SUMMARY 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 represents one of the most persistent mysteries 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.

Lamin B1 is essential for neuronal migration and progenitor proliferation during the development of the cerebral cortex. The observation of distinct phenotypes of Lmnb1- and Lmnb2-knockout mice and the differences in the nuclear morphology of cortical neurons in vivo suggest that lamin B1 and lamin B2 play distinct functions in the developing brain.

Nuclear lamins are components of the nuclear lamina, a structural scaffolding for the cell nucleus. Defects in lamins A and C cause an array of human diseases, including muscular dystrophy, lipodystrophy, and progeria, but no diseases have been linked to the loss of lamins B1 or B2. To explore the functional relevance of lamin B2, we generated lamin B2-deficient mice and found that they have severe brain abnormalities resembling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum. This neuronal layering abnormality is due to defective neuronal migration, a process that is dependent on the organized movement of the nucleus within the cell. These studies establish an essential function for lamin B2 in neuronal migration and brain development.brain | lissencephaly | neuronal migration | nuclear envelope | nuclear lamina T he nuclear lamina is an intermediate filament meshwork lying beneath the inner nuclear membrane that provides a structural scaffolding for the nucleus (1). The lamina is also important for other processes, including gene transcription, chromatin organization, nuclear pore distribution, nuclear envelope assembly, and tethering of the nucleus to the cytoskeleton (1, 2). The main components of the nuclear lamina are nuclear lamins, a class of intermediate filament proteins that is generally divided into two groups, A-type (lamins A and C) and B-type (lamins B1 and B2) (3, 4). Lamins A and C are produced from LMNA by alternative splicing, whereas lamins B1 and B2 are encoded by distinct genes, LMNB1 and LMNB2, respectively. Lamins B1 and B2 are expressed in all cells and throughout development, whereas lamins A and C are expressed in differentiated cells, beginning at midgestation (3).Interest in the nuclear lamins has intensified with the discovery that over a dozen human diseases, including muscular dystrophy, cardiomyopathy, lipodystrophy, and progeria, are caused by mutations in LMNA (5-7). To date, more than 340 missense, nonsense, frameshift, and splicing mutations have been identified (5). In contrast, no human diseases have been linked to these types of mutations in LMNB1 and LMNB2, and, so far, the only clear-cut association between B-type lamins and disease has been the finding of LMNB1 gene duplications in autosomal-dominant leukodystrophy (8).The paucity of "lamin B diseases" is probably not due to complete redundancy of lamins B1 and B2, as Lmnb1-deficient mice are small during embryonic development and die soon after birth with defects in lungs and bones (9). Also, Lmnb1-deficient fibroblasts display misshapen cell nuclei, aneuploidy, and early senescence (9). To further examine the functional importance of the B-type lamins, we generated Lmnb2-deficient mice.

Triglyceride-rich lipoproteins (TRLs) undergo lipolysis by lipoprotein lipase (LPL), an enzyme that is transported to the capillary lumen by an endothelial cell protein, GPIHBP1. For LPL-mediated lipolysis to occur, TRLs must bind to the lumen of capillaries. This process is often assumed to involve heparan sulfate proteoglycans (HSPGs), but we suspected that TRL margination might instead require GPIHBP1. Indeed, TRLs marginate along the heart capillaries of wild-type but not Gpihbp1−/− mice, as judged by fluorescence microscopy, quantitative assays with infrared-dye–labeled lipoproteins, and EM tomography. Both cell culture and in vivo studies showed that TRL margination depends on LPL bound to GPIHBP1. Of note, the expression of LPL by endothelial cells in Gpihbp1−/− mice did not restore defective TRL margination, implying that the binding of LPL to HSPGs is ineffective in promoting TRL margination. Our studies show that GPIHBP1-bound LPL is the main determinant of TRL margination.

