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Background: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR . The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25 , are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25 , overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. Conclusions: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

High-density lipoprotein (HDL) is a heterogeneous mixture of blood-circulating multimolecular particles containing many different proteins, lipids, and RNAs. Recent advancements in mass spectrometry-based proteotype analysis show promise for the analysis of proteoforms across large patient cohorts. In order to create the required spectral libraries enabling these data-independent acquisition (DIA) strategies, HDL was isolated from the plasma of more than 300 patients with a multiplicity of physiological HDL states. HDL proteome spectral libraries consisting of 296 protein groups and more than 786 peptidoforms were established, and the performance of the DIA strategy was benchmarked for the detection of HDL proteotype differences between healthy individuals and a cohort of patients suffering from diabetes mellitus type 2 and/or coronary heart disease. Bioinformatic interrogation of the data using the generated spectral libraries showed that the DIA approach enabled robust HDL proteotype determination. HDL peptidoform analysis enabled by using spectral libraries allowed for the identification of post-translational modifications, such as in APOA1, which could affect HDL functionality. From a technical point of view, data analysis further shows that protein and peptide quantities are currently more discriminative between different HDL proteotypes than peptidoforms without further enrichment. Together, DIA-based HDL proteotyping enables the robust digitization of HDL proteotypes as a basis for the analysis of larger clinical cohorts.

BackgroundHigh-density lipoprotein (HDL) is a heterogenous mixture of blood-circulating multimolecular particles containing many different proteins, lipids, and RNAs. Recent advancements in mass spectrometry-based proteotype analysis strategies enable the sensitive and reproducible quantification of proteins across large patient cohorts.MethodsHDL particles were isolated from plasma of more than 300 healthy individuals or patients with a multiplicity of physiological HDL states. From these, peptides were extracted and HDL proteome spectral libraries were generated. This is a prerequisite for using data-independent acquisition (DIA) strategies to analyze HDL particles from clinical cohorts using mass spectrometry.ResultsThe resulting HDL proteome spectral libraries consist of 296 protein groups and 341 peptidoforms of potential biological significance identified with high confidence. We used the HDL proteome libraries to evaluate HDL proteotype differences in between healthy individuals and patients suffering from diabetes mellitus type 2 (T2DM) and/or coronary heart disease (CHD). Bioinformatic interrogation of the data revealed significant quantitative differences in the HDL proteotypes including a significant depletion of phosphatidylinositol-glycan-specific phospholipase D (PHLD) from disease-derived HDL particles.ConclusionThe DIA-based HDL proteotyping strategy enabled sensitive and reproducible digitization of HDL proteotypes derived from patient cohorts and provides new insights into the composition of HDL particles as a rational basis to decode structure-function-disease relationships of HDL.List of human genes and protein names discussed in the paper- APOA1 (Apolipoprotein A-I)- APOA2 (Apolipoprotein A-II)- APOE (Apolipoprotein E)- APOC3 (Apolipoprotein C3)- CLUS (Clusterin)- PHLD (Phosphatidylinositol-glycan-specific phospholipase D)- PON1 (Serum paraoxonase/arylesterase 1)- PON3 (Serum paraoxonase/lactonase 3)- PSPB (Pulmonary surfactant-associated protein B)- RAB1B (Ras-related protein Rab-1B)- RAB6A (Ras-related protein Rab-6A)- RB11A/B (Ras-related protein Rab-11A/B)- RP1BL (Ras-related protein Rap-1b-like protein)- RAB10 (Ras-related protein Rab-10)- SAA1 (Serum amyloid A-1 protein)- SAA2 (Serum amyloid A-2 protein)- SAA4 (Serum amyloid A-4 protein)- SCRB1 (Scavenger receptor class B member 1)

