Flavonoids regulate LDLR through different mechanisms tied to their specific structures.

Flavonoids, polyphenolic compounds found in plant-based diets, are associated with reduced risk of cardiovascular disease and longevity. These components are reported to reduce plasma levels of low-density lipoprotein (LDL) through an upregulation of the LDL receptor (LDLR), but the mechanism is still largely unknown. In this study, we have systematically screened the effect of 12 flavonoids from six different flavonoid subclasses on the effect on LDLR. This paper provides an in-depth analysis on how these flavonoids affect LDLR regulation and functionality. We found that most but not all of the tested flavonoids increased LDLR mRNA levels. Surprisingly, this increase was attributed to different regulatory mechanisms, such as enhanced LDLR promoter activity, LDLR mRNA stabilization, or LDLR protein stabilization, of which specific effectual parts of the flavonoid molecular structure could be assigned. These types of comparative analysis of various flavonoids enhance clarity and deepen the understanding of how the different structures of flavonoids affect LDLR regulation. Our data offer useful insights that may guide future research in developing therapeutic approaches for cardiovascular health.

Functional characterization of missense variants affecting the extracellular domains of ABCA1 using a fluorescence-based assay.

Excess cholesterol originating from nonhepatic tissues is transported within HDL particles to the liver for metabolism and excretion. Cholesterol efflux is initiated by lipid-free or lipid-poor apolipoprotein A1 interacting with the transmembrane protein ABCA1, a key player in cholesterol homeostasis. Defective ABCA1 results in reduced serum levels of HDL cholesterol, deposition of cholesterol in arteries, and an increased risk of early onset CVD. Over 300 genetic variants in ABCA1 have been reported, many of which are associated with reduced HDL cholesterol levels. Only a few of these have been functionally characterized. In this study, we have analyzed 51 previously unclassified missense variants affecting the extracellular domains of ABCA1 using a sensitive, easy, and low-cost fluorescence-based assay. Among these, only 12 variants showed a distinct loss-of-function phenotype, asserting their direct association with severe HDL disorders. These findings emphasize the crucial role of functional characterization of genetic variants in pathogenicity assessment and precision medicine. The functional rescue of ABCA1 loss-of-function variants through proteasomal inhibition or by the use of the chemical chaperone 4-phenylbutyric acid was genotype specific. Genotype-specific responses were also observed for the ability of apolipoprotein A1 to stabilize the different ABCA1 variants. In view of personalized medicine, this could potentially form the basis for novel therapeutic strategies.

Variants in the CETP gene affect levels of HDL cholesterol by reducing the amount, and not the specific lipid transfer activity, of secreted CETP.

BACKGROUND: Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters in plasma from high density lipoprotein (HDL) to very low density lipoprotein and low density lipoprotein. Loss-of-function variants in the CETP gene cause elevated levels of HDL cholesterol. In this study, we have determined the functional consequences of 24 missense variants in the CETP gene. The 24 missense variants studied were the ones reported in the Human Gene Mutation Database and in the literature to affect HDL cholesterol levels, as well as two novel variants identified at the Unit for Cardiac and Cardiovascular Genetics, Oslo University Hospital in subjects with hyperalphalipoproteinemia. METHODS: HEK293 cells were transiently transfected with mutant CETP plasmids. The amounts of CETP protein in lysates and media were determined by Western blot analysis, and the lipid transfer activities of the CETP variants were determined by a fluorescence-based assay. RESULTS: Four of the CETP variants were not secreted. Five of the variants were secreted less than 15% compared to the WT-CETP, while the other 15 variants were secreted in varying amounts. There was a linear relationship between the levels of secreted protein and the lipid transfer activities (r = 0.96, p<0.001). Thus, the secreted variants had similar specific lipid transfer activities. CONCLUSION: The effect of the 24 missense variants in the CETP gene on the lipid transfer activity was mediated predominantly by their impact on the secretion of the CETP protein. The four variants that prevented CETP secretion cause autosomal dominant hyperalphalipoproteinemia. The five variants that markedly reduced secretion of the respective variants cause mild hyperalphalipoproteinemia. The majority of the remaining 15 variants had minor effects on the secretion of CETP, and are considered neutral genetic variants.

Lysosomal acid lipase does not have a propeptide and should not be considered being a proprotein.

