J L Goldstein

The availability of compactin (ML-236B), a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl Coenzyme A reductase, has permitted the demonstration of a hitherto unsuspected aspect of mevalonate metabolism and isoprenoid synthesis in cultured mammalian cells. 3-Hydroxy-3-methylglutaryl Coenzyme A reductase, the enzyme that synthesizes mevalonate, appears to be regulated through a multivalent feedback mechanism. Full suppression of the reductase requires the presence of at least two regulators: 1) cholesterol, which is normally derived exogenously from plasma low density lipoprotein (LDL), and 2) a nonsterol product, which is normally synthesized endogenously from mevalonate. Evidence indicates that both of these regulators of the reductase may be essential for the growth of mammalian cells in culture. The multivalent feedback regulation of 3-hydroxy-3-methylglutaryl Coenzyme A reductase, together with secondary regulatory changes in other enzymes of the sterol synthetic pathway, coordinates the branched pathway of mevalonate metabolism so as to assure a constant supply of cholesterol and nonsterol products. These new findings have important implications for the understanding of isoprenoid metabolism and its relation to cell growth.

Cultured fibroblasts derived from patients with homozygous familial hypercholesterolemia, which lack functional low density lipoprotein (LDL) receptors, fail to bind, take up, or degrade the lipoprotein with high affinity; therefore LDL-cholesterol is not made available for suppression of cholesterol synthesis or activation of cholesteryl ester formation. When LDL was given a positive charge by reaction with N,N-dimethyl-1,3-propanediamine (cationized LDL), the rate of degradation of the lipoprotein was increased by more than 100-fold in the homozygous familial hypercholesterolemia fibroblasts. Degradation of cationized LDL was inhibited by chloroquine, suggesting that it occurred in cellular lysosomes. Although the cationized LDL entered the cell through a mechanism independent of the LDL receptor, the cholesterol liberated from the degradation of the lipoprotein became available for suppression of cholesterol synthesis and stimulation of cholesteryl ester formation in the homozygous familial hypercholesterolemia fibroblasts. The rate of degradation of albumin by fibroblasts was also increased by more than 100-fold when this protein was coupled to N,N-dimethyl-1,3-propanediamine. The ability to deliver a protein to lysosomes by giving it a strong positive charge may have potential relevance not only to familial hypercholesterolemia, but also to inborn errors of metabolism that involve deficiencies in lysosomal enzymes.

Rapid internalization of the cell surface low density lipoprotein (LDL) receptor requires the first 22 amino acids of the cytoplasmic domain (residues 790-811), which must include an aromatic residue at position 807. In the human LDL receptor, this position is part of a tetrameric sequence, NPVY. A similar tetramer, NPXY (where X stands for any amino acid), is conserved in LDL receptors from six species (including Xenopus laevis) and in two members of the LDL receptor gene family, human LDL receptor-related protein and rat GP330. To determine whether the NPXY sequence is necessary for coated pit-mediated internalization, we used oligonucleotide-directed mutagenesis to substitute alanines for individual amino acids in the cytoplasmic tail of the human LDL receptor. Substitution of alanine for Asn804, Pro805, or Tyr807 (but not Val806) markedly reduced internalization. Only one other amino acid in the cytoplasmic 22-mer (Phe802) was important for internalization. A review of published data revealed NPXY sequences in cytoplasmic domains of at least 10 other cell surface proteins, including tyrosine kinase-linked receptors of the epidermal growth factor and insulin receptor family, the beta-subunits of three integrin receptors, and the amyloid A4 precursor protein. This occurrence is much more frequent than would be expected by chance alone. The possibility of a conditional role for the NPXY sequence in ligand-independent internalization of these proteins is discussed.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.