In non-hematopoietic cells, PTP-PEST is a critical regulator of adhesion and migration. This effect correlates with the aptitude of PTP-PEST to dephosphorylate cytoskeletal proteins such as Cas, focal adhesion associated-kinase (FAK), Pyk2, and PSTPIP. While not established, a similar function may also exist in immune cells. Additionally, overexpression studies provided an indication that PTP-PEST may be a negative regulator of lymphocyte activation. Interestingly, mutations in a PTP-PEST- and PTP-HSCF-interacting protein, PSTPIP1, were identified in humans with pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome and familial recurrent arthritis, two autoinflammatory diseases. These mutations abrogate the ability of PSTPIP1 to bind PTP-PEST and PTP-HSCF, suggesting that these two PTPs may be negative regulators of inflammation.

Background: PTP-PEST is a phosphatase essential for embryonic viability. Results: PTP-PEST is critical for adhesion and migration of endothelial cells. Its absence in endothelial cells results in mouse embryonic lethality. Conclusion:The embryonic viability seen in constitutive PTP-PEST-deficient mice is due to a defect in endothelial cell functions. Significance: PTP-PEST is a key regulator of endothelial cell functions in vitro and in vivo.