Protein aggregation in different disease types, associated proteins and their distinct aggregation patterns in the cell

© 2014 Dr. Saskia PollingA growing number of diseases, such as type 2 diabetes, certain cancers and many neurodegenerative disorders, have been found to incorporate a level of protein aggregation. These protein aggregation diseases are characterised by the appearance of protein aggregates in the form of deposits, which leads to the common concept that inappropriate aggregation polypeptide sequences is detrimental to cell viability. The aggresome first described the formation of a protein deposit as a cascade of cellular quality control processes that traffic dispersed misfolded proteins into a centralized location. However, since then additional observations indicate that protein quality control machinery may deliberately cluster polypeptides into distinct deposit types, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here the existence of distinct protein deposits is confirmed using three model systems of disease-relevant mutant proteins and peptide sequences. In addition, the intrinsic oligomeric state of these systems was investigated in relation to the presence of IPOD and JUNQ deposits using a sedimentation velocity analysis (SVA) paradigm. Confocal microscopy results indicated that polyglutamine models formed an IPOD, whereas the other models polyalanine and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into a JUNQ deposit. Co-expression studies indicated that the JUNQ and IPOD deposits are spatially distinct structures. More detailed insight in aggregate formation inside the complex cellular context, rather than in simple in vitro system, was provided through novel application of flow cytometry pulse-shape analysis (PulSA) and SVA on these model systems. This approach allowed the characterization of the oligomeric state of proteins inside cell lysate, an environment too complex for many of the techniques used for detailed in vitro characterization. Cells containing deposits were separated from those without using PulSA on a fluorescence-activated cell sorter (FACS), after which SVA studies revealed that while poly(72)glutamine (Q72) was almost entirely monomeric until deposits formed, SOD1 mutants and poly(37)alanine (A37) displayed altered oligomeric states with respect to their non-aggregating counterparts regardless of the presence of protein deposits. This difference in altered oligomeric state was further supported by FRET experiments on A37. In addition, this state was observed as an intrinsic property of A37 in vitro. Mutations leading to JUNQ deposits are proposed to induce a constitutive “misfolded” state, possibly exposing hydrophobic side-chains that attract and eventually overwhelm the protein quality control capacity. When protein quality control is overwhelmed proteins finally accumulate into JUNQ deposits. PolyQ-expanded proteins feature polar side-chains and as such are not “misfolded” in this same sense, but instead are highly prone to forming fibrils and are proposed to differently engage with protein quality control. Understanding the basic knowledge of protein aggregation and deposit formation in the cell will enable us to target therapeutics more effectively, since current models are incomplete

Misfolded polyglutamine, polyalanine, and superoxide dismutase 1 aggregate via distinct pathways in the cell

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively misfolded state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control

Misfolded polyglutamine, polyalanine, and superoxide dismutase 1 aggregate via distinct pathways in the cell

© 2014 by The American Society for Biochemistry and Molecular BiologyThe research outputs in this collection have been funded in whole or in part by the National Health and Medical Research Council (NHMRC).Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate selfaggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion sub-types, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis (SVA). Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the non-aggregating forms, regardless of whether cells had inclusions or not; whereas 72Q was almost exclusively monomeric until inclusions formed. We propose mutations leading to JUNQ inclusions induce a constitutively "misfolded" state exposing hydrophobic sidechains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. PolyQ is not "misfolded" in this same sense due to universal polar sidechains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.10.1074/jbc.M113.52018

Tracking protein aggregation and mislocalization in cells with flow cytometry

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies

Polyalanine expansions drive a shift into alpha-helical clusters without amyloid-fibril formation

Published online 16 November 2015Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form β-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial. We show here that aggregation of polyAla is mechanistically dissimilar to that of polyGln and hence does not exhibit amyloid kinetics. PolyAla assembled spontaneously into α-helical clusters with diverse oligomeric states. Such clustering was pervasive in cells irrespective of visible aggregate formation, and it disrupted the normal physiological oligomeric state of two human proteins natively containing polyAla: ARX and SOX3. This self-assembly pattern indicates that polyAla expansions chronically disrupt protein behavior by imposing a deranged oligomeric status.Saskia Polling, Angelique R Ormsby, Rebecca J Wood, Kristie Lee, Cheryl Shoubridge, James N Hughes, Paul Q Thomas, Michael D W Griffin, Andrew F Hill, Quill Bowden, Till Böcking and Danny M Hatter