Yvonne Newhouse

Identifying polyglutamine protein species in situ that best predict neurodegeneration

SUMMARY Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function on proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies (IBs), IB formation may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that monoclonal antibody (mAb) 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular weight conformational states expanded polyQ assumes and disappears in higher molecular-weight aggregated forms, such as IBs. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Type III hyperlipoproteinemia associated with apolipoprotein E phenotype E3/3. Structure and genetics of an apolipoprotein E3 variant.

A family has been described in which type III hyperlipoproteinemia is associated with apo E phenotype E3/3 (Havel, R. J., L. Kotite, J. P. Kane, P. Tun, and T. Bersot. 1983. J. Clin. Invest. 72:379-387). In the current study, the structure of apo E from the propositus of this family was determined using both protein and DNA analyses. The propositus is heterozygous for two different apo E alleles, one coding for normal apo E3 and one for a previously undescribed variant apo E3 in which arginine replaces cysteine at residue 112 and cysteine replaces arginine at residue 142. Apo E gene analysis of nine other family members spanning four generations indicated that only those five members having type III hyperlipoproteinemia possess the variant apo E3. Like the propositus, all five are heterozygous for this variant, suggesting that the disorder in this family is transmitted in a dominant fashion. The variant apo E3 was defective in its ability to bind to lipoprotein receptors, and this functional defect probably contributes to the expression of type III hyperlipoproteinemia in this family.

New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E.

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimers disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, 13C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the α-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich inN- and O-sulfo groups such as that found in the liver and the brain.

Model of Biologically Active Apolipoprotein E Bound to Dipalmitoylphosphatidylcholine

Apolipoprotein (apo)E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 Å resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex.

Apolipoprotein E•dipalmitoylphosphatidylcholine particles are ellipsoidal in solution

Apolipoprotein E (apoE) is a major protein component of cholesterol-transporting lipoprotein particles in the central nervous system and in plasma. Polymorphisms of apoE are associated with cardiovascular disease and with a predisposition to Alzheimers disease and other forms of neurodegeneration. For full biological activity, apoE must be bound to a lipoprotein particle. Complexes of apoE and phospholipid mimic many of these activities. In contrast to a widely accepted discoidal model of apoA-I bound to dimyristoylphosphatidylcholine, which is based on solution studies, an X-ray diffraction study of apoE bound to dipalmitoylphosphatidylcholine (DPPC) indicated that apoE•DPPC particles are quasi-spheroidal and that the packing of the phospholipid core is similar to a micelle. Using small-angle X-ray scattering, we show that apoE•DPPC particles in solution are ellipsoidal and that the shape of the phospholipid core is compatible with a twisted-bilayer model. The proposed model is consistent with the results of mass spectrometric analysis of products of limited proteolysis. These revealed that the nonlipid-bound regions of apoE in the particle are consistent with an α-helical hairpin.

An optimized negative-staining protocol of electron microscopy for apoE4•POPC lipoprotein

Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE•phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4•palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4•POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.

Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli

The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced inEscherichia coli a soluble fragment containing the ligand-binding domain (residues 1–292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide probes

A rapid procedure for determining apolipoprotein E genotype from genomic DNA has been developed. In this procedure, DNA is amplified by the polymerase chain reaction, and allele-specific oligonucleotide probes are used to detect the cysteine-arginine interchanges at residues 112 and 158 that distinguish the three common isoforms of apolipoprotein E. The method was tested with 68 subjects, representing the six common phenotypes, and yielded results consistent with the known phenotype.

Crystallization and diffraction properties of the Fab fragment of 3B5H10, an antibody specific for disease-causing polyglutamine stretches

Because it binds soluble forms of proteins with disease-associated polyglutamine expansions, the antibody 3B5H10 is a powerful tool for studying polyglutamine-related diseases. Crystals of the 3B5H10 Fab (47 kDa) were obtained by vapor diffusion at room temperature from PEG 3350. However, the initial crystals gave highly anisotropic diffraction patterns. After optimization of the crystallization conditions and cryoprotectants, a nearly isotropic diffraction pattern at 2.6 A resolution was achieved for crystals with unit-cell parameters a = 133.26, b = 79.52, c = 41.49 A and space group P2(1)2(1)2. Dehydrated crystals diffracted isotropically to 1.9 A with unit-cell parameters a = 123.65, b = 78.25, c = 42.26 A, beta = 90.3 degrees and space group P2(1).

Structure of a monoclonal 2E8 Fab antibody fragment specific for the low-density lipoprotein-receptor binding region of apolipoprotein E refined at 1.9 Å

The crystal structure of the Fab fragment of 2E8, the monoclonal IgG1,kappa antibody specific for the low-density lipoprotein (LDL) receptor-binding region of apolipoprotein E (apoE), has been solved by molecular replacement and refined at 1.9 A resolution (PDB entry 12E8). Two 2E8 Fab molecules in the asymmetric unit are related by noncrystallographic symmetry and are hydrogen bonded through a beta-sheet-like intermolecular contact between the heavy-chain complementarity-determining regions 3 (CDRH3) of each molecule. The structure has been refined to an R value of 0.22 (Rfree = 0.27). The initially ill-defined heavy-chain constant domain (CH1) of 2E8 has been retraced with the aid of automatic refinement, confirming the beta-sheet tracing independently of any starting models. As a resolution better than 2 A is not common for Fab fragments, this model represents a well defined Fab structure and should prove useful in MR solution of other Fab fragments. Furthermore, in the absence of an LDL-receptor structure, the homology of the 2E8 CDRH2 to the ligand-binding domain of the LDL receptor has been exploited to model the apoE-LDL-receptor interaction.