Food-derived peptides with hypocholesterolemic activity: Production, transepithelial transport and cellular mechanisms
Jianqiang LiCarlotta BollatiLorenza d’AdduzioMelissa FanzagaIvan Cruz‐ChamorroAnna ArnoldiCesare R. SirtoriCarmen Lammi
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Abstract:
In recent years, food-derived peptides have gained much attention for their potential health benefits. Some short and medium-sized peptides released from food proteins after their enzymatic hydrolysis may exhibit hypocholesterolemic activity. Hypocholesterolemic peptides act either by targeting exogenous cholesterol in the gastrointestinal (GI) tract or by modulating endogenous cholesterol levels via cholesterol metabolism pathways in the liver after being absorbed. This paper provides a comprehensive review of current pieces of evidence regarding the production, transepithelial transport, and cellular mechanisms underlying the hypocholesterolemic activities of food-derived peptides. The molecular mechanisms of hypocholesterolemic peptides involve bile acid binding, inhibition of cholesterol micellar solubility, statin-like effects through the modulation of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCoAR), as well as the targeting of interactions between proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR), sterol regulatory element-binding protein 2 (SREBP-2), and hepatocyte nuclear factor 1α (HNF-1α) pathways. Furthermore, some peptides exhibit multiple biological activities, such as anti-inflammatory and antioxidant activities, besides cholesterol-lowering properties, thereby safeguarding cellular components against high levels of cholesterol-induced damage. However, since only a few studies have evaluated the in vivo effects of hypocholesterolemic peptides, further studies carried out in animal models or human are necessary to exploit these ingredients in the prevention and management of hypercholesterolemia.Keywords:
PCSK9
Kexin
Intervention with drugs to reduce Low Density Lipoprotein-cholesterol (LDL-C) has proven to decrease the risk of subsequent cardiovascular events, including mortality. Our goal is to develop novel, small molecule, LDL-C lowering drugs by targeting the Low Density Lipoprotein Receptor (LDLR) degradation pathway, which is modulated by the protease proprotein convertase subtilisin-like kexin type 9 (PCSK9). PCSK9 has been shown to bind to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR; it enhances the degradation of the LDLR by an unknown. We successfully integrated virtual screening methods and cell based assays into a simple, efficient procedure and identified compounds that interfere with the PCSK9-LDLR interaction. Using the atomic coordinates of the crystal structure of the PCSK9/LDLR-EGF-A complex, about a million drug-like compounds were docked in silico into the region of PCSK9 where LDLR-EGF-A binds. We selected 179 top scoring compounds that showed the best fit in the binding site as well as the greatest number of interactions with PCSK9. These compounds were tested in (a) our PCSK9/LDLR dependent HEK293 cell-based recombinant assay and (b) the endogenously expressed LDLR in HepG2/PCSK9 dependent cell based assay for their ability to upregulate the LDLR. From these screening campaigns, eleven compounds consistently exhibited concentration dependent increase in the LDLR as comparable to control, with IC 50 s in the low micromolar range. Three of the top picks were shown to exhibit a 5 to 10 fold up-regulation at 1.6 μM in two cell lines. In addition, these compounds also exhibited an increase in the fluorescently labeled DiI-LDL uptake in the nanomolar range in situ . Furthermore, in a two week animal study, subcutaneous injection of 4 mg/kg of one of these compounds resulted in a 32% reduction of the total cholesterol level in mice fed a high fat/high cholesterol diet as compared to 27% cholesterol reduction obtained with 40 mg/kg Atorvastatin. To our knowledge, no small molecule antagonist against PCSK9 has been reported, only mAbs. Thus, identifying such an oral small molecule PCSK9 inhibitor represents a tremendous advance and opportunity for drug development.
PCSK9
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Proprotein Convertases
Subtilisin
Low-density lipoprotein
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Background— Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation. Methods and Results— To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely because of the impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apolipoprotein B–containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine PCSK9 levels by 4.3-fold in wild-type mice and not at all in LDLR −/− mice, in which murine PCSK9 levels were already 10-fold higher than in wild-type mice. In addition, LDLR +/− mice had a 2.7-fold elevation in murine PCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of human LDLR in transgenic mice caused a 70% decrease in serum murine PCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in wild-type mice (half-life, 5.2 minutes), faster clearance in human LDLR transgenics (2.9 minutes), and much slower clearance in LDLR −/− recipients (50.5 minutes). Supportive results were obtained with an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression. Conclusions— Our results support a scenario in which LDLR represents the main route of elimination of PCSK9 and a reciprocal regulation between these 2 proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.
