Brandon S. J. Davies

GPIHBP1 Is Responsible for the Entry of Lipoprotein Lipase into Capillaries

The lipolytic processing of triglyceride-rich lipoproteins by lipoprotein lipase (LPL) is the central event in plasma lipid metabolism, providing lipids for storage in adipose tissue and fuel for vital organs such as the heart. LPL is synthesized and secreted by myocytes and adipocytes, but then finds its way into the lumen of capillaries, where it hydrolyzes lipoprotein triglycerides. The mechanism by which LPL reaches the lumen of capillaries has remained an unresolved problem of plasma lipid metabolism. Here, we show that GPIHBP1 is responsible for the transport of LPL into capillaries. In Gpihbp1-deficient mice, LPL is mislocalized to the interstitial spaces surrounding myocytes and adipocytes. Also, we show that GPIHBP1 is located at the basolateral surface of capillary endothelial cells and actively transports LPL across endothelial cells. Our experiments define the function of GPIHBP1 in triglyceride metabolism and provide a mechanism for the transport of LPL into capillaries.

Chylomicronemia With a Mutant GPIHBP1 (Q115P) That Cannot Bind Lipoprotein Lipase

Objective—GPIHBP1 is an endothelial cell protein that binds lipoprotein lipase (LPL) and chylomicrons. Because GPIHBP1 deficiency causes chylomicronemia in mice, we sought to determine whether some cases of chylomicronemia in humans could be attributable to defective GPIHBP1 proteins. Methods and Results—Patients with severe hypertriglyceridemia (n=60, with plasma triglycerides above the 95th percentile for age and gender) were screened for mutations in GPIHBP1. A homozygous GPIHBP1 mutation (c.344A>C) that changed a highly conserved glutamine at residue 115 to a proline (p.Q115P) was identified in a 33-year-old male with lifelong chylomicronemia. The patient had failure-to-thrive as a child but had no history of pancreatitis. He had no mutations in LPL, APOA5, or APOC2. The Q115P substitution did not affect the ability of GPIHBP1 to reach the cell surface. However, unlike wild-type GPIHBP1, GPIHBP1-Q115P lacked the ability to bind LPL or chylomicrons (d < 1.006 g/mL lipoproteins from Gpihbp1−/− mice). Mouse GPIHBP1 with the corresponding mutation (Q114P) also could not bind LPL. Conclusions—A homozygous missense mutation in GPIHBP1 (Q115P) was identified in a patient with chylomicronemia. The mutation eliminated the ability of GPIHBP1 to bind LPL and chylomicrons, strongly suggesting that it caused the patient’s chylomicronemia.

Regulation of prelamin A but not lamin C by miR-9, a brain-specific microRNA

Lamins A and C, alternatively spliced products of the LMNA gene, are key components of the nuclear lamina. The two isoforms are found in similar amounts in most tissues, but we observed an unexpected pattern of expression in the brain. Western blot and immunohistochemistry studies showed that lamin C is abundant in the mouse brain, whereas lamin A and its precursor prelamin A are restricted to endothelial cells and meningeal cells and are absent in neurons and glia. Prelamin A transcript levels were low in the brain, but this finding could not be explained by alternative splicing. In lamin A-only knockin mice, where alternative splicing is absent and all the output of the gene is channeled into prelamin A transcripts, large amounts of lamin A were found in peripheral tissues, but there was very little lamin A in the brain. Also, in knockin mice expressing exclusively progerin (a toxic form of prelamin A found in Hutchinson–Gilford progeria syndrome), the levels of progerin in the brain were extremely low. Further studies showed that prelamin A expression, but not lamin C expression, is down-regulated by a brain-specific microRNA, miR-9. Expression of miR-9 in cultured cells reduced lamin A expression, and this effect was abolished when the miR-9–binding site in the prelamin A 3′ UTR was mutated. The down-regulation of prelamin A expression in the brain could explain why mouse models of Hutchinson–Gilford progeria syndrome are free of central nervous system pathology.