Lysosomal acid lipase (LAL) plays an important role in lipid metabolism by performing hydrolysis of triglycerides and cholesteryl esters in the lysosome. Based upon characteristics of LAL purified from human liver, it has been proposed that LAL is a proprotein with a 55 residue propeptide that may be essential for proper folding, intracellular transport, or enzymatic function. However, the biological significance of such a propeptide has not been fully elucidated. In this study, we have performed a series of studies in cultured HepG2 and HeLa cells to determine the role of the putative propeptide. However, by Western blot analysis and subcellular fractionation, we have not been able to identify a cleaved LAL lacking the N-terminal 55 residues. Moreover, mutating residues surrounding the putative cleavage site at Lys76 ↓ in order to disrupt a proteinase recognition sequence, did not affect LAL activity. Furthermore, forcing cleavage at Lys76 ↓ by introducing the optimal furin cleavage site RRRR↓EL between residues 76 and 77, did not affect LAL activity. These data, in addition to bioinformatics analyses, indicate that LAL is not a proprotein. Thus, it is possible that the previously reported cleavage at Lys76 ↓ could have resulted from exposure to proteolytic enzymes during the multistep purification procedure.

Bone morphogenetic protein 1 cleaves the linker region between ligand-binding repeats 4 and 5 of the LDL receptor and makes the LDL receptor non-functional.

The cell-surface low-density lipoprotein receptor (LDLR) internalizes low-density lipoprotein (LDL) by receptor-mediated endocytosis and plays a key role in the regulation of plasma cholesterol levels. The ligand-binding domain of the LDLR contains seven ligand-binding repeats of approximately 40 residues each. Between ligand-binding repeats 4 and 5, there is a 10-residue linker region that is subject to enzymatic cleavage. The cleaved LDLR is unable to bind LDL. In this study, we have screened a series of enzyme inhibitors in order to identify the enzyme that cleaves the linker region. These studies have identified bone morphogenetic protein 1 (BMP1) as being the cleavage enzyme. This conclusion is based upon the use of the specific BMP1 inhibitor UK 383367, silencing of the BMP1 gene by the use of siRNA or CRISPR/Cas9 technology and overexpression of wild-type BMP1 or the loss-of-function mutant E214A-BMP1. We have also shown that the propeptide of BMP1 has to be cleaved at RSRR120↓ by furin-like proprotein convertases for BMP1 to have an activity towards the LDLR. Targeting BMP1 could represent a novel strategy to increase the number of functioning LDLRs in order to lower plasma LDL cholesterol levels. However, a concern by using BMP1 inhibitors as cholesterol-lowering drugs could be the risk of side effects based on the important role of BMP1 in collagen assembly.

Strategies to prevent cleavage of the linker region between ligand-binding repeats 4 and 5 of the LDL receptor.

A main strategy for lowering plasma low-density lipoprotein (LDL) cholesterol levels is to increase the number of cell-surface LDL receptors (LDLRs). This can be achieved by increasing the synthesis or preventing the degradation of the LDLR. One mechanism by which an LDLR becomes non-functional is enzymatic cleavage within the 10 residue linker region between ligand-binding repeats 4 and 5. The cleaved LDLR has only three ligand-binding repeats and is unable to bind LDL. In this study, we have performed cell culture experiments to identify strategies to prevent this cleavage. As a part of these studies, we found that Asp193 within the linker region is critical for cleavage to occur. Moreover, both 14-mer synthetic peptides and antibodies directed against the linker region prevented cleavage. As a consequence, more functional LDLRs were observed on the cell surface. The observation that the cleaved LDLR was present in extracts from the human adrenal gland indicates that cleavage of the linker region takes place in vivo. Thus, preventing cleavage of the LDLR by pharmacological measures could represent a novel lipid-lowering strategy.

Inhibitors of AKT kinase increase LDL receptor mRNA expression by two different mechanisms.

Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206 and triciribine, two highly selective AKT inhibitors increase the level of low density lipoprotein receptor (LDLR) mRNA which leads to increased amount of cell-surface LDLRs. However, whereas MK-2206 induces transcription of the LDLR gene, triciribine stabilizes LDLR mRNA, raising the possibility that the two inhibitors may actually affect other kinases than AKT. In this study, we aimed to ascertain the role of AKT in regulation of LDLR mRNA expression by examining the effect of five additional AKT inhibitors on LDLR mRNA levels. Here we show that in cultured HepG2 cells, AKT inhibitors ARQ-092, AKT inhibitor VIII, perifosine, AT7867 and CCT128930 increase LDLR mRNA levels by inducing the activity of LDLR promoter. CCT128930 also increased the stability of LDLR mRNA. To study the role of AKT isoforms on LDLR mRNA levels, we examined the effect of siRNA-mediated knockdown of AKT1 or AKT2 on LDLR promoter activity and LDLR mRNA stability. Whereas knockdown of either AKT1 or AKT2 led to upregulation of LDLR promoter activity, only knockdown of AKT2 had a stabilizing effect on LDLR mRNA. Taken together, these results provide strong evidence for involvement of AKT in regulation of LDLR mRNA expression, and point towards the AKT isoform specificity for upregulation of LDLR mRNA expression.

