Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in<i>Leishmania major</i>

Mukherjee, Sumi; Moitra, Samrat; Xu, Wei; Hernandez, Veronic; Zhang, Kai

doi:10.1101/2020.06.14.150953

Cited by 6 publications

(10 citation statements)

References 77 publications

(43 reference statements)

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“…As previously described [22], c14dm ̅ parasites were mostly dead after 24 h of heat shock, while both WT and c14dm ̅ /+C14DM parasites were mostly alive by that time and maintained 18S rRNA level at around 80% (Figure 4E and D). These results suggest that in addition to increased membrane fluidity and accumulation of superoxide, a defective heat shock response (failure to properly induce HSP gene expression and accelerated degradation of other RNAs) contributes to the extreme heat sensitivity displayed by c14dm ̅ (Figure 4E) [22,23].…”

Section: Rna Degrades Faster During Heat Shock and The Induction Of Heat Shock Response Is Compromised In C14dm ̅mentioning

confidence: 95%

“…Mean fluorescence intensities (MFI) were determined by flow cytometry using an Attune NxT Acoustic Flow Cytometer to confirm the induction of mitochondrial stress prior to cell collection for downstream analysis. Cell viability was determined by measuring the incorporation of 5.5 µg/ml of propidium iodide (PI) via flow cytometry as described [23]. Neither antimycin A nor Lglutathione treatments significantly affected cell viability based on PI staining (5% PI positive).…”

Section: Leishmania Culturing and Treatmentsmentioning

confidence: 99%

“…HSP83 is known to be upregulated in response to heat shock [32]. The c14dm ̅ mutant displays extreme sensitivity to stress conditions such as heat shock and starvation [22,23]. The ability to cope with stress is essential for Leishmania survival in mammals where they encounter dramatic changes in temperature, nutrient availability and pH [33].…”

Section: Rna Degrades Faster During Heat Shock and The Induction Of Heat Shock Response Is Compromised In C14dm ̅mentioning

confidence: 99%

“…C14dm ̅ cells have elevated level of reactive oxygen species (ROS) mostly in the form of mitochondrial superoxide [23]. In order to examine if oxidative stress contributes to the RNA reduction, we cultivated c14dm ̅ cells in the presence of L-glutathione and performed total RNA extractions.…”

Section: Reduced Rna Levels In C14dm ̅ Is Not Caused By the Accumulation Of Oxidantsmentioning

confidence: 99%

“…This mutant cannot remove the C14-methyl group from lanosterol or other sterol intermediates. The defect in sterol synthesis leads to increased membrane fluidity, mitochondrion dysfunction, superoxide accumulation, hypersensitivity to heat and severely reduced virulence in mice [22,23].…”

Section: Introductionmentioning

confidence: 99%

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Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Karamysheva¹,

Moitra²,

Perez³

et al. 2021

Preprint

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Leishmania species are parasitic protozoans which cause leishmaniasis affecting millions of people worldwide. Sterols are important components of plasma and organellar membranes and serve as precursors for the synthesis of signaling molecules. In contrast to animals, Leishmania do not produce cholesterol but instead synthesize ergostane-based sterols. C14-demethylase is a key enzyme involved in the biosynthesis of sterols in Leishmania parasites and an important drug target. Its inactivation leads to multiple defects including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis plays an unexpected role in global gene regulation in Leishmania parasites.

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Section: Rna Degrades Faster During Heat Shock and The Induction Of Heat Shock Response Is Compromised In C14dm ̅mentioning

confidence: 95%

Section: Leishmania Culturing and Treatmentsmentioning

confidence: 99%

Section: Rna Degrades Faster During Heat Shock and The Induction Of Heat Shock Response Is Compromised In C14dm ̅mentioning

confidence: 99%

Section: Reduced Rna Levels In C14dm ̅ Is Not Caused By the Accumulation Of Oxidantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Karamysheva¹,

Moitra²,

Perez³

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

et al. 2021

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Leishmania parasites are trypanosomatid protozoans that cause leishmaniasis affecting millions of people worldwide. Sterols are important components of the plasma and organellar membranes. They also serve as precursors for the synthesis of signaling molecules. Unlike animals, Leishmania does not synthesize cholesterol but makes ergostane-based sterols instead. C-14-demethylase is a key enzyme involved in the biosynthesis of sterols and an important drug target. In Leishmania parasites, the inactivation of C-14-demethylase leads to multiple defects, including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C-14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis in Leishmania plays an unexpected role in global gene regulation.

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Molecular Characterization of Sterol C4-Methyl Oxidase in Leishmania major

Ning,

Basu,

Hsu

et al. 2024

IJMS

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Sterol biosynthesis requires the oxidative removal of two methyl groups from the C-4 position by sterol C-4-demethylase and one methyl group from the C-14 position by sterol C-14-demethylase. In Leishmania donovani, a CYP5122A1 (Cytochrome P450 family 5122A1) protein was recently identified as the bona fide sterol C-4 methyl oxidase catalyzing the initial steps of C-4-demethylation. Besides CYP5122A1, Leishmania parasites possess orthologs to ERG25 (ergosterol pathway gene 25), the canonical sterol C-4 methyl oxidase in Saccharomyces cerevisiae. To determine the contribution of CYP5122A1 and ERG25 in sterol biosynthesis, we assessed the essentiality of these genes in Leishmania major, which causes cutaneous leishmaniasis. Like in L. donovani, CYP5122A1 in L. major could only be deleted in the presence of a complementing episome. Even with strong negative selection, L. major chromosomal CYP5122A1-null mutants retained the complementing episome in both promastigote and amastigote stages, demonstrating its essentiality. In contrast, the L. major ERG25-null mutants were fully viable and replicative in culture and virulent in mice. Deletion and overexpression of ERG25 did not affect the sterol composition, indicating that ERG25 is not required for C-4-demethylation. These findings suggest that CYP5122A1 is the dominant and possibly only sterol C-4 methyl oxidase in Leishmania, and inhibitors of CYP5122A1 may have strong therapeutic potential against multiple Leishmania species.

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Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance inLeishmania major

Cited by 6 publications

References 77 publications

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Molecular Characterization of Sterol C4-Methyl Oxidase in Leishmania major

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