Mutation of conserved cysteines in the Ly6 domain of GPIHBP1 in familial chylomicronemia

We investigated a family from northern Sweden in which three of four siblings have congenital chylomicronemia. LPL activity and mass in pre- and postheparin plasma were low, and LPL release into plasma after heparin injection was delayed. LPL activity and mass in adipose tissue biopsies appeared normal. [35S]Methionine incorporation studies on adipose tissue showed that newly synthesized LPL was normal in size and normally glycosylated. Breast milk from the affected female subjects contained normal to elevated LPL mass and activity levels. The milk had a lower than normal milk lipid content, and the fatty acid composition was compatible with the milk lipids being derived from de novo lipogenesis, rather than from the plasma lipoproteins. Given the delayed release of LPL into the plasma after heparin, we suspected that the chylomicronemia might be caused by mutations in GPIHBP1. Indeed, all three affected siblings were compound heterozygotes for missense mutations involving highly conserved cysteines in the Ly6 domain of GPIHBP1 (C65S and C68G). The mutant GPIHBP1 proteins reached the surface of transfected Chinese hamster ovary cells but were defective in their ability to bind LPL (as judged by both cell-based and cell-free LPL binding assays). Thus, the conserved cysteines in the Ly6 domain are crucial for GPIHBP1 function.

Dual Activators of the Sterol Biosynthetic Pathway of Saccharomyces cerevisiae: Similar Activation/Regulatory Domains but Different Response Mechanisms

ABSTRACT Genes encoding biosynthetic enzymes that make ergosterol, the major fungal membrane sterol, are regulated, in part, at the transcriptional level. Two transcription factors, Upc2p and Ecm22p, bind to the promoters of most ergosterol biosynthetic (ERG) genes, including ERG2 and ERG3, and activate these genes upon sterol depletion. We have identified the transcriptional activation domains of Upc2p and Ecm22p and found that UPC2-1, a mutation that allows cells to take up sterols aerobically, increased the potency of the activation domain. The equivalent mutation in ECM22 also greatly enhanced transcriptional activation. The C-terminal regions of Upc2p and Ecm22p, which contained activation domains, also conferred regulation in response to sterol levels. Hence, the activation and regulatory domains of these proteins overlapped. However, the two proteins differed markedly in how they respond to an increased need for sterols. Upon inducing conditions, Upc2p levels increased, and chromatin immunoprecipitation experiments revealed more Upc2p at promoters even when the activation/regulatory domains were tethered to a different DNA-binding domain. However, induction resulted in decreased Ecm22p levels and a corresponding decrease in the amount of Ecm22p bound to promoters. Thus, these two activators differ in their contributions to the regulation of their targets.

A Role for Sterol Levels in Oxygen Sensing in Saccharomyces cerevisiae

Upc2p and Ecm22p are a pair of transcription factors responsible for the basal and induced expression of genes encoding enzymes of ergosterol biosynthesis in yeast (ERG genes). Upc2p plays a second role as a regulator of hypoxically expressed genes. Both sterols and heme depend upon molecular oxygen for their synthesis, and thus the levels of both have the potential to act as indicators of the oxygen environment of cells. Hap1p is a heme-dependent transcription factor that both Upc2 and Ecm22p depend upon for basal level expression of ERG genes. However, induction of both ERG genes and the hypoxically expressed DAN/TIR genes by Upc2p and Ecm22p occurred in response to sterol depletion rather than to heme depletion. Indeed, upon sterol depletion, Upc2p no longer required Hap1p to activate ERG genes. Mot3p, a broadly acting repressor/activator protein, was previously shown to repress ERG gene expression, but the mechanism was unclear. We established that Mot3p bound directly to Ecm22p and repressed Ecm22p- but not Upc2p-mediated gene induction.

The Posttranslational Processing of Prelamin A and Disease

Human geneticists have shown that some progeroid syndromes are caused by mutations that interfere with the conversion of farnesyl-prelamin A to mature lamin A. For example, Hutchinson-Gilford progeria syndrome is caused by LMNA mutations that lead to the accumulation of a farnesylated version of prelamin A. In this review, we discuss the posttranslational modifications of prelamin A and their relevance to the pathogenesis and treatment of progeroid syndromes.

