Cyclosporin-A-induced prion protein aggresomes are dynamic quality-control cellular compartments

Abstract

SummaryDespite the activity of cellular quality-control mechanisms, subsets of mature and newly synthesized polypeptides fail to fold properly and form insoluble aggregates. In some cases, protein aggregation leads to the development of human neurodegenerative maladies, including Alzheimer's and prion diseases. Aggregates of misfolded prion protein (PrP), which appear in cells after exposure to the drug cyclosporin A (CsA), and disease-linked PrP mutants have been found to accumulate in juxtanuclear deposition sites termed 'aggresomes'. Recently, it was shown that cells can contain at least two types of deposition sites for misfolded proteins: a dynamic quality-control compartment, which was termed 'JUNQ', and a site for terminally aggregated proteins called 'IPOD'. Here, we show that CsA-induced PrP aggresomes are dynamic structures that form despite intact proteasome activity, recruit chaperones and dynamically exchange PrP molecules with the cytosol. These findings define the CsA-PrP aggresome as a JUNQ-like dynamic qualitycontrol compartment that mediates the refolding or degradation of misfolded proteins. Together, our data suggest that the formation of PrP aggresomes protects cells from proteotoxic stress.

Protein Quality Control in Health and Disease

“…We fused PrP and yellow fluorescent protein (PrP-YFP), induced the formation of PrP-containing aggresomes, and followed the dynamics of the chimeric, fluorescently tagged protein aggregates within these deposits. Using live-imaging techniques, we found that PrP-YFP molecules are highly mobile within the aggresomes, and these structures exhibit a high rate of molecular exchange with the cytosol (Ben-Gedalya et al 2011). Yet, although these indirect observations support the notion that the aggregate deposition sites are protective entities, it was required to directly test the relationships between these sites and cell survival.…”

“…3). They both are located proximal to the nucleus, contain highly mobile protein aggregates, exhibit a high rate of molecular exchange with the cytosol, and recruit molecular chaperones and proteasomes (Garcia-Mata et al 1999;Ben-Gedalya et al 2011). Interestingly, vimentin fibers are also important in the regulation of JUNQ and were found to control an asymmetric inheritance of JUNQ in mammalian cells (Ogrodnik et al 2014).…”

Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin

“…Misfolded proteins with differing properties do not always localize to the same IB and require different signals for triage between degradation, aggregation, and autophagy pathways (21). In particular, in recent work, we examined the JUNQ IB, which colocalizes with Hsp70 and sHsps and contains proteasomes (15,22). Coexpressing a toxic aggregation species [e.g., fALSassociated protein Superoxide Dismutase (SOD1G93A mutant)] decreased the mobility of soluble misfolded proteins, such as von Hippen-Lindau (VHL) protein, Ubc9ts, or luciferase in the JUNQ.…”

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

“…Confirm correct DNA sequence and ensure that the GFP moiety is in frame with the protein of interest. Perform sequencing using standard recombinant DNA strategies 21 . NOTE: Many institutions have core DNA facilities, which the investigator is encouraged to contact for additional information.…”

“…The glycosylphosphatidylinositol (GPI) anchored protein PrP 20 serves as an excellent example. A construct containing the yellow fluorescent protein (YFP) attached to the amino terminus of PrP (YFP-PrP) 21,22 , a generous gift from Dr. Ehud Cohen, from the Hebrew University of Jerusalem. Prior to protease treatment, the fluorescent signal from the extracellular YFP was bright (Figure 4).…”