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

2019 ◽  
Vol 28 (22) ◽  
pp. 3734-3741 ◽  
Author(s):  
Thea Bismo Strøm ◽  
Katrine Bjune ◽  
Luís Teixeira da Costa ◽  
Trond P Leren
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Lipid Lowering ◽  
Ldl Receptors ◽  
Linker Region ◽  
Cholesterol Levels

Abstract 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.

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

2019 ◽  
Vol 29 (8) ◽  
pp. 1229-1238
Author(s):  
Thea Bismo Strøm ◽  
Katrine Bjune ◽  
Trond P Leren
Keyword(s):  
Ligand Binding ◽  
Bone Morphogenetic Protein ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Linker Region ◽  
Cholesterol Levels ◽  
Morphogenetic Protein ◽  
Bone Morphogenetic Protein 1

Abstract 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.

scholarly journals Measurement of the absolute number of functioning low-density lipoprotein receptors in vivo using a monoclonal antibody

1995 ◽  
Vol 305 (3) ◽  
pp. 897-904 ◽  
Author(s):  
C Fitzsimmons ◽  
R Bush ◽  
D Hele ◽  
C Godliman ◽  
E Gherardi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Body Weight ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Absolute Number ◽  
Low Density ◽  
Ldl Receptors ◽  
The Absolute

MAC188 S/S is a monoclonal antibody which can be used in vivo to measure the absolute number of functioning low-density lipoprotein (LDL) receptors in a rabbit. The antibody binds to the extra-cellular domain of the LDL receptor and binding is not blocked by the presence of LDL. When the antibody-receptor complex is internalized, receptor recycling is inhibited for several hours. Thus when saturating doses of MAC188 S/S are administered intravenously, the amount of antibody removed from the blood (minus non-specific removal) is determined solely by the total number of LDL receptors in an animal. In this study MAC188 S/S was used to measure the number of LDL receptors in control rabbits and in animals treated with 17 alpha-ethinyl oestradiol. After treatment (which caused a 47% decrease in plasma cholesterol), receptor-mediated removal of MAC188 S/S from the blood was saturated in both groups following injection of 3.0 mg of antibody per kg body weight. Based on the amount of antibody removed via the LDL receptor at this dose, the total number of accessible LDL receptors was calculated as (2.0 +/- 0.3) x 10(15) receptors per kg body weight in control rabbits and (4.0 +/- 0.4) x 10(15) receptors per kg body weight in oestrogen-treated animals. The number of receptors in various organs was also determined. The monoclonal antibody approach therefore, allows accurate determination of LDL receptor numbers in animals with markedly different concentrations of circulating LDL, conditions in which the use of endogenous ligand would be subject to significant errors.

scholarly journals GOLIATH regulates LDLR availability and plasma LDL cholesterol levels

2020 ◽  
Author(s):  
Bethan L. Clifford ◽  
Kelsey E. Jarrett ◽  
Joan Cheng ◽  
Angela Cheng ◽  
Marcus Seldin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ldl Cholesterol ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Lipoprotein Receptors ◽  
Cholesterol Levels ◽  
Genome Wide

AbstractIncreasing the availability of hepatic low-density lipoprotein receptors (LDLR) remains a major clinical target for reducing circulating plasma LDL cholesterol (LDL-C) levels. Here, we identify the molecular mechanism underlying genome-wide significant associations in the GOLIATH locus with plasma LDL-C levels. We demonstrate that GOLIATH is an E3 ubiquitin ligase that ubiquitinates the LDL Receptor resulting in redistribution away from the plasma membrane. Overexpression of GOLIATH decreases hepatic LDLR and increases plasma LDL-C levels. Silencing of Goliath using antisense oligonucleotides, germline deletion, or AAV-CRISPR in vivo strategies increases hepatic LDLR abundance and availability, thus decreasing plasma LDL-C. In vitro ubiquitination assays demonstrate RING-dependent regulation of LDLR abundance at the plasma membrane. Our studies identify GOLIATH as a novel post-translational regulator of LDL-C levels via modulation of LDLR availability, which is likely important for understanding the complex regulation of hepatic LDLR.

scholarly journals Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex.

1988 ◽  
Vol 106 (6) ◽  
pp. 1831-1841 ◽  
Author(s):  
R K Pathak ◽  
R K Merkle ◽  
R D Cummings ◽  
J L Goldstein ◽  
M S Brown ◽  
...  
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Ldl Receptors ◽  
Naturally Occurring ◽  
Rough Er ◽  
Mutant Receptors ◽  
Carbohydrate Chains

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.