Loss of pancreatic β-cell mass and function as a result of sustained ER stress is a core step in the pathogenesis of diabetes mellitus type 2. The complex control of β-cells and insulin production involves hedgehog (Hh) signaling pathways as well as cholesterol-mediated effects. In fact, data from studies in humans and animal models suggest that HDL protects against the development of diabetes through inhibition of ER stress and β-cell apoptosis. We investigated the mechanism by which HDL inhibits ER stress and apoptosis induced by thapsigargin, a sarco/ER Ca(2+)-ATPase inhibitor, in β-cells of a rat insulinoma cell line, INS1e. We further explored effects on the Hh signaling receptor Smoothened (SMO) with pharmacologic agonists and inhibitors. Interference with sterol synthesis or efflux enhanced β-cell apoptosis and abrogated the anti-apoptotic activity of HDL. During ER stress, HDL facilitated the efflux of specific oxysterols, including 24-hydroxycholesterol (OHC). Supplementation of reconstituted HDL with 24-OHC enhanced and, in cells lacking ABCG1 or the 24-OHC synthesizing enzyme CYP46A1, restored the protective activity of HDL. Inhibition of SMO countered the beneficial effects of HDL and also LDL, and SMO agonists decreased β-cell apoptosis in the absence of ABCG1 or CYP46A1. The translocation of the SMO-activated transcription factor glioma-associated oncogene GLI-1 was inhibited by ER stress but restored by both HDL and 24-OHC. In conclusion, the protective effect of HDL to counter ER stress and β-cell death involves the transport, generation, and mobilization of oxysterols for activation of the Hh signaling receptor SMO

High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and antidiabetogenic activities on cells. To resolve structure-function-disease relationships of HDL, we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition, as well as functionality in 5 cell types. The integration of 40 clinical characteristics, 34 nuclear magnetic resonance (NMR) features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modeling revealed, first, that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition, and function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity (CEC) was no proxy of other functions. Third, 3 potentially novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation-induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.

The ATP-binding cassette transporters ABCA1 and ABCG1 as well as scavenger receptor BI (SR-BI) mediate the efflux of lipids from macrophages to apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). We used RNA interference in RAW264.7 macrophages to study the interactions of ABCA1, ABCG1, and SR-BI with lipid-free apoA-I, native and reconstituted HDL with apoA-I:phosphatidylcholine ratios of either 1:40 (rHDL(1:40)) or 1:100 (rHDL(1:100)). Knock-down of ABCA1 inhibits the cellular binding at 4 degrees C of lipid-free apoA-I but not of HDL whereas suppression of ABCG1 or SR-BI reduces the binding of HDL but not lipid-free apoA-I. The degree of lipidation influences the interactions of rHDL with ABCG1 and SR-BI. Knock-down of ABCG1 inhibits more effectively the binding and cholesterol efflux capacities of lipid-poorer rHDL(1:40) whereas knock-down of SR-BI has a more profound effect on the binding and cholesterol efflux capacities of lipid-richer rHDL(1:100). Moreover, knock-down of ABCG1 but not SR-BI interferes with the association of lipid-free apoA-I during prolonged incubation at 37 degrees C. Finally, knock-down of ABCG1 inhibits the binding of initially lipid-free apoA-I which has been preconditioned by cells with high ABCA1 activity. The gained ability of initially lipid-free apoA-I to interact with ABCG1 is accompanied by its shift from electrophoretic pre-beta- to alpha-mobility. Taken together, these data suggest that the interaction of lipid-free apoA-I with ABCA1 generates a particle that immediately interacts with ABCG1 but not with SR-BI. Furthermore, the degree of lipidation influences the interaction of HDL with ABCG1 or SR-BI.

Accumulation of lipid-loaded macrophages (foam cells) within the vessel wall is an early hallmark of atherosclerosis. High-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) can efficiently promote cholesterol efflux from macrophages. Therefore, the interaction of HDL and apoA-I with macrophages appears to be important in the initial steps of reverse cholesterol transport, i.e. the transport of excess cholesterol from foam cells to the liver. However, although several cellular apoA-I and HDL receptors and transporters have been identified, it is as yet controversial how these interactions lead to cholesterol efflux from foam cells. In this study, we show that RAW264.7 macrophages bind HDL and apoA-I in a compatible manner. Furthermore, cell surface biotinylation experiments revealed that apoA-I but not HDL is specifically internalised. Binding of HDL to macrophages is decreased by reducing the expression of scavenger receptor BI (SR-BI) with cyclic adenosine monophosphate (cAMP), acetylated low-density lipoprotein (acLDL) or RNA interference. In contrast, apoA-I cell association and internalisation is modulated in parallel with ATP-binding cassette transporter A1 (ABCA1) expression which is altered by stimulating cells with cAMP and acLDL or expressing short hairpin RNA (shRNA) against ABCA1. Consistent with this, cell surface trapping of ABCA1 with cyclosporin A (CsA) results in increased apoA-I binding but reduced internalisation. Furthermore, blocking apoA-I uptake inhibits cholesterol efflux to apoA-I but not to HDL. Taken together, these data suggest that apoA-I- but not HDL-mediated cholesterol efflux may involve retroendocytosis.