HIV protease inhibitors (HIV-PIs) are key components of highly active antiretroviral therapy, but they have been associated with adverse side effects, including partial lipodystrophy and metabolic syndrome. We recently demonstrated that a commonly used HIV-PI, lopinavir, inhibits ZMPSTE24, thereby blocking lamin A biogenesis and leading to an accumulation of prelamin A. ZMPSTE24 deficiency in humans causes an accumulation of prelamin A and leads to lipodystrophy and other disease phenotypes. Thus, an accumulation of prelamin A in the setting of HIV-PIs represents a plausible mechanism for some drug side effects. Here we show, with metabolic labeling studies, that lopinavir leads to the accumulation of the farnesylated form of prelamin A. We also tested whether a new and chemically distinct HIV-PI, darunavir, inhibits ZMPSTE24. We found that darunavir does not inhibit the biochemical activity of ZMP-STE24, nor does it lead to an accumulation of farnesyl-prelamin A in cells. This property of darunavir is potentially attractive. However, all HIV-PIs, including darunavir, are generally administered with ritonavir, an HIV-PI that is used to block the metabolism of other HIV-PIs. Ritonavir, like lopinavir, inhibits ZMP-STE24 and leads to an accumulation of prelamin A.HIV protease inhibitors (HIV-PIs) 3 are designed to inhibit the HIV aspartyl protease, which is required for generating viral core proteins (1). HIV-PIs have become essential elements of modern antiretroviral regimens, but they have been associated with significant side effects, including partial lipodystrophy and metabolic syndrome (2-4). Similar disease phenotypes have been observed in association with missense mutations in LMNA (the gene for lamins A and C) (5, 6) and with genetic defects associated with defective conversion of prelamin A to mature lamin A (7-9).In 2003, Caron et al. (10) reported that a pair of HIV-PIs, indinavir and nelfinavir, appeared to lead to the accumulation of small amounts of prelamin A in a preadipocyte cell line. This finding was intriguing, but the biochemical mechanism was obscure. Potentially, this finding could have been due to the inhibition of any of four different enzymatic steps in prelamin A metabolism. The biogenesis of mature lamin A from prelamin A involves 1) farnesylation of a C-terminal cysteine by protein farnesyltransferase; 2) the removal of the last three amino acids of prelamin A (a redundant enzymatic activity of Ras converting enzyme 1 (RCE1) and ZMPSTE24); 3) the methylation of a newly exposed farnesylcysteine by isoprenylcysteine carboxyl methyltransferase (ICMT); and 4) the removal of the last 15 residues of the protein, including the farnesylcysteine methyl ester, by ZMPSTE24 (11). Steps 2-4 are utterly dependent on the first post-translational processing step, protein farnesylation.Recently, our laboratories showed that several HIV-PIs, but notably lopinavir, lead to substantial prelamin A accumulation in cultured cells at therapeutically relevant concentrations, and we went on to identify the ...

