Yeast Models of Prion-Like Proteins That Cause Amyotrophic Lateral Sclerosis Reveal Pathogenic Mechanisms

Many proteins involved in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) are remarkably similar to proteins that form prions in the yeast Saccharomyces cerevisiae. These ALS-associated proteins are not orthologs of yeast prion proteins, but are similar in having long, intrinsically disordered domains that are rich in hydrophilic amino acids. These so-called prion-like domains are particularly aggregation-prone and are hypothesized to participate in the mislocalization and misfolding processes that occur in the motor neurons of ALS patients. Methods developed for characterizing yeast prions have been adapted to studying ALS-linked proteins containing prion-like domains. These yeast models have yielded major discoveries, including identification of new ALS genetic risk factors, new ALS-causing gene mutations and insights into how disease mutations enhance protein aggregation

The Role of Post-Translational Modifications on Prion-Like Aggregation and Liquid-Phase Separation of FUS

Subcellular mislocalization and aggregation of the human FUS protein occurs in neurons of patients with subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. FUS is one of several RNA-binding proteins that can functionally self-associate into distinct liquid-phase droplet structures. It is postulated that aberrant interactions within the dense phase-separated state can potentiate FUS’s transition into solid prion-like aggregates that cause disease. FUS is post-translationally modified at numerous positions, which affect both its localization and aggregation propensity. These modifications may influence FUS-linked pathology and serve as therapeutic targets

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

<div><p>Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.</p></div

Cellular proteins trapped with Htt polyQ aggregates are disproportionately composed of long intrinsically-disordered (ID) domains.

<p>(A) Comparisons of the percentages of proteins with long ID domains of the 52 yeast proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.t001" target="_blank">Table 1</a> (reproducibly found by TAPI to be tightly associated with Htt-Q103-GFP aggregates) versus 100 randomly-selected yeast proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.s002" target="_blank">S1 File</a>). Most of the identified proteins have long ID domains of at least 100 amino acids. (B) Comparisons of the percentages of proteins with long ID domains of the 91 rat proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.t002" target="_blank">Table 2</a> (reproducibly found by TAPI to be tightly associated with GFP-Htt-Q74 aggregates) versus 200 randomly-selected rat proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.s003" target="_blank">S2 File</a>). ID domains are defined as regions of 30 or more amino acids with IUPred scores of 0.5 or greater [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.ref047" target="_blank">47</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.ref098" target="_blank">98</a>]. Chi-Square Fisher’s Exact test (Graphpad software) was used to determine significance between TAPI-identified proteins and proteome control sets.</p

Western blotting confirms that TAPI-identified proteins are trapped in large, detergent-resistant Htt-polyQ aggregates.

<p>(A) Immunoblotting reveals that Htt-Q103-GFP, but not Htt-Q25-GFP, forms large detergent-resistant aggregates that fractionate in the pellet (partial TAPI purification; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#sec002" target="_blank">methods</a>) and remain at the top of an acrylamide gel under SDS-PAGE conditions. (B) Immunoblotting confirms that HA-tagged Bmh1p, Def1p, Ent2p, and Sgt2p (proteins identified by mass spec) get trapped in the detergent-resistant aggregates that can be seen stuck at the top of the gels in the pellet fractions. As a negative control, HA-tagged His3p (not identified by mass spec) shows no susceptibility to co-aggregation with Htt-Q103-GFP. Note that Def1p was not easily visualized in the supernatant fraction because it is prone to degradation (data not shown). Samples were spun at 45,000 rpm, except Ent2p (10,000 rpm). <b>S</b> = supernatant; <b>P</b> = pellet fraction.</p

Polyglutamine-expanded Huntingtin exon 1 forms aggregates in PC-12 cells that can be isolated by TAPI.

<p>(A) Fluorescence microscopy of PC-12 cells expressing doxycycline inducible transgene GFP-tagged Huntingtin exon 1 (Htt) with normal (Q23) or expanded polygluamine tract (Q74). (B) Western blot of GFP-Htt-Q23 and GFP-Htt-Q74 showing high molecular weight aggregates can be isolated from Htt-Q74 expressing PC-12 cells. Lysate = input; TAPI = purified aggregates.</p

Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 91) in <i>Rattus norvegicus</i>.

<p>Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 91) in <i>Rattus norvegicus</i>.</p