Lawrence P. Aggerbeck

Molecular analysis and intestinal expression of SAR1 genes and proteins in Anderson's disease (Chylomicron retention disease)

BackgroundAndersons disease (AD) or chylomicron retention disease (CMRD) is a very rare hereditary lipid malabsorption syndrome. In order to discover novel mutations in the SAR1B gene and to evaluate the expression, as compared to healthy subjects, of the Sar1 gene and protein paralogues in the intestine, we investigated three previously undescribed individuals with the disease.MethodsThe SAR1B, SAR1A and PCSK9 genes were sequenced. The expression of the SAR1B and SAR1A genes in intestinal biopsies of both normal individuals and patients was measured by RTqPCR. Immunohistochemistry using antibodies to recombinant Sar1 protein was used to evaluate the expression and localization of the Sar1 paralogues in the duodenal biopsies.ResultsTwo patients had a novel SAR1B mutation (p.Asp48ThrfsX17). The third patient, who had a previously described SAR1B mutation (p.Leu28ArgfsX7), also had a p.Leu21dup variant of the PCSK9 gene. The expression of the SAR1B gene in duodenal biopsies from an AD/CMRD patient was significantly decreased whereas the expression of the SAR1A gene was significantly increased, as compared to healthy individuals. The Sar1 proteins were present in decreased amounts in enterocytes in duodenal biopsies from the patients as compared to those from healthy subjects.ConclusionsAlthough the proteins encoded by the SAR1A and SAR1B genes are 90% identical, the increased expression of the SAR1A gene in AD/CMRD does not appear to compensate for the lack of the SAR1B protein. The PCSK9 variant, although reported to be associated with low levels of cholesterol, does not appear to exert any additional effect in this patient. The results provide further insight into the tissue-specific nature of AD/CMRD.

Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix− nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this “nodule-specific transcriptome” were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.

The use of high-performance liquid chromatography for the determination of size and molecular weight of proteins: a caution and a list of membrane proteins suitable as standards

We propose a list of 15 water-soluble globular proteins and 13 forms of detergent-soluble membrane proteins of known Stokes radii for the calibration of high-performance liquid chromatography columns. It is shown that it is advisable to use different sets of standards for these two types of proteins as the detergent-solubilized membrane proteins may behave differently, being excluded or retarded, depending upon the gel support. A smooth, although nonlinear, relationship between Stokes radii and erf-1(1--KD) is observed while a large scatter of points exists if the calibration is expressed as the molecular weight as a function of KD.

Structure of serum low-density lipoprotein: I. A solution X-ray scattering study of a hyperlipidemic monkey low-density lipoprotein☆

A serum low-density lipoprotein isolated from Rhesus monkeys that had been fed a coconut oil/cholesterol diet was studied by solution X-ray scattering experiments at variable contrast, at two temperatures, respectively, below (21 °C) and above (41 °C) the transition temperature. The experiments were carried out on an absolute scale, in the range (900 A−1) < s < (20 A−1) in water containing variable amounts of sodium bromide. Previous experiments (Aggerbeck et al., 1978) had shown that in this system the solution can be considered to be ideal and mono-disperse and that a volume can be ascribed to each macromolecule in solution inside of which the electron density distribution is independent of the density of the solvent. The structure analysis proceeds stepwise. First the morphological parameters are determined, namely those parameters (radius of gyration, volume, etc.) which are defined by some properties of the intensity and autocorrelation functions at their origin—value, slope or curvature; at the same time the three characteristic functions are determined. Next the autocorrelation function is plotted and analysed in the two-dimensional space (distance to the origin; solvent density). These steps lead to the following conclusions, which apply to both the low and the high-temperature forms: the particle is fairly isometric in shape; the outer surface is deeply convoluted; the outer region is sparsely occupied by a hydrated protein; the particle lacks a coarse centre of symmetry; the peripheral protein region is condensed in a small number of globules. It is also concluded that the temperature-induced structural transition involves both the external protein region and the core. All these conclusions are based upon a straightforward analysis of the X-ray scattering data. Pursuing the analysis beyond this stage requires making some assumption about the structure of the particle. These assumptions are focused on symmetry: it is argued, on the basis of formal considerations, of an analysis of the X-ray scattering data, and of the electron microscope observations (see Gulick-Krzywicki et al. (1979), accompanying paper), that the particle is likely to display a tetrahedral symmetry. Assuming that the symmetry belongs to point group 23, precise models are worked out for the shape of the predominantly lipid core and for the structure of the protein region. Moreover, at 21 °C the organization of the cholesteryl esters and the triglycerides inside the core is analyzed in terms of the organization of steroid-rich and hydrocarbon-rich regions of the cholesteryl esters, and it is shown to be more complicated than a concentric spherical multilayer. The models proposed are in excellent agreement with the X-ray scattering curves as well as with the chemical and physical properties of the particle; they also agree with the electron microscope observations. The most conspicuous features of the models and of the temperature-induced structural transitions are the following: the core of the particle, of low average electron density, is of almost spherical shape; this core contains the lipids and some protein; the amount of protein in the core is higher at 21 °C than at 41 °C; the core is surrounded by four protein globules, located at tetrahedral positions; each of these globules is compact at 21 °C and spreads out at 41 °C; at 21 °C the core displays a highly organized structure (tentatively described as a cubic organization of spherical elements); at 41 °C the core is less precisely organized although its structure is still more ordered than a liquid. The models are not intended to provide more than a coarse description of the structure; problems like the molecular weight of the apolipoprotein, the precise structure of the lipid in the core, the location of the protein which is not incorporated in the globules, are left open. Several formal aspects of the data processing adopted in this work are discussed; the information content of the data is shown to be redundant with respect to the models.

A Novel Abetalipoproteinemia Genotype IDENTIFICATION OF A MISSENSE MUTATION IN THE 97-kDa SUBUNIT OF THE MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN THAT PREVENTS COMPLEX FORMATION WITH PROTEIN DISULFIDE ISOMERASE

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297 → Gln, Asp384 → Ala, and Arg540 → His. Transient expression of each mutant showed that only the Arg540 → His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540 → His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.

Anderson’s Disease Exclusion of Apolipoprotein and Intracellular Lipid Transport Genes

Andersons disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48-containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/mu(2)) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/mu(2), 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.

SMARCA2 and other genome-wide supported schizophrenia-associated genes: regulation by REST/NRSF, network organization and primate-specific evolution

The SMARCA2 gene, which encodes BRM in the SWI/SNF chromatin-remodeling complex, was recently identified as being associated with schizophrenia (SZ) in a genome-wide approach. Polymorphisms in SMARCA2, associated with the disease, produce changes in the expression of the gene and/or in the encoded amino acid sequence. We show here that an SWI/SNF-centered network including the Smarca2 gene is modified by the down-regulation of REST/NRSF in a mouse neuronal cell line. REST/NRSF down-regulation also modifies the levels of Smarce1, Smarcd3 and SWI/SNF interactors (Hdac1, RcoR1 and Mecp2). Smarca2 down-regulation generates an abnormal dendritic spine morphology that is an intermediate phenotype of SZ. We further found that 8 (CSF2RA, HIST1H2BJ, NOTCH4, NRGN, SHOX, SMARCA2, TCF4 and ZNF804A) out of 10 genome-wide supported SZ-associated genes are part of an interacting network (including SMARCA2), 5 members of which encode transcription regulators. The expression of 3 (TCF4, SMARCA2 and CSF2RA) of the 10 genome-wide supported SZ-associated genes is modified when the REST/NRSF-SWI/SNF chromatin-remodeling complex is experimentally manipulated in mouse cell lines and in transgenic mouse models. The REST/NRSF-SWI/SNF deregulation also results in the differential expression of genes that are clustered in chromosomes suggesting the induction of genome-wide epigenetic changes. Finally, we found that SMARCA2 interactors and the genome-wide supported SZ-associated genes are considerably enriched in genes displaying positive selection in primates and in the human lineage which suggests the occurrence of novel protein interactions in primates. Altogether, these data identify the SWI/SNF chromatin-remodeling complex as a key component of the genetic architecture of SZ.

The aryl hydrocarbon receptor system.

Abstract The aryl hydrocarbon receptor (AhR) recognizes a large number of xenobiotics, such as polyaromatic hydrocarbons (PAHs) and dioxins, and it activates several metabolic and detoxification pathways. Recent evidence suggests that this receptor also has important endogenous functions subsequent to activation by natural dietary compounds and/or endogenous metabolites. This receptor, thus, has physiological functions that extend beyond specific instances of detoxification. Understanding the roles played by this receptor might be enhanced by a systems biology approach. Indeed, the AhR “ligandome” is very complex and the different classes of ligands involved could induce widely diverse effects. The protein “interactome” of the AhR comprises several tens of proteins and it is altered by the binding of ligands to the receptor. Furthermore, large-scale studies have shown cell and tissue-specific patterns of regulated gene expression which may depend upon the type of ligand, although these aspects need further substantiation. Finally, the AhR biological effects are extensive and include detoxification, cellular proliferation and migration, immune regulation and neuronal effects. A holistic approach should provide a better understanding of the biology of this receptor in addition to providing new avenues for the identification of specific toxicity mechanisms.

Structure of serum low-density lipoprotein: II. A freeze-etching electron microscopy study

Normal human and diet-induced hyperlipidemic Rhesus monkey serum low-density lipoprotein structure was investigated by freeze-etching electron microscopy employing a novel rapid freezing technique. Using turnip yellow mosaic virus as a standard, the technique was shown to be capable of providing information regarding the general architecture of particles in solution. Human and monkey serum low-density lipoprotein particle morphology appeared to deviate markedly from that of a perfect sphere. Instead, the outer layer of the particles appeared to consist of a small number of globules. The number and dimensions of these globules as well as their arrangement are in remarkable agreement with the tetrahedral model proposed by Luzzati et al. (1979) in the preceding paper for the low temperature form of the Rhesus monkey low-density lipoprotein.

[27] Studies of lipoproteins by freeze-fracture and etching electron microscopy

Publisher Summary Freeze-drying, freeze-fracturing and etching, and low-temperature electron microscopy of frozen hydrated specimens are the methods used for studying cryofixed material. This chapter describes a simple and inexpensive procedure for the study of plasma lipoproteins using a Balzers freeze-etching unit. The technique consists of four essential steps: cryofixation of the sample solution, either in the presence or absence of antifreeze agents, fracture of the cryofixed sample solution followed by etching of the fractured surface, and finally heavy metal replication of the fractured and etched surface. Freeze-fracture electron microscopy has become the preeminent technique for the structural study of cell membranes and lipid phases following subsequent improvements, and has been applied to the study of biological macromolecules in solution. Although the freeze-fracture/etching technique may provide detailed information on lipoprotein morphology, the chemical nature of the various components observed on the replica cannot be determined by the freeze-fracture/etching technique alone. Preliminary studies suggest that this technique may be very useful for the study of serum lipoproteins.