Degradation of LDLR protein mediated by 'gain of function' PCSK9 mutants in normal and ARH cells.

Dominant gain-of-function mutations in proprotein convertase subtilisin kexin type 9 (PCSK9) cause familial hypercholesterolaemia (FH) and result in accelerated atherosclerosis and premature coronary heart disease. It is believed that PCSK9 binds to LDL-receptor (LDLR) protein and prevents its recycling to the cell surface; gain-of-function PCSK9 mutants enhance LDLR degradation. Several new variants of PCSK9 have been identified, but their effect on PCSK9 activity has not been determined. We describe a new procedure for assessing the activity of four putative gain-of-function mutations identified in FH patients (D129N, D374H, N425S, R496W). All four mutant proteins were secreted normally from transfected HEK293T cells. Immortalized lymphocytes from normolipaemic controls were incubated with conditioned medium from transfected cells and cell-surface LDLR protein was determined by FACS. D374H was as potent as D374Y in reducing cell-surface LDLR, while the other three mutations were more potent than wild type, but less so than the D374 mutants; this correlated with total serum cholesterol in the patients. Substitution of different amino acids at 374 showed that aspartate in this position was critical; even glutamate at residue 374 increased LDLR degradation. When the assay was carried out with ARH-negative lymphocytes that are unable to internalise the LDLR, D374Y-PCSK9 was able to reduce cell-surface LDLR by 35%, compared with approximately 70% for normal lymphocytes. Thus, PCSK9-mediated LDLR degradation is not entirely dependent on ARH function. We propose a novel ARH-independent pathway for PCSK9 activity on LDLR.

Adaptor protein disabled-2 modulates low density lipoprotein receptor synthesis in fibroblasts from patients with autosomal recessive hypercholesterolaemia.

Autosomal recessive hypercholesterolaemia (ARH), characterized clinically by severe inherited hypercholesterolaemia, is caused by recessive null mutations in LDLRAP1 (formerly ARH). Immortalized lymphocytes and monocyte-macrophages, and presumably hepatocytes, from ARH patients fail to take up and degrade plasma low density lipoproteins (LDL) because they lack LDLRAP1, a cargo-specific adaptor required for clathrin-mediated endocytosis of the LDL receptor. Surprisingly, LDL-receptor function is normal in ARH patients' skin fibroblasts in culture. Disabled-2 (Dab2) has been implicated previously in clathrin-mediated internalization of LDL-receptor family members, and we show here that Dab2 is highly expressed in skin fibroblasts, but not in lymphocytes. SiRNA-depletion of Dab2 profoundly reduced LDL-receptor activity in ARH fibroblasts as a result of profound reduction in LDL-receptor protein, but not mRNA; heterologous expression of murine Dab2 reversed this effect. In contrast, LDL-receptor protein content was unchanged in Dab-2-depleted control cells. Incorporation of 35S-labelled amino acids into LDL receptor protein revealed a corresponding apparent reduction in accumulation of newly synthesized LDL-receptor protein on depletion of Dab2 in ARH, but not in control, cells. This reduction in LDL-receptor protein in Dab2-depleted ARH cells could not be reversed by treatment of the cells with proteasomal or lysosomal inhibitors. Thus, we propose a novel role for Dab2 in ARH fibroblasts, where it is apparently required to allow normal translation of LDL receptor mRNA.

Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1.

Familial hypercholesterolemia is an autosomal dominant disorder with a gene-dosage effect that is usually caused by mutations in the LDL receptor gene that disrupt normal clearance of LDL. In the homozygous form, it results in a distinctive clinical phenotype, characterized by inherited hypercholesterolemia, cholesterol deposition in tendons, and severe premature coronary disease. We described previously two families with autosomal recessive hypercholesterolemia that is not due to mutations in the LDL receptor gene but is characterized by defective LDL receptor-dependent internalization and degradation of LDL by transformed lymphocytes from the patients. We mapped the defective gene to chromosome 1p36 and now show that the disorder in these and a third English family is due to novel mutations in ARH1, a newly identified gene encoding an adaptor-like protein. Cultured skin fibroblasts from affected individuals exhibit normal LDL receptor activity, but their monocyte-derived macrophages are similar to transformed lymphocytes, being unable to internalize and degrade LDL. Retroviral expression of normal human ARH1 restores LDL receptor internalization in transformed lymphocytes from an affected individual, as demonstrated by uptake and degradation of (125)I-labeled LDL and confocal microscopy of cells labeled with anti-LDL-receptor Ab.