GPIHBP1—an endothelial cell molecule important for the lipolytic processing of chylomicrons

Purpose of review To summarize recent data indicating that glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) plays a key role in the lipolytic processing of chylomicrons. Recent findings Lipoprotein lipase hydrolyses triglycerides in chylomicrons at the luminal surface of the capillaries in heart, adipose tissue, and skeletal muscle. The endothelial cell molecule that facilitates the lipolytic processing of chylomicrons has never been clearly defined. Mice lacking GPIHBP1 manifest chylomicronemia, with plasma triglyceride levels as high as 5000 mg/dl. In wild-type mice, GPIHBP1 is expressed on the luminal surface of capillaries in heart, adipose tissue, and skeletal muscle. Cells transfected with GPIHBP1 bind both chylomicrons and lipoprotein lipase avidly. Summary The chylomicronemia in Gpihbp1-deficient mice, the fact that GPIHBP1 is located within the lumen of capillaries, and the fact that GPIHBP1 binds lipoprotein lipase and chylomicrons suggest that GPIHBP1 is a key platform for the lipolytic processing of triglyceride-rich lipoproteins.

The Acidic Domain of GPIHBP1 Is Important for the Binding of Lipoprotein Lipase and Chylomicrons

GPIHBP1, a glycosylphosphatidylinositol-anchored endothelial cell protein of the lymphocyte antigen 6 (Ly6) family, plays a key role in the lipolysis of triglyceride-rich lipoproteins (e.g. chylomicrons). GPIHBP1 is expressed along the luminal surface of endothelial cells of heart, skeletal muscle, and adipose tissue, and GPIHBP1-expressing cells bind lipoprotein lipase (LPL) and chylomicrons avidly. GPIHBP1 contains an amino-terminal acidic domain (amino acids 24-48) that is enriched in aspartate and glutamate residues, and we previously speculated that this domain might be important in binding ligands. To explore the functional importance of the acidic domain, we tested the ability of polyaspartate or polyglutamate peptides to block the binding of ligands to pgsA-745 Chinese hamster ovary cells that overexpress GPIHBP1. Both polyaspartate and polyglutamate blocked LPL and chylomicron binding to GPIHBP1. Also, a rabbit antiserum against the acidic domain of GPIHBP1 blocked LPL and chylomicron binding to GPIHBP1-expressing cells. Replacing the acidic amino acids within GPIHBP1 residues 38-48 with alanine eliminated the ability of GPIHBP1 to bind LPL and chylomicrons. Finally, mutation of the positively charged heparin-binding domains within LPL and apolipoprotein AV abolished the ability of these proteins to bind to GPIHBP1. These studies indicate that the acidic domain of GPIHBP1 is important and that electrostatic interactions play a key role in ligand binding.

GPIHBP1, an endothelial cell transporter for lipoprotein lipase

Interest in lipolysis and the metabolism of triglyceride-rich lipoproteins was recently reignited by the discovery of severe hypertriglyceridemia (chylomicronemia) in glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1)-deficient mice. GPIHBP1 is expressed exclusively in capillary endothelial cells and binds lipoprotein lipase (LPL) avidly. These findings prompted speculation that GPIHBP1 serves as a binding site for LPL in the capillary lumen, creating “a platform for lipolysis.” More recent studies have identified a second and more intriguing role for GPIHBP1—picking up LPL in the subendothelial spaces and transporting it across endothelial cells to the capillary lumen. Here, we review the studies that revealed that GPIHBP1 is the LPL transporter and discuss which amino acid sequences are required for GPIHBP1–LPL interactions. We also discuss the human genetics of LPL transport, focusing on cases of chylomicronemia caused by GPIHBP1 mutations that abolish GPIHBP1’s ability to bind LPL, and LPL mutations that prevent LPL binding to GPIHBP1.