Protocol for assessing mitochondrial cholesterol transport and protein molten globule state in steroidogenic and nonsteroidogenic systems

Abstract

Steroid hormones are essential for the survival of all mammals for carbohydrate metabolism, stress management, and sexual reproduction. Here, we present a protocol for assessing mitochondrial cholesterol transport and protein molten globule state via measurement of pregnenolone or progesterone synthesis in steroidogenic and nonsteroidogenic cellular systems. We describe steps for cell culture, transfection, and measurement of steroidogenic activity from nonsteroidogenic cells. We then detail procedures for metabolic conversion. For complete details on the use and execution of this protocol, please refer to Bose,¹ Bose et al.,² Pawlak et al.,³ and Prasad et al.⁴.

Keywords: Cell Biology; Health Sciences; Mass Spectrometry; Metabolism; Molecular Biology; Protein Biochemistry.

Graphical abstract

Figure 1

First step of steroid hormone synthesis Chemical reaction of the catalysis of cholesterol to pregnenolone by cytochrome P450 side chain cleavage enzyme (P450scc), where the side chain of cholesterol at C21-22 position is cleaved generating pregnenolone and 6-amino caproic acid. In the next step catalysis of pregnenolone to progesterone is completed by 3-beta hydroxy steroid dehydrogenase (3βHSD2) enzyme in the mitochondrial matrix.

Figure 2

Diagram showing the transfection procedure Schematic presentation showing an ideal experiment for the determination of activity from the media and expression by western blotting from the nonsteroidogenic cells transfected in a six well plate. Normalization vector (pKS) was added appropriately to have a total length of DNA same in all wells. 22-R Hydroxy cholesterol is a positive control describing the maximum capacity of a cell to metabolize cholesterol.

Figure 4

Development of a standard graph for activity measurement (A) Standard graph for evaluation of activity of an unknown sample. (B) Percentage of radioactive antibody bound with the known amounts of steroid, pregnenolone. X-axis is the concentration of the steroids and Y-axis is the percentage bound. The red solid square is the data point with a best fit shown in black solid line. The standards were available from the vendor/manufacturer. The activity of experimental samples is determined from the standard graph.

Figure 3

Schematic radioimmuno assay (RIA) procedure A detailed step-by-step presentation for the measurement of steroidogenic activity by RIA.

Figure 5

Determination of a protein molten globule state (A) Cell-free synthesized ³⁵S-SCC was imported into the isolated mitochondria in presence and absence of the indicated concentrations of urea and analyzed through SDS-PAGE. (B) Semi quantitative analysis of the imported SCC fractions from panel A. (C and D) Import analysis of ³⁵S-SCC for 30 min (C) and 2 h (D) into the isolated mitochondria in presence of the indicated combination of digitonin and sorbitol or independently. The import process was analyzed through SDS-PAGE. (E) Schematic presentation showing the block of intermediate 54-kDa SCC form further processing to 51-kDa, confirming the formation of molten globule state.

Figure 6

Characterization of steroids (A) Thin layer chromatography (TLC) of the steroids, ³H-pregnenolone to ³H-progesterone synthesized in the presence of the indicated inhibitors were separated through a mixture of solvent gradient. The specific steroid is extracted from the TLC plate and subjected to GC-MS analysis. (B) TLC analysis of ¹⁴C-cholesterol to ¹⁴C-pregnenolone. With permission from the publisher this figure is reproduced from our previous publication “Prasad et al. J Biol Chem 287:9534-9546, 2012”.

Figure 7

GC/MS analysis (A) Extracted-ion chromatogram of m/z 316.2, (B) MS spectrum at retention time 21.02 min corresponding to the MS spectrum of pregnenolone.

Figure 8

Flow chart showing stepwise processing of the dry molten globule and activity assessment procedure