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

The ID domains of Sgt2p and Fus mediate their localization to Htt-polyQ aggregates in yeast cells.

<p>(A, B) Western blots of lysates from yeast strain W303 expressing HttQ25-GFP or HttQ103-GFP in combination with HA-tagged Sgt2p or Sgt2ΔID (A = αGFP; B = αHA). (C, D) Western blots of cells expressing HttQ25-GFP or HttQ103-GFP in combination with FUS or FUSΔID (C = αGFP; D = FUS & α-β actin). Because FUS is quickly degraded in non-denaturing conditions, input controls using urea lysis of cells were included to show initial protein loads. (E, F) Western blots of FUS or FUS(4FL) in HttQ25-GFP-expressing or HttQ103-GFP-expressing cells.</p

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

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

Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 52) in <i>Saccharomyces cerevisiae</i>.

<p>Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 52) in <i>Saccharomyces cerevisiae</i>.</p

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

<p>(A) Fluorescence microscopy of yeast expressing GFP-tagged Huntingtin exon 1 (Htt) with a normal (Q25) or an expanded polyglutamine tract (Q103). (B) Western blot of GFP-Htt-Q25 and GFP-Htt-Q103 showing that high molecular weight aggregates can be isolated from Htt-Q103 expressing yeast cells. Lysate = input; TAPI = purified aggregate.</p

Confirmation of polyQ-associated proteins from PC-12 cells identified by TAPI.

<p>(A) Western blotting shows that the addition of doxycycline to the PC-12 cell model induces the expression of HttQ74-GFP, resulting in aggregates that can be purified by TAPI. The kinase ERK is probed as a negative control; ERK was never identified by mass spectrometry, so is not expected to co-fractionate with polyQ aggregates. (B) Western blot analysis of TAPI-purified polyQ aggregates from PC-12 cells confirms the presence of several disease-associated proteins only in the Htt-Q74 samples. All proteins migrated near their predicted molecular weights. For control, the TAPI procedure was conducted in parallel on the induced Htt-Q23 cell line (FUS, TDP-43, UBQLN2, HNRNPA1) or the un-induced Htt-Q74 cell line (CLINT1, HSPA8, RAD23B, SGTA). (C) Confocal microscopy shows localization of identified proteins to Htt-Q74 aggregates in PC12 cells. (left) RAD23B, nominally a DNA repair protein, localizes to nuclear Htt-Q74 inclusions but not cytoplasmic inclusions. (middle) FUS, an RNA-binding protein localizes to nuclear and cytoplasmic Htt-Q74 inclusions. (right) CLINT1, a clatherin-interacting protein, is observed in cytoplasmic Htt-Q74 aggregates. Arrows indicate foci with co-localized proteins. Green = GFP; Magenta = CLINT1, FUS or RAD23B in merge.</p

Htt-polyQ aggregate-associated proteins found in inclusions and aggregates in various neurodegenerative diseases.

<p>Htt-polyQ aggregate-associated proteins found in inclusions and aggregates in various neurodegenerative diseases.</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