Árpád Karsai

Stepwise dynamics of epitaxially growing single amyloid fibrils

The assembly mechanisms of amyloid fibrils, tissue deposits in a variety of degenerative diseases, is poorly understood. With a simply modified application of the atomic force microscope, we monitored the growth, on mica surface, of individual fibrils of the amyloid β25–35 peptide with near-subunit spatial and subsecond temporal resolution. Fibril assembly was polarized and discontinuous. Bursts of rapid (up to 300-nm−1) growth phases that extended the fibril by ≈7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid β1–42 fibrils growing on graphite, suggesting that the discontinuous assembly mechanisms may be a general feature of epitaxial amyloid growth. Amyloid assembly may thus involve fluctuation between a fast-growing and a blocked state in which the fibril is kinetically trapped because of intrinsic structural features. The used scanning-force kymography method may be adapted to analyze the assembly dynamics of a wide range of linear biopolymers.

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.

Oriented epitaxial growth of amyloid fibrils of the N27C mutant β25–35 peptide

Amyloid fibrils are present in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. Amyloid fibrils may be used in nanotechnology applications, because of their self-assembly properties and stability, if their growth and orientation can be controlled. Recently, we have shown that amyloid β25–35 (Aβ25–35) forms a highly oriented, K+-dependent network on mica. Here, we analyzed the properties of Aβ25–35_N27C, the cysteine residue of which may be used for subsequent chemical modifications. We find that Aβ25–35_N27C forms epitaxially growing fibrils on mica, which evolve into a trigonally oriented branched network. The binding is apparently more sensitive to cation concentration than that of the wild-type peptide. By nanomanipulating Aβ25–35_N27C fibrils with a gold-coated AFM tip, we show that the sulfhydryl of Cys27 is reactive and accessible from the solution. The oriented network of Aβ25–35_N27C fibrils can therefore be specifically labeled and may be used for constructing nanobiotechnological devices.

Lithium induces phosphoglucomutase activity in various tissues of rats and in bipolar patients

Phosphoglucomutase catalyses the reversible conversion of glucose-6-P and glucose-1-P. Lithium is a potent inhibitor of phosphoglucomutase in vitro, however, it is not known if phosphoglucomutase was significantly inhibited by Li+ in Li+-treated bipolar patients. Here, we demonstrate that phosphoglucomutase inhibition by chronic Li+ treatment causes alterations of glucose-phosphate levels in various tissues of rats. Also, phosphoglucomutase inhibition results in compensatory elevation of phosphoglucomutase activity in rat tissues and in leukocytes isolated from Li+-treated bipolar patients. The increase of uninhibited phosphoglucomutase activity in leukocytes of Li+-treated bipolar patients is due to the increased expression of the PGM1 gene.

Thermally-Induced Effects in Oriented Network of Amyloid β25–35 fibrils

Amyloid fibrils are filamentous protein deposits in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. They may be used in nanotechnology applications because of their self-assembly properties and stability. Recently we have shown that amyloid beta 25–35 (Aβ25–35) forms a highly oriented, K+-dependent network on mica, and its mutant form (Aβ25–35_N27C) may be chemically addressed for functionalization in dedicated applications. In the present work we investigated thermally-induced changes in the morphology of the oriented Aβ25–35 fibril network. The fibrils maintained a high orientation stability in the temperature range of 30–70 °C, suggesting that orientational rearrangement of Aβ25–35 fibrils on mica is an unfavorable process. Above ∼45 °C a gradual decrease in fibril length and dissociation from the surface could be observed. Furthermore, at high temperatures (45–70 °C) the average fibril thickness increased, indicating changes in the underlying structure or structural dynamics. Possibly, a thermally induced transition in the Aβ25–35 peptide around 45 °C leads to structural changes in the fibril as well. The temperature-dependent changes need to be considered in the use of amyloid fibrils in nanotechnology applications.