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动脉粥样硬化患者心血管的疾病在世界上是死亡和病态的主要原因。低密度脂蛋白胆固醇(LDL-C ) 的血浆层次断然与动脉粥样硬化的风险被相关。在有血胆脂醇过多的病人的高血浆 LDL 集中在动脉的内部墙中导致 LDL 的逐渐增加，它变得氧化并且支持泡沫房间的形成，因而开始动脉粥样硬化。血浆 LDL 主要通过 LDL 受体(LDLR ) 被清除小径。在 LDLR 的变化引起熟悉的血胆脂醇过多并且增加早熟的冠的心疾病的风险。LDLR 的表示经由甾醇在 transcriptional 水平被调整规章的元素绑定蛋白质 2 (SREBP-2 ) 并且在主要通过 proprotein convertase subtilisin/kexin-type 的 posttranslational 层次 9 (PCSK9 ) 并且 LDLR (偶像) 的可诱导的 degrader。在这评论，我们在 PCSK9 的研究总结最近的进展。
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Low-density lipoprotein
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role controlling the levels of low-density lipoprotein (LDL) particles that circulate in the bloodstream. Several gain-of-function and loss-of-function mutations in the PCSK9 gene, which occur naturally, have been identified and linked to hypercholesterolemia and hypocholesterolemia, respectively. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of the LDL receptor (LDLR) protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels primarily by increasing hepatic expression of LDLR protein and thereby accelerating clearance of circulating LDL cholesterol. Since the loss of a functional PCSK9 in human is not associated with apparent deleterious effects, this protease is becoming an attractive target for lowering plasma LDL cholesterol levels either alone or in combination with statins.
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Hypercholesterolemia characterized by excessively elevated levels of plasma low-density lipoprotein-cholesterol (LDLc) increases the risk of atherosclerosis, causing cardiovascular disease and cerebrovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9), which is secreted from liver, interacts with LDL-receptor (LDLR) that uptakes LDLc from plasma into hepatocytes. The PCSK9-LDLR interaction leads degradation of the LDLR on hepatocytes, resulting in elevation of plasma LDLc levels. Although anti-PCSK9 antibodies that inhibit PCSK9-LDLR interaction have been used for the treatment of hypercholesterolemia, the antibody agents are very expensive because they are produced using mammalian cells. To overcome this problem, it is necessary to develop PCSK9/LDLR interaction inhibitors that are produced by chemical synthesis to be low-cost in hypercholesterolemia treatment.
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Since the discovery in 2003 that gain-of-function mutations in the gene encoding proprotein convertase subtilisin–kexin type 9 (PCSK9) cause autosomal dominant hypercholesterolemia, which was followed by the identification in 2005 of loss-of-function mutations in PCSK9 as a cause of lower low-density lipoprotein (LDL) cholesterol levels, interest in the PCSK9 pathway has exploded. PCSK9 is a secreted serine protease that binds to the extracellular domain of the LDL receptor and targets the LDL receptor to the lysosomal compartment for degradation.1 Consequently, PCSK9 prevents recycling of the LDL receptor to the cell surface, thereby attenuating LDL clearance. PCSK9 is present in human . . .
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Elevated LDL-cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and degradation. Recent findings from our laboratory also suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is able to induce degradation of the low density lipoprotein receptor-related protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors. Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to reduced PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR regulation, but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification of PCSK9 targets should allow a better understanding of the consequences of PCSK9 inhibition for lowering LDLc and tumor metastasis.
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Proprotein Convertases
Subtilisin
Proprotein Convertases
Low-density lipoprotein
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PCSK9 (proprotein convertase subtilisin/kexin type 9) mediates the post-translational degradation of the LDL receptor (LDLR) and, as a result, modulates serum levels of LDL-cholesterol (LDL-C). Individuals with gain-of-function mutations in the PCSK9 gene exhibit high serum levels of LDL-C, while those with loss-of-function mutations have low serum levels of LDL-C and are protected from heart disease. Similarly, mice lacking the expression of PCSK9 exhibit higher levels of LDLR in the liver and reduced serum cholesterol, while the overexpression of PCSK9 reduces LDLR and results in increased serum cholesterol. Thus, as a novel, validated target for controlling serum levels of LDL-C, PCSK9 has attracted research attention. The biological inhibition of PCSK9 appears feasible, and preclinical programs based on RNAi targeting of the protein are at an advanced stage. In contrast, the development of conventional small-molecule therapeutics to inhibit PCSK9 continues to present challenges.
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