pregnenolone  (Valiant Co Ltd)

Journal: iScience

Article Title: Dry molten globule conformational state of CYP11A1 (SCC) regulates the first step of steroidogenesis in the mitochondrial matrix

doi: 10.1016/j.isci.2024.110039

Figure Lengend Snippet: Partially unfolded and folded SCC association with the matrix side of the IMM (A) Schematic presentation of cholesterol to pregnenolone catalysis by SCC. (B) Top, mitochondrial import and processing of SCC. 35 S- cell free synthesized SCC was imported into the mitochondria isolated from pig adrenals, placenta, and MA-10 cells. The 61-kDa SCC was imported into 57- and 51-kDa proteins. Bottom, western blot of the mitochondria applied for import experiments with a VDAC2 antibody showing the presence of identical amounts of mitochondria applied to each reaction. (C) Top, integration of imported SCC proteins. After import of 35 S-SCC with isolated mitochondria, the imported (P) fraction was separated from non-imported fraction (S) by washing followed by centrifugation. In some cases, following import the fractions were extracted with Na 2 CO 3 at pH 11.5 followed by separation with a centrifugation. Bottom, western blot of the membrane shown in the top panel with a VDAC2 antibody. (D) Incubation with varying concentrations of proteinase K (PK) following import of 35 S-SCC into the freshly isolated mitochondria for 30 min. (E) Semiquantitative analysis of the integration of the 57-kDa (Solid line in black with round circle, ⸺○⸺) and 51-kDa (broken small solid lines in blue with square, --□- -) SCC and its protection from PK from (D). The Y axis is the band intensity in arbitrary phosphorimager units (AU), and X axis is PK concentration. (F) Following mitochondrial import of 35 S-SCC, mitochondria were fractionated into OMM, IMM, and matrix fractions followed by incubation with 0.4% Triton X- and with two different concentrations of PK (500 ng/mL [1X] and 50 ng/mL [0.1X]). (G) Western blot of the mitochondrial fractions with antibodies specific for the IMM resident, Tim23, (Top) and matrix resident, HSP70, (bottom). (H) Schematic presentation showing SCC import into mitochondria and its association with the mitochondrial membrane first as a 57-kDa protein, which is then folded completely and remained integrated with the IMM facing the matrix. Data in (E) are presented as the mean ± S.E. of three independent experiments p < 0.05.

Article Snippet: The medium was collected after 48 h, and the accumulated pregnenolone was measured from the media collected by radioimmunoassay (RIA kit, MP Biomedicals, Solon, OH, USA).

Techniques: Synthesized, Isolation, Western Blot, Centrifugation, Membrane, Incubation, Concentration Assay

Journal: iScience

Article Title: Dry molten globule conformational state of CYP11A1 (SCC) regulates the first step of steroidogenesis in the mitochondrial matrix

doi: 10.1016/j.isci.2024.110039

Figure Lengend Snippet: Hydrogen ions shield processing at the intermediate state (A and B) Import of 35 S-SCC for 30 min (A) and 2 h (B) in the presence of sorbitol and digitonin together or independently before or after equilibrating freshly isolated mitochondria from MA-10 cells. (C) Schematic presentation of arresting 57-kDa intermediate state at the matrix with the osmolyte barrier for electrolytes. (D) Left, Import of 35 S-SCC for 2 h in the presence of various concentrations of AEBSF. Bottom, western blotting with a VDAC2 antibody. (E) Semiquantitative analysis of the processing of 57- and 51-kDa SCC in the presence of different concentrations of AEBSF from the top of (D). (F) Top left, Mitochondrial import 35 S-labeled SCC for 1 h in the presence of atractoloside (ATL), oligomycin (Oligo), and valinomycin (Val) for 1 h. Top right, After incubation for 1 h, the inhibitors were removed by dialysis against import buffer followed by washing and centrifugation, and imported for 1 h under identical conditions. Bottom left, western blotting analysis a VDAC2 antibody. (G) SCC activity (pregnenolone synthesis) in the presence of inhibitors and after removal of inhibitors in (F). (H) Schematic cartoon showing the requirement two hydrogen ions for the processing of the intermediate state to an active folded state. Data presented in (E and G) as the mean ± S.E. of three independent experiments and p < 0.05.

Article Snippet: The medium was collected after 48 h, and the accumulated pregnenolone was measured from the media collected by radioimmunoassay (RIA kit, MP Biomedicals, Solon, OH, USA).

Techniques: Isolation, Western Blot, Labeling, Incubation, Centrifugation, Activity Assay

Journal: iScience

Article Title: Dry molten globule conformational state of CYP11A1 (SCC) regulates the first step of steroidogenesis in the mitochondrial matrix

doi: 10.1016/j.isci.2024.110039

Figure Lengend Snippet: Minimotifs are generated from the C-terminus (A) Design of the different C-terminal SCC deletional mutants. (B and C) Import of the 35 S-SCC and the deletional constructs from the (A) for 1 h in the presence of varying concentrations of PK. (D and E) Intensity of protection of the 51-kDa (D) and 57-kDa (E) imported proteins in the presence of varying concentration of PK and its relation to the length of the C-terminal amino acids. The X axis is the different concentrations of PK, while Y axis represents represent the intensity of protection. (F) SCC activity (pregnenolone) co-transfected with the truncated C-terminal deletional mutants with ferredoxin, ferredoxin reductase, and StAR cDNA in COS-1 cells. The X axis is the wild-type SCC and SCC mutants, while Y axis represents the amounts of pregnenolone synthesized after 42 h of transfection. Data in (D–F) are presented as the mean ± S.E. of three independent experiments and p < 0.05. (G) Schematic presentation of the formation of unstable folding in the absence of far C-terminal amino acids, resulting a state easily susceptible to proteolysis.

Article Snippet: The medium was collected after 48 h, and the accumulated pregnenolone was measured from the media collected by radioimmunoassay (RIA kit, MP Biomedicals, Solon, OH, USA).

Techniques: Generated, Construct, Concentration Assay, Activity Assay, Transfection, Synthesized