Erik Lundgren

Only amyloidogenic intermediates of transthyretin induce apoptosis

In diseases like Alzheimers disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.

Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy.

The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six β-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F′ and H-H′ with a prolongation of these β-strands and, interestingly, with a possible shift in β-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.

Probing solvent accessibility of amyloid fibrils by solution NMR spectroscopy

Amyloid is the result of an anomalous protein and peptide aggregation, leading to the formation of insoluble fibril deposits. At present, 18 human diseases have been associated with amyloid deposits—e.g., Alzheimers disease and Prion-transmissible Spongiform Encephalopathies. The molecular structure of amyloid is to a large extent unknown, because of lack of high-resolution structural information within the amyloid state. However, from other experimental data it has been established that amyloid fibrils predominantly consist of β-strands arranged perpendicular to the fibril axis. Identification of residues involved in these secondary structural elements is therefore of vital importance to rationally designing appropriate inhibitors. We have designed a hydrogen/deuterium exchange NMR experiment that can be applied on mature amyloid to enable identification of the residues located inside the fibril core. Using a highly amyloidogenic peptide, corresponding to residues 25–35 within the Alzheimer Aβ(1–43) peptide, we could establish that residues 28–35 constitute the amyloid core, with residues 31 and 32 being the most protected. In addition, quantitative values for the solvent accessibility for each involved residue could be obtained. Based on our data, two models of peptide assembly are proposed. The method provides a general way to identify the core of amyloid structures and thereby pinpoint areas suitable for design of inhibitors.

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity

In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 °C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.

Modifications of transthyretin in amyloid fibrils: analysis of amyloid from homozygous and heterozygous individuals with the Met30 mutation.

The finding of individuals homozygous for FAP I (familial amyloidotic polyneuropathy, transthyretin TTRMet30) with amyloid deposits in the vitreous body, gave us access to a unique material lacking wild type transthyretin and contaminating proteins. Amyloid TTR is modified in several ways. Besides the full‐length protein and its dimer form, two smaller bands were identified by SDS‐PAGE and protein sequencing. One corresponded to a peptide starting at amino acid Thr49, the other was a mixture of two peptides starting at positions 1 and 3 in a 3:1 ratio. Upon reduction the amount of the TTR dimer decreased, the monomer amount increased, and the resulting monomers became available for carboxymethylation. Moreover, the mobility of the small band, which includes Cys10, increased upon reduction. This cysteine seemed to be involved in an interchain disulfide bridge both between intact TTR molecules and between small fragments. The same pattern was found in heterozygous fibril material although smaller amounts of the truncated peptides were found. Fibrils were formed both from normal and mutated TTR in heterozygotes. The significance of our results for amyloid formation is discussed.

The gene for Best's macular dystrophy is located at 11q13 in a Swedish family

A large Swedish family with more than 250 cases of Bests macular dystrophy has been clinically and genetically studied. The gene was traced to a couple born in central Sweden in the 17th century. Highly significant evidence for genetic linkage to DNA markers on chromosome 11q13 was detected. A lod score of 15.12 was obtained at recombination fraction 0.01 with DNA marker INT2 (also called FGF3). The retinally expressed gene ROM1, which maps to the same chromosomal region is a candidate for this genetic disease.

The beta-slip: a novel concept in transthyretin amyloidosis.

Transthyretin is a tetrameric plasma protein associated with two forms of amyloid disease. The structure of the highly amyloidogenic transthyretin triple mutant TTRG53S/E54D/L55S determined at 2.3 A resolution reveals a novel conformation: the beta-slip. A three-residue shift in beta strand D places Leu-58 at the position normally occupied by Leu-55 now mutated to serine. The beta-slip is best defined in two of the four monomers, where it makes new protein-protein interactions to an area normally involved in complex formation with retinol-binding protein. This interaction creates unique packing arrangements, where two protein helices combine to form a double helix in agreement with fiber diffraction and electron microscopy data. Based on these findings, a novel model for transthyretin amyloid formation is presented.

Homozygosity for the transthyretin-met30-gene in two Swedish sibs with familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant inherited disorder. Recent biochemical studies have revealed that amyloid protein in FAP of Japanese, Swedish and Portuguese origin mainly consists of a variant transthyretin (TTR) (formerly called prealbumin) with one amino acid substitution of methionine for valine at position 30. In a 56‐year‐old man with typical polyneuropathy, gastrointestinal problems and vitreous amyloid, we diagnosed homozygosity for the TTR‐met30‐gene using RFLP analysis. In a family study, a sister presented the same homozygous RFLP pattern; however, in a careful clinical investigation we were not able to demonstrate any of the typical symptoms of FAP, nor could we demonstrate amyloid deposits in a biopsy skin specimen. This is the first report of homozygosity for the TTR‐met30‐gene, and it shows that the mutation of the protein involved in amyloid formation may be necessary but is clearly not sufficient for the clinical symptoms.

Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein.

Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.

Antibodies to specific EBNA-1 domains and HLA DRB1⁎1501 interact as risk factors for multiple sclerosis☆

Epitope reactivity of multiple sclerosis (MS) plasma antibodies against the Epstein-Barr virus protein EBNA-1 and its association with HLA DRB1*1501 status was investigated in a case-referent study. Based on EBNA-1 fragment reactivity and the effect of peptide blocking, four 29-36 amino acid long EBNA-1 fragments were selected for detailed studies. MS cases had increased antibody reactivity against several EBNA-1 domains, of which antibodies against EBNA-1 (amino acid 385-420) in HLA DRB1*1501 positive individuals were associated with a 24-fold risk increase for MS. The data need confirmation in a larger sample but suggest a role for this epitope in the autoimmune pathogenesis of MS.