Amyloid cannot resist identification

Abstract

PersPective 464Prion volume 7 issue 6 T he capacity to polymerize into amyloid fibrils is common to many proteins. While some proteins naturally form these fibrils to serve functional roles, amyloid is usually associated with pathogenic processes in which specific proteins aberrantly aggregate within cells or tissues. Though the contribution of amyloid fibrils to actual disease pathogenesis is not always clear, one possibility is that the titration of essential proteins from solution into aggregates contributes to the cellular degeneration common to many amyloid diseases. Using mammalian and yeast model systems, we recently showed that the common biophysical properties of amyloid aggregatesincluding strong resistance to dissolution-enable stringent purification and identification of both amyloid-forming and amyloid-associated proteins directly from cells. Strikingly, many proteins that were previously implicated in formation or clearance of intracellular aggregates, including several stress granule components, were found to co-aggregate with amyloid formed by a polyglutamineexpanded huntingtin fragment. This direct evaluation of proteins within aggregates can help identify new amyloid-forming proteins, as well as proteins that can indirectly contribute to disease mechanisms.

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

“…As observed previously, HttQ25-GFP is soluble during expression, whereas HttQ103-GFP forms toxic cytoplasmic SDS-resistant aggregates [ 35 ] ( Fig 1A ) that have amyloid-like tinctorial properties and can be trapped at the top of an SDS acrylamide gel [ 9 ] ( Fig 1B ; S1B Fig ). Proteins that are specifically associated with Htt amyloid-like aggregates were isolated using the TAPI method [ 9 , 28 ], which traps the large detergent-resistant species in acrylamide gel matrix for subsequent extraction and identification. As demonstrated in Fig 1B , the TAPI method isolates amyloid-like aggregates of HttQ103-GFP, whereas the non-aggregate-forming HttQ25-GFP does not form species large enough, or sufficiently detergent-resistant, for isolation.…”

“…Previous studies suggest that RBPs may localize to aggregates because RNA co-aggregates with amyloid-forming proteins [ 55 ]. However the TAPI method involves extensive RNase treatment [ 9 , 28 ]; while we cannot conclude that RNA is completely absent, the vast majority of RNA is eliminated prior to aggregate isolation ( S1B Fig ). As most RNA-dependent interactions should be lost, a preponderance of RBPs in the HttQ103-GFP aggregate is likely independent of RNA-mediated interactions.…”

“…Also, antibody-based screening is limited to a predetermined set of functioning antibodies and thus may overlook unexpected interactions. Our mass spectrometry-coupled approach, called TAPI, is specific to the most chemically-resistant forms of protein aggregates and eliminates the need to excise individual bands from acrylamide gels [ 9 , 28 ]. The high stringency of TAPI—due to DNase, RNase, and detergent treatment along with SDS-gel electrophoresis of aggregates—minimizes the identification of non-specific and loosely-associated proteins.…”

“…Recently, we established a mass spectrometry-based approach for non-targeted identification of intracellular amyloid-forming and amyloid-associated proteins [ 9 , 28 ]. Our method, called TAPI ( T echnique for A myloid P urification and I dentification), exploits the biophysical characteristics of amyloid, namely detergent resistance and high molecular weight, to isolate amyloid aggregates from cell lysates.…”

Hepatic Expression of Serum Amyloid A1 Is Induced by Traumatic Brain Injury and Modulated by Telmisartan

“…*P < 0.05, **P < 0.005, and ***P < 0.001. ajp.amjpathol.org -The American Journal of Pathology characteristic that distinguishes amyloid aggregates from other protein complexes. 54 We did not detect highmolecular-weight SDS-resistant species of SAA1; instead, we only observed accumulation of 30-kDa SAA1 species that might represent SAA1 dimers or trimers ( Figure 4A). These species were equally present in preparations from control and post-CCI mice.…”