Leishmania infantum: tuning digitonin fractionation for comparative proteomic of the mitochondrial protein content

Hide, Mallorie; Ritleng, A. S.; Brizard, Jean-Paul; Monte-Allegre, A.; Sereno, Denis

doi:10.1007/s00436-008-1062-9

Cited by 13 publications

(9 citation statements)

References 9 publications

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“…At low concentrations of digitonin the cholesterol rich plasma membrane can be effectively solubilized with little to no solubilization or permeabilization of intracellular membranes that are lower in cholesterol such as the endoplasmic reticulum (ER) and mitochondrial membranes. Similar concentration dependent effects on the degree of cell lysis by digitonin have also been shown in studies on parasites [3,4]. …”

Section: Resultssupporting

confidence: 76%

Crude subcellular fractionation of cultured mammalian cell lines

2009

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BackgroundThe expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples.FindingsWe have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step.ConclusionWe describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

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Section: Resultssupporting

confidence: 76%

Crude subcellular fractionation of cultured mammalian cell lines

2009

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“…Similar to WT and c14dm‾ /+C14DM parasites, cytochrome c in c14dm‾ remained in the mitochondrial enriched fractions when the digitonin extraction was performed at room temperature ( S1B Fig ). As a control, digitonin treatment at 37 °C disrupted the outer mitochondrial membrane, causing cytochrome c release into the cytosol [ 36 ] ( S1B Fig ). Thus, despite of its effects on ΔΨm and mitochondrial size, genetic ablation of C14DM does not affect mitochondrial membrane integrity at least with regard to cytochrome c localization under ambient conditions.…”

Section: Resultsmentioning

confidence: 99%

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

Mukherjee

Moitra

et al. 2020

PLoS Pathog

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Sterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm�). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm�mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm� mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm�. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.

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“…Similar to WT and c14dm - /+C14DM parasites, cytochrome c in c14dm - remained in the mitochondrial enriched fractions when the digitonin extraction was performed at room temperature (Fig S1B). As a control, digitonin treatment at 37 *C disrupted the outer mitochondrial membrane, causing cytochrome c release into the cytosol [36] (Fig S1B). Thus, despite of its effects on ΔΨm and mitochondrial size, genetic ablation of C14DM does not affect mitochondrial membrane integrity at least with regard to cytochrome c localization under ambient conditions.…”

Section: Resultsmentioning

confidence: 99%

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

Mukherjee¹,

Moitra

Xu³

et al. 2020

Preprint

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ABSTRACTSterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm-). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm- mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm- mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm-. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.AUTHOR SUMMARYSterols are well recognized for their stabilizing effects on the plasma membrane, but their functions in intracellular organelles are under explored, which hampers the development of sterol synthesis inhibitors as drugs. Our previous studies have demonstrated significant plasma membrane instability in the sterol biosynthetic mutant c14dm- in Leishmania major, a pathogenic protozoan responsible for cutaneous leishmaniasis causing 1-1.5 million infections a year. While the plasma membrane defects have undoubtedly contributed to the reduced virulence exhibited by c14dm- mutants, it was not clear whether other cellular processes were also affected. In this study, we revealed profound mitochondrial dysfunctions and elevated level of reactive oxygen species in c14dm- mutants. These sterol mutants rely heavily on glycolysis to generate energy and are extremely sensitive to glucose restriction. In addition, the accumulation of oxidants appears to be responsible (at least in part) for the previously observed heat sensitivity in c14dm- mutants. Thus, genetic or chemical inactivation of C14DM can influence the functions of cellular organelles beyond the plasma membrane. These findings shed light on the mechanism of action for azole compounds and provide new insight into the roles of sterol biosynthesis in Leishmania parasites.

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Leishmania infantum: tuning digitonin fractionation for comparative proteomic of the mitochondrial protein content

Cited by 13 publications

References 9 publications

Crude subcellular fractionation of cultured mammalian cell lines

Crude subcellular fractionation of cultured mammalian cell lines

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

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

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