The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-ß peptide fibrils and inhibits secondary nucleation.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD ß-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first ß-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-ß (Aß42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aß, the CTD constructs instead bind to Aß42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two ß-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

Retardation of Aß42 fibril formation by apolipoprotein A-I and recombinant HDL particles.

The double nucleation mechanism of amyloid ß (Aß) peptide aggregation is retained from buffer to cerebrospinal fluid (CSF) but with reduced rate of all microscopic processes. Here, we used a bottom-up approach to identify retarding factors in CSF. We investigated the Aß42 fibril formation as a function of time in the absence and presence of apolipoprotein A-I (ApoA-I), recombinant high-density lipoprotein (rHDL) particles, or lipid vesicles. A retardation was observed in the presence of ApoA-I or rHDL particles, most pronounced with ApoA-I, but not with lipid vesicles. Global kinetic analysis implies that rHDL interferes with secondary nucleation. The effect of ApoA-I could best be described as an interference with secondary and to a smaller extent primary nucleation. Using surface plasmon resonance and microfluidics diffusional sizing analyses, we find that both rHDL and ApoA-I interact with Aß42 fibrils but not Aß42 monomer, thus the effect on kinetics seems to involve interference with the catalytic surface for secondary nucleation. The Aß42 fibrils were imaged using cryogenic-electron microscopy and found to be longer when formed in the presence of ApoA-I or rHDL, compared to formation in buffer. A retarding effect, as observed in CSF, could be replicated using a simpler system, from key components present in CSF but purified from a CSF-free host. However, the effect of CSF is stronger implying the presence of additional retarding factors.

On the Aggregation of Apolipoprotein A-I.

In vivo, apolipoprotein A-I (ApoA-I) is commonly found together with lipids in so-called lipoprotein particles. The protein has also been associated with several diseases-such as atherosclerosis and amyloidosis-where insoluble aggregates containing ApoA-I are deposited in various organs or arteries. The deposited ApoA-I has been found in the form of amyloid fibrils, suggesting that amyloid formation may be involved in the development of these diseases. In the present study we investigated ApoA-I aggregation into amyloid fibrils and other aggregate morphologies. We studied the aggregation of wildtype ApoA-I as well as a disease-associated mutant, ApoA-I K107Δ, under different solution conditions. The aggregation was followed using thioflavin T fluorescence intensity. For selected samples the aggregates formed were characterized in terms of size, secondary structure content, and morphology using circular dichroism spectroscopy, dynamic light scattering, atomic force microscopy and cryo transmission electron microscopy. We find that ApoA-I may form globular protein-only condensates, in which the α-helical conformation of the protein is retained. The protein in its unmodified form appears resistant to amyloid formation; however, the conversion into amyloid fibrils rich in ß-sheet is facilitated by oxidation or mutation. In particular, the K107Δ mutant shows higher amyloid formation propensity, and the end state appears to be a co-existence of ß-sheet rich amyloid fibrils and α-helix-rich condensates.

Purification and HDL-like particle formation of apolipoprotein A-I after co-expression with the EDDIE mutant of Npro autoprotease.

Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in E. coli. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus intact. ApoA-I was then purified using a five-step procedure composed of anion exchange chromatography, immobilized metal ion affinity chromatography, hydrophobic interaction chromatography, boiling and size exclusion chromatography. This led to protein of high purity as confirmed with SDS-PAGE and mass spectrometry. The purified ApoA-I formed discoidal objects in the presence of zwitterionic phospholipid DMPC, showing its retained function of interacting with lipids. The protocol was also tested by expression and purification of two ApoA-I mutants, both of which could be purified in the same manner as the wildtype, showing the robustness of the protocol.

Autocatalytic amplification of Alzheimer-associated Aß42 peptide aggregation in human cerebrospinal fluid.

Alzheimer's disease is linked to amyloid ß (Aß) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aß aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aß42 in human CSF through kinetic experiments at several Aß42 monomer concentrations (0.8-10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF.