Ingrid Dacklin

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.

Misfolded transthyretin causes behavioral changes in a Drosophila model for transthyretin-associated amyloidosis

Familial amyloidotic polyneuropathy is an autosomal dominant neurodegenerative disorder caused by accumulation of mutated transthyretin (TTR) amyloid fibrils in different organs and prevalently around peripheral nerves. We have constructed transgenic flies, expressing the clinical amyloidogenic variant TTRL55P and the engineered variant TTR‐A (TTRV14N/V16E) as well as the wild‐type protein, all in secreted form. Within a few weeks, both mutants but not the wild‐type TTR demonstrated a time‐dependent aggregation of misfolded molecules. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability and shortened life span. In contrast, expression of wild‐type TTR had no discernible effect on either longevity or behavior. These results suggest that Drosophila can be used as a disease‐model to study TTR amyloid formation, and to screen for pharmacological agents and modifying genes.

The "edge strand" hypothesis: Prediction and test of a mutational "hot-spot" on the transthyretin molecule associated with FAP amyloidogenesis

The amyloid fibrils of familial amyloidotic polyneuropathy (FAP) are composed of genetic variants of transthyretin (TTR). Over 40 amino-acid variants have been identified associated with amyloidosis. We have performed an analysis of the effect of the different substitutions, such as location and changes in side-chain volume, hydrophilicity and charge on the TTR molecule. We have also analyzed the distribution of variants along the peptide chain. While we could demonstrate no pattern of change associated with the bulk properties of the amyloidogenic variant residues, the distribution analysis suggested a significant correlation with the position of variant residues along the polypeptide chain. We identified a significant peak in the distribution of variants which, in the folded protein chain, was associated with the edge strands (residues 45-58) of the two s-sheets that form the structural framework of the TTR molecule. In contrast, the regions of the molecule associated with the subunit interactions are almost devoid of amyloidogenic variants. To test this finding, we constructed two variant TTRs using recombinant techniques, one with a triple substitution and one with a triple deletion in the edge strand region. On expression, the variants spontaneously formed material that produced a characteristic redshift upon Congo red staining, was composed of fibrils 80-110A in diameter and gave s-structure X-ray patterns suggesting the formation of amyloid fibrils. We conclude that the effect of amyloidogenic variants may be to alter the structure or stability of the edge strands of the s-sheets of TTR, thus promoting the growth of inter-molecular s-sheet structures characteristic of amyloid.

Uptake of aggregating transthyretin by fat body in a drosophila model for TTR-associated amyloidosis

Background A functional link has been established between the severe neurodegenerative disorder Familial amyloidotic polyneuropathy and the enhanced propensity of the plasma protein transthyretin (TTR) to form aggregates in patients with single point mutations in the TTR gene. Previous work has led to the establishment of an experimental model based on transgenic expression of normal or mutant forms of human TTR in Drosophila flies. Remarkably, the severity of the phenotype was greater in flies that expressed a single copy than with two copies of the mutated gene. Methodology/Principal Findings In this study, we analyze the distribution of normal and mutant TTR in transgenic flies, and the ultrastructure of TTR-positive tissues to clarify if aggregates and/or amyloid filaments are formed. We report the formation of intracellular aggregates of 20 nm spherules and amyloid filaments in thoracic adipose tissue and in brain glia, two tissues that do not express the transgene. The formation of aggregates of nanospherules increased with age and was more considerable in flies with two copies of mutated TTR. Treatment of human neuronal cells with protein extracts prepared from TTR flies of different age showed that the extracts from older flies were less toxic than those from younger flies. Conclusions/Significance These findings suggest that the uptake of TTR from the circulation and its subsequent segregation into cytoplasmic quasi-crystalline arrays of nanospherules is part of a mechanism that neutralizes the toxic effect of TTR.

Inhibition of TTR aggregation-induced cell death--a new role for serum amyloid P component.

Background Serum amyloid P component (SAP) is a glycoprotein that is universally found associated with different types of amyloid deposits. It has been suggested that it stabilizes amyloid fibrils and therefore protects them from proteolytic degradation. Methodology/Principal Findings In this paper, we show that SAP binds not only to mature amyloid fibrils but also to early aggregates of amyloidogenic mutants of the plasma protein transthyretin (TTR). It does not inhibit fibril formation of TTR mutants, which spontaneously form amyloid in vitro at physiological pH. We found that SAP prevents cell death induced by mutant TTR, while several other molecules that are also known to decorate amyloid fibrils do not have such effect. Using a Drosophila model for TTR-associated amyloidosis, we found a new role for SAP as a protective factor in inhibition of TTR-induced toxicity. Overexpression of mutated TTR leads to a neurological phenotype with changes in wing posture. SAP-transgenic flies were crossed with mutated TTR-expressing flies and the results clearly confirmed a protective effect of SAP on TTR-induced phenotype, with an almost complete reduction in abnormal wing posture. Furthermore, we found in vivo that binding of SAP to mutated TTR counteracts the otherwise detrimental effects of aggregation of amyloidogenic TTR on retinal structure. Conclusions/Significance Together, these two approaches firmly establish the protective effect of SAP on TTR-induced cell death and degenerative phenotypes, and suggest a novel role for SAP through which the toxicity of early amyloidogenic aggregates is attenuated.

Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster

Abstract Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL) with the dual capacity to act both as a sulphur carrier and posttranslational protein modifier. Here we characterize the Drosophila melanogaster homologues of Urm1 (CG33276) and its E1 activating enzyme Uba4 (CG13090), and show that they function together to induce protein urmylation in vivo. Urm1 conjugation to target proteins in general, and to the evolutionary conserved substrate Peroxiredoxin 5 (Prx5) specifically, is dependent on Uba4. A complete loss of Urm1 is lethal in flies, although a small number of adult zygotic Urm1n123 mutant escapers can be recovered. These escapers display a decreased general fitness and shortened lifespan, but in contrast to their S. cerevisiae counterparts, they are resistant to oxidative stress. Providing a molecular explanation, we demonstrate that cytoprotective JNK signaling is increased in Urm1 deficient animals. In agreement, molecular and genetic evidence suggest that elevated activity of the JNK downstream target genes Jafrac1 and gstD1 strongly contributes to the tolerance against oxidative stress displayed by Urm1n123 null mutants. In conclusion, Urm1 is a UBL that is involved in the regulation of JNK signaling and the response against oxidative stress in the fruit fly.

Mapping protein conformations in fibril structures using monoclonal antibodies.

Publisher Summary Sixteen proteins are known to aggregate and form amyloid fibrils. These have very similar biophysical and biochemical properties, independent of which proteins form the building blocks. Fibrils have an ordered structure containing β strands arranged perpendicular to the fibril direction. The three-dimensional structure is known for many amyloid-forming proteins, mostly from X-ray diffraction studies. Much less is known about the structure of the involved proteins in fibrils, mainly because available techniques for high-resolution studies are not sufficiently informative for fibrillar structures. The methods described in this chapter are designed to develop monoclonal antibodies specific for structural changes associated with amyloid formation. There is no obvious correlation between the native structure of amyloidogenic proteins and β structure-rich amyloid fibrils. Two necessary steps can be envisaged as a general mechanism for the formation of amyloid, independent of the protein involved. The first step is a change in conformation leading to β structures, even in proteins with a dominance of α helices, as exemplified by prion proteins and lysozyme. The second step is self-aggregation with the formation of fibrils. This sequence of events necessarily means that the structural changes in the first stage open up new surfaces, which are important for the aggregation step.

A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster

By covalently conjugating to target proteins, ubiquitin-like modifiers (UBLs) act as important regulators of target protein localization and activity, thereby playing a critical role in the orchestration of cellular biology. The most ancient and one of the least studied UBLs is Urm1, a dual-function protein that in parallel to performing similar functions as its prokaryotic ancestors in tRNA modification, also has adopted the capacity to conjugate to cellular proteins analogous to ubiquitin and other UBL modifiers. In order to increase the understanding of Urm1 and its role in multicellular organisms, we have used affinity purification followed by mass spectrometry to identify putative targets of Urm1 conjugation (urmylation) at three developmental stages of the Drosophila melanogaster lifecycle. Altogether we have recovered 79 Urm1-interacting proteins in Drosophila, which include the already established Urm1 binding partners Prx5 and Uba4, together with 77 candidate urmylation targets that are completely novel in the fly. Among these, the majority was exclusively identified during either embryogenesis, larval stages or in adult flies. We further present biochemical evidence that four of these proteins are covalently conjugated by Urm1, whereas the fifth verified Urm1-binding protein appears to interact with Urm1 via non-covalent means. Besides recapitulating the previously established roles of Urm1 in tRNA modification and during oxidative stress, functional clustering of the newly identified Urm1-associated proteins further positions Urm1 in protein networks that control other types of cellular stress, such as immunological threats and DNA damage. In addition, the functional characteristics of several of the candidate targets strongly match the phenotypes displayed by Urm1n123 null animals, including embryonic lethality, reduced fertility and shortened lifespan. In conclusion, this identification of candidate targets of urmylation significantly increases the knowledge of Urm1 and presents an excellent starting point for unravelling the role of Urm1 in the context of a complex living organism.

The HTLV-1 oncoprotein Tax is modified by the ubiquitin related modifier 1 (Urm1)

BackgroundAdult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic human T-cell lymphotropic virus 1 infection, triggered by the virally encoded oncoprotein Tax. The transforming activity and subcellular localization of Tax is strongly influenced by posttranslational modifications, among which ubiquitylation and SUMOylation have been identified as key regulators of the nuclear/cytoplasmic shuttling of Tax, as well as its ability to activate NF-κB signaling.ResultsAdding to the complex posttranslational modification landscape of Tax, we here demonstrate that Tax also interacts with the ubiquitin-related modifier 1 (Urm1). Conjugation of Urm1 to Tax results in a redistribution of Tax to the cytoplasm and major increase in the transcription of the NF-ĸB targets Rantes and interleukin-6. Utilizing a tax-transgenic Drosophila model, we show that the Urm1-dependent subcellular targeting of Tax is evolutionary conserved, and that the presence of Urm1 is strongly correlated with the transcriptional output of Diptericin, an antimicrobial peptide and established downstream target of NF-κB in flies.ConclusionsThese data put forward Urm1 as a novel Tax modifier that modulates its oncogenic activity and hence represents a potential novel target for developing new strategies for treating ATL.