Carol C. Shoulders

The structure and evolution of the human β-globin gene family

Argiris Efstratiadis Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115 James W. Posakony, Tom Maniatis, Richard M. Lawn* and Catherine O’Connell+ Division of Biology California Institute of Technology Pasadena, California 91125 Richard A. Spritz, Jon K. DeRiel,# Bernard G. Forget and Sherman M. Weissman Departments of Genetics and Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510 Jerry L. Slightom, Ann E. Blechl and Oliver Smithies Laboratory of Genetics University of Wisconsin Madison, Wisconsin 53706 Francisco E. Baralle, Carol C. Shoulders and Nicholas J. ProudfootQ MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England Summary We present the results of a detailed comparison of the primary structure of human p-like globin genes and their flanking sequences. Among the se- quences located 5’ to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 2 1 and 77 + 10 bp, respectively, 5’ to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calcula- tion of the divergence times of individual @like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence com- parisons. In addition, the evolutionary relationship of the embryonic e-globin gene to the other human P-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of B-like globin genes during evolution.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders.

Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.

Impaired fatty acid metabolism in familial combined hyperlipidemia. A mechanism associating hepatic apolipoprotein B overproduction and insulin resistance.

To establish whether insulin resistance and/or postprandial fatty acid metabolism might contribute to familial combined hyperlipidemia (FCH) we have examined parameters of insulin resistance and lipid metabolism in six FCH kindreds. Probands and relatives (n = 56) were divided into three tertiles on the basis of fasting plasma triglycerides (TG). Individuals in the highest tertile (TG > 2.5 mM; n = 14) were older and had increased body mass index, systolic blood pressure, and fasting plasma insulin concentrations compared with individuals in the lowest tertile (n = 24). The former also presented with decreased HDL cholesterol and increased total plasma cholesterol, HDL-TG, and apoprotein B, E, and CIII concentrations. Insulin concentrations were positively correlated with plasma apo B, apo CIII, apo E, and TG, and inversely with HDL cholesterol. Fasting nonesterified fatty acids (NEFA) were elevated in FCH subjects compared to six unrelated controls and five subjects with familial hypertriglyceridemia. Prolonged and exaggerated postprandial plasma NEFA concentrations were found in five hypertriglyceridemic FCH probands. In FCH the X2 minor allele of the AI-CIII-AIV gene cluster was associated with increased fasting plasma TG, apo CIII, apo AI, and NEFA concentrations and decreased postheparin lipolytic activities. The clustering of risk factors associated with insulin resistance in FCH indicates a common metabolic basis for the FCH phenotype and the syndrome of insulin resistance probably mediated by an impaired fatty acid metabolism.

The primary structure of the human ϵ-globin gene

Abstract We describe the complete nucleotide sequence of the human ϵ-globin gene including 387 nucleotides of 5′ flanking sequence and 301 nucleotides of 3′ flanking sequence. The arrangement of coding, noncoding and intervening sequences in this gene is entirely consistent with its identification as the embryonic β -like globin gene.

Linkage and Association Between Distinct Variants of the APOA1/C3/A4/A5 Gene Cluster and Familial Combined Hyperlipidemia

Objective—Combined hyperlipidemia is a common disorder, characterized by a highly atherogenic lipoprotein profile and a substantially increased risk of coronary heart disease. The purpose of this study was to establish whether variations of apolipoprotein A5 (APOA5), a newly discovered gene of lipid metabolism located 30 kbp downstream of the APOA1/C3/A4 gene cluster, contributes to the transmission of familial combined hyperlipidemia (FCHL). Methods and Results—We performed linkage and association tests on 128 families. Two independent alleles, APOA5c.56G and APOC3c.386G, of the APOA1/C3/A4/A5 gene cluster were overtransmitted in FCHL (P =0.004 and 0.007, respectively). This was paired with reduced transmission of the common APOA1/C3/A4/A5 haplotype (frequency 0.4461) to affected subjects (P =0.012). The APOA5c.56G genotype accounted for 7.3% to 13.8% of the variance in plasma triglyceride levels in probands (P <0.004). The APOC3c.386G genotypes accounted for 4.4% to 5.1% of the variance in triglyceride levels in FCHL spouses (P <0.007), suggesting that this allele marks a FCHL quantitative trait as well as representing a susceptibility locus for the condition. Conclusions—A combined linkage and association analysis establishes that variation at the APOA1/C3/A4/A5 gene cluster contributes to FCHL transmission in a substantial proportion of northern European families.

