Fig 2: MUT-deficient human fibroblasts have increased LDLR expression and function and decreased sterol content.a Catabolism of odd-chain fatty acids, cholesterol, and amino acids. MMAB catalyzes the final step in the conversion of vitamin B12 into adenosylcobalamin (AdoCbl), a coenzyme for methylmalonyl-coA mutase (MUT). Mutations in MMAB or MUT lead to the metabolic disorder, methylmalonic aciduria. b, c Propionylcarntitine (b) and methylmalonic acid (MMA) (c) content in Huh7 cells transfected with a non-silencing siRNA (NS) or siRNA MMAB (siMMAB) for 60 h. Data are mean ± s.e.m. n = 3–5 biological replicates per group. Statistical comparisons by unpaired Welch’s two-sample t test. d Relative levels of propionylcarnitine, succinate and acetyl-CoA in WT and MUT-deficient fibroblasts incubated in DMEM containing 10% LPDS for 24 h. Data are mean ± s.e.m. of five independent experiments. Statistical comparisons by unpaired Welch’s two-sample t test. e Representative images of DiI-LDL uptake in human fibroblasts deficient of methylmalonyl-coA mutase (MUT) or wild-type (WT) controls and incubated with 30 µg/ml DiI-LDL for 2 h at 37 °C. Red, DiI-LDL. Scale, 200 µm. Quantification of DiI-LDL uptake by FACS is shown to the right. Data are mean ± s.e.m. of three independent experiments. Statistical comparisons by unpaired two-sided Student’s t test. f, g qRT-PCR (f) and Western blot (g) analysis of SREBP2 responsive genes in WT and MUT fibroblasts incubated in LPDS for 24 h. HSP90 was used as a loading control. Quantification of blot is shown to the right. Data are mean ± s.e.m. of 3–5 independent experiments in duplicate, respectively. Statistical comparisons by unpaired two-sided Student’s t test. h Sterol content of WT and MUT fibroblasts incubated in DMEM containing 10% LPDS for 24 h. Lipids were extracted and analyzed by GC-MS. “Total sterols” were calculated as the sum of all sterols that could be detected. Data are mean ± s.e.m. of three independent experiments. Statistical comparisons by unpaired two-sided Student’s t test. Source data are provided as a Source Data file.

Fig 3: Primary RNAi screen validation.a Functional map of top 250 hits from primary RNAi screen. Top hits identified in the primary screens (Fig. 1a) were uploaded into DAVID for functional annotation cluster analysis. Individual genes were assigned to the highest enriched functional annotation cluster in which they were present (see Supplementary Data 1). Regulators of LDLR activity in functional clusters that interact or share functional annotations are depicted in colored circles. Selected genes not found within functional annotation clusters are indicated as white hexagons. Lines between genes in the same and different functional clusters indicate STRING interaction score. Approximate cellular locations of each functional gene cluster are pictured. b Average robust z scores of top six hits validated in a secondary screening assay using siRNAs from independent libraries. Gray bars, positive and negative screening controls; white bars, Ambion siRNA library used in primary screen; black bars, Dharmacon siRNA library used in secondary screen. Data are mean ± s.e.m. n = 3 technical replicates for Ambion siRNA library and 12-32 technical replicates for Dharmacon siRNA library. Dashed line: robust z score = 1.5 or -1.5. Source data are provided as a Source Data file.

Fig 4: LDLR expression of the different variants at cell surface in CHO-ldlA7 transfected cells.For confocal analysis of LDLR at cellular membrane and LDL uptake, CHO-ldlA7 transfected cells were immunostained as described in Material and Methods with anti-hLDLR antibody and anti-ApoB100 antibody. Texas Red and Alexa Fluor 488 labeled secondary antibodies were used to visualize LDLR or LDL, respectively. Dapi was used to stain nuclei. The images show a representative individual cell.

Fig 5: Feedback control of cholesterol biosynthesis through modulation of adenosylcobalamin metabolism.a Schematic showing negative feedback regulation of cholesterol biosynthesis by MMAB. Loss of MMAB leads to reduced conversion of cob(I)alamin to AdoCbl (1), which impairs the function of methylmalonyl-CoA mutase (MUT) activity and results in the incomplete breakdown of certain amino acids, cholesterol and odd-chain fatty acids (2). This defect leads to the accumulation of propionic acid, which reduces cholesterol biosynthesis through indirect mechanisms (3), and the accumulation of methylmalonic acid, which reduces cholesterol biosynthesis by inhibiting HMGCR (4). Sterol depletion leads to the subsequent upregulation of SREBP2-mediated gene expression (5), thereby providing an additional control point by which cholesterol biosynthesis is regulated by its end product. BCAAs branched chain amino acids, FAs fatty acids, MMA methylmalonic acid. b Schematic showing the role of the adenosylcobalamin pathway in regulating hepatic LDLR expression and circulating LDL-C levels.