Ricardo H. Pires

Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

Background Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated. Methodology/Principal Findings We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway. Conclusions/Significance Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders.

Structure and assembly–disassembly properties of wild-type transthyretin amyloid protofibrils observed with atomic force microscopy†

Transthyretin (TTR) is an important human transport protein present in the serum and the cerebrospinal fluid. Aggregation of TTR in the form of amyloid fibrils is associated with neurodegeneration, but the mechanisms of cytotoxicity are likely to stem from the presence of intermediate assembly states. Characterization of these intermediate species is therefore essential to understand the etiology and pathogenesis of TTR‐related amyloidoses. In the present work we used atomic force microscopy to investigate the morphological features of wild‐type (WT) TTR amyloid protofibrils that appear in the early stages of aggregation. TTR protofibrils obtained by mild acidification appeared as flexible filaments with variable length and were able to bind amyloid markers (thioflavin T and Congo red). Surface topology and contour‐length distribution displayed a periodic pattern of ∼15 nm, suggesting that the protofibrils assemble via an end‐binding oligomer fusion mechanism. The average height and periodic substructure found in protofibrils is compatible with the double‐helical model of the TTR amyloid protofilament. Over time protofibrils aggregated into bundles and did not form mature amyloid‐like fibrils. Unlike amyloid fibrils that are typically stable under physiological conditions, the bundles dissociated into component protofibrils with axially compacted and radially dilated structure when exposed to phosphate‐buffered saline solution. Thus, WT TTR can form metastable filamentous aggregates that may represent an important transient state along the pathway towards the formation of cytotoxic TTR species. Copyright

Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates

Homologous recombination (HR) is a key process in the repair of double‐stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Blooms syndrome RecQ‐family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR‐intermediate DNA structures, by using single‐molecule visualization (electron microscopic and atomic force microscopic single‐particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full‐length BLM, we used a previously characterized truncated construct (BLM642–1290) as a monomeric control. Contrary to previous models proposing a ring‐forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single‐stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.—Gyimesi, M., Pires, R.H., Billington, N., Sarlós, K., Kocsis, Z.S., Módos, K., Kellermayer, M. S. Z., Kovács, M., Visualization of human Blooms syndrome helicase molecules bound to homologous recombination intermediates. FASEB J. 27, 4954–4964 (2013). www.fasebj.org

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ, in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.

Single-Molecule Studies of Amyloidogenic Proteins

Single-molecule biophysical tools have developed in the past two decades from jaw-dropping attractions to essential laboratory tools. In recent years, these methods have been applied to the exploration of the structure, mechanics, and mechanically driven conformational changes of amyloidogenic protein systems as well. Amyloidogenic proteins are a rich and diverse group of molecules capable of forming amyloid fibrils, and many of them are implicated in severe degenerative diseases. Because many of the diseases cause not only health problems but societal burden as well, experimental research aiming at obtaining quicker and more precise diagnosis, a better understanding of the molecular mechanisms, and more efficient therapies has ever been intensifying. In spite of seeing greater structural detail in amyloid fibrils, the intrafibrillar dynamics, the nature of the structural changes related to the amyloidogenic transition, and the role that mechanical properties might play are still not understood well. Single-molecule methods offer a unique insight into the behavior of amyloidogenic proteins and amyloid fibrils. This chapter focuses primarily on single-molecule mechanics approaches. Single-molecule methods are described and the adaptation of the techniques in the investigation of a number of amyloidogenic systems is discussed.