Structure and Function of Proprotein Convertase Subtilisin/kexin Type 9 (PCSK9) in Hyperlipidemia and Atherosclerosis

2019 ◽  
Vol 20 (10) ◽  
pp. 1029-1040 ◽  
Author(s):  
Xinjie Lu
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Structure And Function ◽  
Lipid Lowering ◽  
Lipid Lowering Therapy ◽  
Proprotein Convertase ◽  
Novel Target ◽  
New Treatments ◽  
And Function

Background:One of the important factors in Low-Density Lipoprotein (LDL) metabolism is the LDL receptor (LDLR) by its capacity to bind and subsequently clear cholesterol derived from LDL (LDL-C) in the circulation. Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is a newly discovered serine protease that destroys LDLR in the liver and thereby controls the levels of LDL in plasma. Inhibition of PCSK9-mediated degradation of LDLR has, therefore, become a novel target for lipid-lowering therapy.Methods:We review the current understanding of the structure and function of PCSK9 as well as its implications for the treatment of hyperlipidemia and atherosclerosis.Results:New treatments such as monoclonal antibodies against PCSK9 may be useful agents to lower plasma levels of LDL and hence prevent atherosclerosis.Conclusion:PCSK9's mechanism of action is not yet fully clarified. However, treatments that target PCSK9 have shown striking early efficacy and promise to improve the lives of countless patients with hyperlipidemia and atherosclerosis.

scholarly journals Interaction of 4-hydroxynonenal-modified low-density lipoproteins with the fibroblast apolipoprotein B/E receptor

1986 ◽  
Vol 234 (1) ◽  
pp. 245-248 ◽  
Author(s):  
W Jessup ◽  
G Jurgens ◽  
J Lang ◽  
H Esterbauer ◽  
R T Dean
Keyword(s):  
Apolipoprotein B ◽  
Negative Charge ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipid Peroxidation Product ◽  
Modified Low Density Lipoproteins ◽  
Peroxidation Product

The incorporation of the lipid peroxidation product 4-hydroxynonenal into low-density lipoprotein (LDL) increases the negative charge of the particle, and decreases its affinity for the fibroblast LDL receptor. It is suggested that this modification may occur in vivo, and might promote atherogenesis.

Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

1986 ◽  
Vol 6 (9) ◽  
pp. 3268-3277
Author(s):  
R D Sege ◽  
K F Kozarsky ◽  
M Krieger
Keyword(s):  
Gamma Irradiation ◽  
Cho Cells ◽  
Gamma Ray ◽  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Ldl Receptors

The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

Secondary prevention care and effect: Total and low-density lipoprotein cholesterol levels and lipid-lowering drug use in women and men after incident myocardial infarction – The Tromsø Study 1994–2016

2018 ◽  
Vol 17 (6) ◽  
pp. 563-570 ◽  
Author(s):  
Laila A Hopstock ◽  
Anne Elise Eggen ◽  
Maja-Lisa Løchen ◽  
Ellisiv B Mathiesen ◽  
Inger Njølstad ◽  
...  
Keyword(s):  
Ldl Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipid Lowering ◽  
Low Density ◽  
Lipid Levels ◽  
Treatment Target ◽  
Cholesterol Levels ◽  
Lipid Lowering Drug ◽  
Lowering Drug

Background: Secondary prevention guidelines after myocardial infarction (MI) are gender neutral, but underutilisation of treatment in women has been reported. Design: We investigated the change in total and low-density lipoprotein (LDL) cholesterol levels and lipid-lowering drug (LLD) use after first-ever MI in a population-based study. Methods: We followed 10,005 participants (54% women) attending the Tromsø Study 1994–1995 and 8483 participants (55% women) attending the Tromsø Study 2007–2008 for first-ever MI up to their participation in 2007–2008 and 2015–2016, respectively. We used linear and logistic regression models to investigate sex differences in change in lipid levels. Results: A total of 395 (MI cohort I) and 132 participants (MI cohort II) had a first-ever MI during 1994–2008 and 2007–2013, respectively. Mean change in total cholesterol was −2.34 mmol/L (SD 1.15) in MI cohort I, and in LDL cholesterol was −1.63 mmol/L (SD 1.12) in MI cohort II. Men had a larger decrease in lipid levels compared to women: the linear regression coefficient for change was −0.33 (95% confidence interval [CI] −0.51 to −0.14) for total cholesterol and −0.21 (95% CI −0.37 to −0.04) for LDL cholesterol, adjusted for baseline lipid value, age and cohort. Men had 73% higher odds (95% CI 1.15−2.61) of treatment target achievement compared to women, adjusted for baseline lipid value, age and cohort. LLD use was reported in 85% of women and 92% of men in MI cohort I, and 80% in women and 89% in men in MI cohort II. Conclusions: Compared to men, women had significantly less decrease in lipid levels after MI, and a smaller proportion of women achieved the treatment target.

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