Characterization of the mechanisms by which missense mutations in the lysosomal acid lipase gene disrupt enzymatic activity.

Hydrolysis of cholesteryl esters and triglycerides in the lysosome is performed by lysosomal acid lipase (LAL). In this study we have investigated how 23 previously identified missense mutations in the LAL gene (LIPA) (OMIM# 613497) affect the structure of the protein and thereby disrupt LAL activity. Moreover, we have performed transfection studies to study intracellular transport of the 23 mutants. Our main finding was that most pathogenic mutations result in defective enzyme activity by affecting the normal folding of LAL. Whereas, most of the mutations leading to reduced stability of the cap domain did not alter intracellular transport, nearly all mutations that affect the stability of the core domain gave rise to a protein that was not efficiently transported from the endoplasmic reticulum (ER) to the Golgi apparatus. As a consequence, ER stress was generated that is assumed to result in ER-associated degradation of the mutant proteins. The two LAL mutants Q85K and S289C were selected to study whether secretion-defective mutants could be rescued from ER-associated degradation by the use of chemical chaperones. Of the five chemical chaperones tested, only the proteasomal inhibitor MG132 markedly increased the amount of mutant LAL secreted. However, essentially no increased enzymatic activity was observed in the media. These data indicate that the use of chemical chaperones to promote the exit of folding-defective LAL mutants from the ER, may not have a great therapeutic potential as long as these mutants appear to remain enzymatically inactive.

Triciribine increases LDLR expression and LDL uptake through stabilization of LDLR mRNA.

Low-density lipoprotein receptor (LDLR) is a key regulator of the metabolism of plasma low-density lipoprotein cholesterol (LDL-C), the elevated levels of which are associated with an increased risk of cardiovascular disease. Therefore, enhancing LDLR expression represents a potent treatment strategy for hypercholesterolemia. Here, we report that in cultured human hepatoma cells, triciribine, a highly selective AKT inhibitor, increases the stability of LDLR mRNA, an event that translates into upregulation of cell-surface LDLR levels and induction of cellular LDL uptake. This effect of triciribine requires ERK activity and is partially dependent on the intervening sequence between the AU-rich elements ARE3 and ARE4 in LDLR 3'UTR. We also show that triciribine downregulates the expression of PCSK9 mRNA and blunts the secretion of its protein. Notably, triciribine was found to potentiate the effect of mevastatin on LDLR protein levels and activity. We also show that primary human hepatocytes respond to triciribine by increasing the expression of LDLR. Furthermore, a pilot experiment with mice revealed that a two-weeks treatment with triciribine significantly induced the hepatic expression of LDLR protein. These results identify triciribine as a novel LDLR-elevating agent and warrant further examination of its potential as a hypocholesterolemic drug either as monotherapy or in combination with statins.

MK-2206, an allosteric inhibitor of AKT, stimulates LDLR expression and LDL uptake: A potential hypocholesterolemic agent.

BACKGROUND AND AIMS: Induction of low-density lipoprotein receptor (LDLR) plays a significant role in reduction of plasma LDL-cholesterol (LDL-C) levels. Therefore, strategies that enhance the protein level of LDLR provide an attractive therapeutic target for the treatment of hypercholesterolemia. With this aim in mind, we concentrated our effort on studying the role of AKT kinase in regulation of LDLR levels and proceeded to examine the effect of MK-2206, an allosteric and highly selective AKT inhibitor, on LDLR expression. METHODS: Cultured human hepatoma cells were used to examine the effect of MK-2206 on the proteolytic processing of sterol regulatory element-binding protein-2 (SREBP-2), the expression of LDLR and cellular internalization of LDL. We also examined the effect of MK-2206 on LDLR levels in primary human hepatocytes. RESULTS: MK-2206 induced the proteolytic processing of SREBP-2, upregulated LDLR expression and stimulated LDL uptake. In contrast to statins, induction of LDLR levels by MK-2206 did not rely on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibition. As a result, cotreatment of cells with MK-2206 and mevastatin potentiated the impact of mevastatin on LDLR. Importantly, MK-2206 stimulated the expression of LDLR by primary human hepatocytes. CONCLUSIONS: MK-2206 is a novel LDLR-inducing agent that, either alone or in combination with statins, exerts a stimulating effect on cellular LDL uptake.