A fluorescence based sensor assay that monitors general protein aggregation in human cells

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ-peptide. Exposure to proteome destabilizers lead to re-localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide-genetic screens to identify genes and pathways involved in protein misfolding and aggregation.publishe

Amyloidogenic cross-seeding of Tau protein: Transient emergence of structural variants of fibrils

<div><p>Amyloid aggregates of Tau protein have been implicated in etiology of many neurodegenerative disorders including Alzheimer's disease (AD). When amyloid growth is induced by seeding with preformed fibrils assembled from the same protein, structural characteristics of the seed are usually imprinted in daughter generations of fibrils. This so-called conformational memory effect may be compromised when the seeding involves proteins with non-identical sequences leading to the emergence of distinct structural variants of fibrils (amyloid ‘<i>strains</i>’). Here, we investigate cross-seeding of full-length human Tau (FL Tau) with fibrils assembled from K18 and K18ΔK280 fragments of Tau in the presence of poly-L-glutamate (poly-Glu) as an enhancer of Tau aggregation. To study cross-seeding between Tau polypeptides and the role of the conformational memory effect in induction of Tau amyloid polymorphism, kinetic assays, transmission electron microscopy, infrared spectroscopy and limited proteolysis have been employed. The fastest fibrillization was observed for FL Tau monomers seeded with preformed K18 amyloid yielding daughter fibrils with unique trypsin digestion patterns. Morphological features of daughter FL Tau fibrils induced by K18 and K18ΔK280 seeds were reminiscent of the mother fibrils (i.e. straight paired fibrils and paired helical filaments (PHFs), respectively) but disappeared in the following generations which became similar to unpaired FL Tau amyloid fibrils formed <i>de novo</i>. The structural evolution observed in our study was accompanied by disappearance of the unique proteolysis profile originated from K18. Our findings may have implications for understanding molecular mechanisms of the emergence and stability of Tau amyloid strains.</p></div

Limited proteolysis of G1, G2 and G3 generations of Tau fibrils by trypsin.

<p>SDS-PAGE analysis showing proteolytic patterns of enzymatically digested fibrils (formed during 72-h fibrillization). The lanes correspond to products of trypsin-digested G1..G3 daughter fibrils of FL Tau induced by homologous seeding (FL Tau generations) and cross-seeding (K18 / K18ΔK280 generations). M lane shows molecular weight marker.</p

Formation of G1, G2 and G3 generations of Tau amyloid fibrils.

<p>(A) A scheme of preparation of mother (G0) <i>de novo</i> formed fibrils from monomers of FL Tau, K18 and K18ΔK280, and daughter fibril generations of FL Tau (G1, G2 and G3) obtained through seeding of FL Tau monomers with various templates. Kinetics of the self-assembly of G1 (B), G2 (C), and G3 (D) fibrils probed by ThT fluorescence. Insets show the same plots with magnified intensity scales.</p

TEM images of G0, G1, G2 and G3 generations of Tau amyloid fibrils.

<p>Red arrowheads indicate morphological variations in the fibrils: paired straight structures formed by K18 and propagated on to G1 and G2 generations of FL Tau fibrils (panels B, E, H), or PHF-like structures formed by K18ΔK280 and propagated on to FL Tau fibrils (panels C, F). Representative images were chosen. Scale bar of 200 nm applies to all micrographs.</p