Role of Cholesterol and its Biosynthetic Precursors on Membrane Organization and Dynamics: A Fluorescence Approach.

Cholesterol is the most representative sterol present in membranes of higher eukaryotes, and is the end product of a long and multistep biosynthetic pathway. Lathosterol and zymosterol are biosynthetic precursors of cholesterol in Kandutsch-Russell and Bloch pathways, respectively. Lathosterol differs with cholesterol merely in the position of the double bond in the sterol ring, whereas zymosterol differs with cholesterol in position and number of double bonds. In this work, we have monitored the effect of cholesterol and its biosynthetic precursors (lathosterol and zymosterol) on membrane organization and dynamics in fluid and gel phase membranes. Toward this goal, we have utilized two fluorescent membrane probes, DPH and its cationic derivative TMA-DPH. Our results using these probes show that cholesterol and its biosynthetic precursors (lathosterol and zymosterol) exhibit similar trend in maintaining membrane organization and dynamics (as reported by fluorescence anisotropy and apparent rotational correlation time), in fluid phase POPC membranes. Notably, although lathosterol and zymosterol show similar trend in maintaining membrane organization and dynamics, the corresponding change for cholesterol is different in gel phase DPPC membranes. These results demonstrate that the position and number of double bonds in sterols is an important determinant in maintaining membrane physical properties. Our results assume significance since accumulation of precursors of cholesterol have been reported to be associated with severe pathological conditions.

Characterization of the Sterol 24-C-Methyltransferase Genes Reveals a Network of Alternative Sterol Biosynthetic Pathways in Mucor lusitanicus.

Certain members of the order Mucorales can cause a life-threatening, often-fatal systemic infection called mucormycosis. Mucormycosis has a high mortality rate, which can reach 96 to 100% depending on the underlying condition of the patient. Mucorales species are intrinsically resistant to most antifungal agents, such as most of the azoles, which makes mucormycosis treatment challenging. The main target of azoles is the lanosterol 14α-demethylase (Erg11), which is responsible for an essential step in the biosynthesis of ergosterol, the main sterol component of the fungal membrane. Mutations in the erg11 gene can be associated with azole resistance; however, resistance can also be mediated by loss of function or mutation of other ergosterol biosynthetic enzymes, such as the sterol 24-C-methyltransferase (Erg6). The genome of Mucor lusitanicus encodes three putative erg6 genes (i.e., erg6a, erg6b, and erg6c). In this study, the role of erg6 genes in azole resistance of Mucor was analyzed by generating and analyzing knockout mutants constructed using the CRISPR-Cas9 technique. Susceptibility testing of the mutants suggested that one of the three genes, erg6b, plays a crucial role in the azole resistance of Mucor. The sterol composition of erg6b knockout mutants was significantly altered compared to that of the original strain, and it revealed the presence of at least four alternative sterol biosynthesis pathways leading to formation of ergosterol and other alternative, nontoxic sterol products. Dynamic operation of these pathways and the switching of biosynthesis from one to the other in response to azole treatment could significantly contribute to avoiding the effects of azoles by these fungi. IMPORTANCE The fungal membrane contains ergosterol instead of cholesterol, which offers a specific point of attack for the defense against pathogenic fungi. Indeed, most antifungal agents target ergosterol or its biosynthesis. Mucormycoses-causing fungi are resistant to most antifungal agents, including most of the azoles. For this reason, the drugs of choice to treat such infections are limited. The exploration of ergosterol biosynthesis is therefore of fundamental importance to understand the azole resistance of mucormycosis-causing fungi and to develop possible new control strategies. Characterization of sterol 24-C-methyltransferase demonstrated its role in the azole resistance and virulence of M. lusitanicus. Moreover, our experiments suggest that there are at least four alternative pathways for the biosynthesis of sterols in Mucor. Switching between pathways may contribute to the maintenance of azole resistance.

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport.

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.

Amiodarone Alters Cholesterol Biosynthesis through Tissue-Dependent Inhibition of Emopamil Binding Protein and Dehydrocholesterol Reductase 24.

Amiodarone is prescribed for the treatment and prevention of irregular heartbeats. Although effective in clinical practice, the long-term use of amiodarone has many unwanted side effects, including cardiac, pulmonary, hepatic, and neurological toxicities. Our objective was to elucidate effects of amiodarone exposure on the cholesterol metabolism in cultured neuronal and non-neuronal cells and in individuals taking amiodarone. We observed that amiodarone increases distinct cholesterol precursors in different cell types in a dose-dependent manner. In liver and kidney cell lines, amiodarone causes increase in desmosterol levels, and in primary cortical neurons and astrocytes, amiodarone increases zymosterol, zymostenol, and 8-dehydrocholesterol (8-DHC). We conclude that amiodarone inhibits two enzymes in the pathway, emopamil binding protein (EBP) and dehydrocholesterol reductase 24 (DHCR24). Cortical neurons and astrocytes are more sensitive to amiodarone than liver and kidney cell lines. We confirmed the inhibition of EBP enzyme by analyzing the sterol intermediates in EBP-deficient Neuro2a cells versus amiodarone-treated control Neuro2a cells. To determine if the cell culture experiments have clinical relevance, we analyzed serum samples from amiodarone users. We found that in patient serum samples containing detectable amount of amiodarone there are elevated levels of the sterol precursors zymosterol, 8-DHC, and desmosterol. This study illustrates the need for close monitoring of blood biochemistry during prolonged amiodarone use to minimize the risk of side effects.

Liquid-Ordered Phase Formation by Mammalian and Yeast Sterols: A Common Feature With Organizational Differences.

Here, biophysical properties of membranes enriched in three metabolically related sterols are analyzed both in vitro and in vivo. Unlike cholesterol and ergosterol, the common metabolic precursor zymosterol is unable to induce the formation of a liquid ordered (l o) phase in model lipid membranes and can easily accommodate in a gel phase. As a result, Zym has a marginal ability to modulate the passive membrane permeability of lipid vesicles with different compositions, contrary to cholesterol and ergosterol. Using fluorescence-lifetime imaging microscopy of an aminostyryl dye in living mammalian and yeast cells we established a close parallel between sterol-dependent membrane biophysical properties in vivo and in vitro. This approach unraveled fundamental differences in yeast and mammalian plasma membrane organization. It is often suggested that, in eukaryotes, areas that are sterol-enriched are also rich in sphingolipids, constituting highly ordered membrane regions. Our results support that while cholesterol is able to interact with saturated lipids, ergosterol seems to interact preferentially with monounsaturated phosphatidylcholines. Taken together, we show that different eukaryotic kingdoms developed unique solutions for the formation of a sterol-rich plasma membrane, a common evolutionary trait that accounts for sterol structural diversity.

Improving the efficacy of hormone therapy in breast cancer: The role of cholesterol metabolism in SERM-mediated autophagy, cell differentiation and death.

Breast cancer (BC) is one of the most common female cancers in the world, with estrogen receptor (ER)-positive BC the most frequent subtype. Tamoxifen (Tam) is an effective drug that competitively binds to the ER and is routinely used for the treatment of ER-positive BC. However, a number of ER-positive BC do not respond to Tam treatment and acquired resistance is often observed, constituting a major challenge for extending patient life expectancy. The mechanisms responsible for these treatment failures remain unclear, indicating the requirement for other targets and better predictors for patient response to Tam. One of Tam's off-targets of interest is the microsomal antiestrogen binding site (AEBS), a multiproteic complex made up of the cholesterol-5,6-epoxide hydrolase (ChEH) enzymes that are involved in the late stages of cholesterol biosynthesis. Tam and other selective ER modulators stimulate oxidative stress and inhibit the ChEH subunits at pharmacological doses, triggering the production and accumulation of cholesterol-5,6-epoxide metabolites responsible for BC cell differentiation and death. However, inhibition of the cholesterogenic activity of the AEBS subunits also induces the accumulation of sterol precursors, which triggers a survival autophagy to impair Tam's efficacy. Altogether, these studies have highlighted the involvement of cholesterol metabolism in the pharmacology of Tam that has provided new clues on how to improve its therapeutic efficacy in both BC and other cancers as well as offering a new rationale for developing more efficient drugs for BC treatment.

The comparison of zymosterol vs cholesterol membrane properties--the effect of zymosterol on lipid monolayers.

In this work systematic investigations on the influence of zymosterol (zymo), which is one of cholesterol precursors, on lipid monolayers were done. The aim of these studies was to perform thorough comparison of zymosterol vs cholesterol membrane activity and fill the gap in the studies on the effect of sterols on membranes. The Langmuir monolayers experiments combined with Brewster angle microscopy studies were performed on binary (SM:zymo, POPC:zymo, GM3:zymo) and ternary (SM:POPC:zymo, SM:GM3:zymo) films differing in the sterol content. The obtained results evidenced differences in the influence of both sterols on lipid monolayers, which was manifested in the parameters calculated based on the isotherms as well is in monolayers morphology. It was found that zymosterol is of condensing, ordering and domain promoting abilities thus this molecule can be included to the group of membrane active sterols. However, zymosterol is much less effective than cholesterol as condensing and ordering agent. These findings were attributed to the differences in the structure of both sterols and their ability to pack tightly with other lipids in the mixed systems.