A Common Binding Site on the Microsomal Triglyceride Transfer Protein for Apolipoprotein B and Protein Disulfide Isomerase

The assembly of triglyceride-rich lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein B (apoB), a microsomal triglyceride transfer protein (MTP), and protein disulfide isomerase (PDI). In the MTP complex, the amino-terminal region of MTP (residues 22–303) interacts with the amino-terminal region of apoB (residues 1–264). Here, we report the identification and characterization of a site on apoB between residues 512 and 721, which interacts with residues 517–603 of MTP. PDI binds in close proximity to this apoB binding site on MTP. The proximity of these binding sites on MTP for PDI and amino acids 512–721 of apoB was evident from studies carried out in a yeast two-hybrid system and by co-immunoprecipitation. The expression of PDI with MTP and apoB16 (residues 1–721) in the baculovirus expression system reduced the amount of MTP co-immunoprecipitated with apoB by 73%. The interaction of residues 512–721 of apoB with MTP facilitates lipoprotein production. Mutations of apoB that markedly reduced this interaction also reduced the level of apoB-containing lipoprotein secretion.

Choline containing metabolites during cell transfection: an insight into magnetic resonance spectroscopy detectable changes

Increases in choline containing metabolites have been associated with a number of disorders, including malignant cell growth. In this study, high resolution magic angle spinning 1H nuclear magnetic resonance spectroscopy was employed to monitor metabolite changes during cell transfection, and an increase in phosphocholine was detected. This increase appears to be correlated with cell membrane disruption associated with the insertion of plasmid DNA into cells, since the level of phosphocholine in mock transfected cells was comparable to that of control cells. These data suggest choline containing metabolite changes detected in vivo using magnetic resonance spectroscopy relate to cell membrane disruption.

Localization of Microsomal Triglyceride Transfer Protein in the Golgi POSSIBLE ROLE IN THE ASSEMBLY OF CHYLOMICRONS

Although a critical role of microsomal transfer protein (MTP) has been recognized in the assembly of nascent apolipoprotein B (apoB)-containing lipoproteins, it remains unclear where and how MTP transfers lipids in the secretory pathway during the maturational process of apoB lipidation. The aims of this study were to determine whether MTP functions in the secretory pathway as well as in the endoplasmic reticulum and whether its large 97-kDa subunit interacts with the small 58-kDa protein disulfide isomerase (PDI) subunit and apoB, particularly in the Golgi apparatus. Using a high resolution immunogold approach combined with specific polyclonal antibodies, the large and small subunits of MTP were observed over the rough endoplasmic reticulum and the Golgi. Double immunocytochemical detection unraveled the colocalization of MTP and PDI as well as MTP and apoB in these same subcellular compartments. To confirm the spatial contact of these proteins, Golgi fractions were isolated, homogenized, and incubated with an anti-MTP large subunit antibody. Immunoprecipitates were applied on SDS-PAGE and then transferred on to nitrocellulose. Immunoblotting the membrane with PDI and apoB antibodies confirmed the colocalization of these proteins with MTP. Furthermore, MTP activity assay disclosed a substantial triglyceride transfer in the Golgi fractions. The occurrence of membrane-associated apoB in the Golgi, coupled with its interaction with active MTP, suggests an important role for the Golgi in the biogenesis of apoB-containing lipoproteins.

The intracellular transport of chylomicrons requires the small GTPase, Sar1b.

Purpose of review The transport of lipoproteins through the secretory pathways of enterocytes and hepatocytes is pivotal for whole-body lipid homeostasis. This review focuses on the assembly and structural evolution of COPII (coat protein) transport carriers that are essential for the transport of chylomicrons from the endoplasmic reticulum to the Golgi apparatus. Recent findings The assembly of endoplasmic reticulum to Golgi transport carriers commences with the coating of specific areas of the endoplasmic reticulum membrane with Sar1-GTP and the Sec23/24 heterodimer. An important advance has been the crystallographic analysis of the Sar1-Sec23/24 complex. The proteins form a bow-tie shaped structure, with a concave face that seems to match the curvature of transport carriers. Mammalian cells produce two isoforms of Sar1, designated Sar1a and Sar1b, both of which are expressed in enterocytes. Sar1b is defective in chylomicron retention disease and Anderson disease, two rare recessive disorders characterized by severe fat malabsorption and a failure to thrive in infancy. Patients with chylomicron retention disease and Anderson disease selectively retain chylomicron-like particles within membrane-bound compartments. By analogy with procollagen, chylomicrons may drive the formation of endoplasmic reticulum to Golgi transport carriers from endoplasmic reticulum sites close to, but separate from, domains of the endoplasmic reticulum coated with Sar1-Sec23/24. The COPII machinery also mediates the transport of VLDL to the Golgi. Summary New insights into the role of the COPII machinery in the intracellular transport of cargo, including chylomicrons and VLDL, may suggest new drug targets for ameliorating the lipid abnormalities of the metabolic syndrome.