Lamin A, a key component of the nuclear lamina, is generated from prelamin A by four post-translational processing steps: farnesylation, endoproteolytic release of the last three amino acids of the protein, methylation of the C-terminal farnesylcysteine, and finally, endoproteolytic release of the last 15 amino acids of the protein (including the farnesylcysteine methyl ester). The last cleavage step, mediated by ZMPSTE24, releases mature lamin A. This processing scheme has been conserved through vertebrate evolution and is widely assumed to be crucial for targeting lamin A to the nuclear envelope. However, its physiologic importance has never been tested. To address this issue, we created mice with a "mature lamin A-only" allele (Lmna LAO ), which contains a stop codon immediately after the last codon of mature lamin A. Thus, Lmna LAO/LAO mice synthesize mature lamin A directly, bypassing prelamin A synthesis and processing. The levels of mature lamin A in Lmna LAO/LAO mice were indistinguishable from those in "prelamin A-only" mice (Lmna PLAO/PLAO ), where all of the lamin A is produced from prelamin A. Lmna LAO/LAO exhibited normal body weights and had no detectable disease phenotypes. A higher frequency of nuclear blebs was observed in Lmna LAO/LAO embryonic fibroblasts; however, the mature lamin A in the tissues of Lmna LAO/LAO mice was positioned normally at the nuclear rim. We conclude that prelamin A processing is dispensable in mice and that direct synthesis of mature lamin A has little if any effect on the targeting of lamin A to the nuclear rim in mouse tissues.Lamin A, one of the principal protein components of the nuclear lamina, is generated from prelamin A by a series of four enzymatic post-translational processing steps (1, 2). First, the cysteine in the C-terminal CAAX motif is farnesylated by protein farnesyltransferase. Second, the last three amino acids of the protein (i.e. the -AAX) are clipped off, a redundant activity of two membrane proteases of the endoplasmic reticulum (ER), 2 RCE1 and ZMPSTE24 (2, 3). Third, the newly exposed farnesylcysteine is methylated by ICMT (4), a membrane methyltransferase of the ER. Finally, the last 15 amino acids of the protein (including the C-terminal farnesylcysteine methyl ester) are clipped off by ZMPSTE24, releasing mature lamin A. Lamin A and lamin C, both "A-type" lamins, are splice variants of LMNA (5, 6). Lamin C does not contain a CAAX motif and therefore does not undergo any of the C-terminal post-translational processing steps.The prelamin A processing pathway has attracted considerable attention from medical geneticists, cell biologists, and pharmacologists (1, 7-11). Hutchinson-Gilford progeria syndrome (HGPS), the classic progeroid disorder of children, is caused by point mutations leading to a 50-amino acid internal deletion within the C-terminal region of prelamin A (7, 8). This deletion does not affect protein farnesylation/methylation but abolishes the final cleavage by ZMPSTE24, resulting in the accumulation of a farnesylated, truncated ...

The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. Mice expressing nonfarnesylated lamin B2 developed normally and were free of disease. In contrast, mice expressing nonfarnesylated lamin B1 died soon after birth, with severe neurodevelopmental defects and striking nuclear abnormalities in neurons. The nuclear lamina in migrating neurons was pulled away from the chromatin so that the chromatin was left "naked" (free from the nuclear lamina). Thus, farnesylation of lamin B1-but not lamin B2-is crucial for brain development and for retaining chromatin within the bounds of the nuclear lamina during neuronal migration.genetically modified mouse models T he nuclear lamina is an intermediate filament meshwork that lies beneath the inner nuclear membrane. This lamina provides structural support for the nucleus and also interacts with nuclear proteins and chromatin, thereby affecting many functions within the cell nucleus (1, 2). In mammals, the main protein components of the nuclear lamina are lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins).Both B-type lamins and prelamin A (the precursor of lamin A) terminate with a CaaX motif, which triggers three posttranslational modifications (3-5): farnesylation of the carboxyl-terminal cysteine (the "C" in the CaaX motif) (6), endoproteolytic cleavage of the last three amino acids (the -aaX) (7,8), and carboxyl methylation of the newly exposed farnesylcysteine (9, 10). Prelamin A undergoes a second endoproteolytic cleavage event, mediated by zinc metalloproteinase STE24 (ZMPSTE24), which removes 15 additional amino acids from the carboxyl terminus, including the farnesylcysteine methyl ester (11)(12)(13)(14). Lamins B1 and B2 do not undergo the second cleavage step and therefore retain their farnesyl lipid anchor.The discovery that Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation yielding an internally truncated farnesyl-prelamin A (15) has focused interest in the farnesylation of nuclear lamins. This interest has been fueled by the finding that disease phenotypes in mouse models of HGPS could be ameliorated by blocking protein farnesylation with a protein farnesyltransferase inhibitor (FTI) (16)(17)(18)(19). Most recently, children with HGPS seemed to show a positive response to FTI treatment (20).The prospect of using an FTI to treat children with HGPS naturally raises the issue of the importance of protein farnesylation for lamin B1 and lamin B2. The B-type lamins are expressed in all mammalian cells and have been h...

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