Ubiquitin-mediated sequestration of normal cellular proteins into polyglutamine aggregates

Abstract

A hallmark of most neurodegenerative diseases, including those caused by polyglutamine expansion, is the formation of ubiquitin (Ub)-positive protein aggregates in affected neurons. This finding suggests that the Ub system may be involved in common mechanisms underlying these otherwise unrelated diseases. Here we report the finding of ataxin-3 (Atx-3), whose mutation is implicated in the neurodegenerative disease spinocerebellar ataxia type 3, in a bioinformatics search of the human genome for components of the Ub system. We show that wild-type Atx-3 is a Ub-binding protein and that the interaction of Atx-3 with Ub is mediated by motifs homologous to those found in a proteasome subunit. Both wild-type Atx-3 and the otherwise unrelated Ub-binding protein p62/Sequestosome-1 have been shown to be sequestered into aggregates in affected neurons in several neurodegenerative diseases, but the mechanism for this recruitment has remained unclear. In this article, we show that functional Ub-binding motifs in Atx-3 and p62 proteins are required for the localization of both proteins into aggregates in a cell-based assay that recapitulates several features of polyglutamine disease. We propose that the Ub-mediated sequestration of essential Ub-binding protein(s) into aggregates may be a common mechanism contributing to the pathogenesis of neurodegenerative diseases.

Fig. 1.

The PUB motifs of the proteasome subunit Rpn10p/S5a and PUBH motifs found in otherwise unrelated proteins. (A) Alignment of the PUB motif found in Rpn10p homologues. Conserved amino acids or side chain features are indicated and colored as follows: blue, very high conservation (>90%); orange, high conservation (65–90%); φ, conserved hydrophobicity. Lowercase letters indicate less frequently occurring residues that nonetheless preserve a side chain feature. (B) Alignment of PUBH motifs from selected human proteins. Numbers to the left indicate the position of the first amino acid in the sequence. Legend is as in A. APC5 is a subunit of the Ub ligase APC. MEKK1 is a protein serine/threonine kinase that also exhibits Ub ligase activity. KIAA1578 and KIAA0794 have other domains that are associated with the Ub system (see text and ref. 9). Conserved PUBH/UIM residues are indicated as in ref .

Fig. 2.

UIM/PUBH motifs mediate noncovalent binding of Atx-3 to Ub chains in vitro.(A) Alignment of UIM/PUBH motifs present in Atx-3 orthologues. Numbers indicate the position of the first amino acid. Conserved amino acids or side chain features are indicated as in Fig. 1. *, The Ser residue that is conserved in PUBH motifs and that was mutated in this study (below). (B) Domain structure of Atx-3. Red boxes represent PUBH motifs and the polyQ tract is blue. Numbers indicate amino acid positions. (C) Atx-3 binding to Ub chains. Effect of truncations and of PUBH motif mutations. N-terminal His₈-tagged Atx-3 protein, its fragments, or a triple PUBH mutant (S236A/S256A/S359A) were used to pull down Ub chains. The equivalent of 20% of Ub chain input is shown in lane 5. Pull-down products were revealed by immunoblot with Abs against Ub. The number of Ub units in the chain mixture is indicated by the subscript in Ub_n.(D) Atx-3 PUBH motifs required for interaction with Ub chains. His-tagged wild-type or mutant Atx-3 (Upper) were used. Legend is as in C.

Fig. 3.

UIM/PUBH motifs mediate noncovalent binding of Atx-3 to Ub chains in vivo.(A) PUBH motif-dependent interaction of Atx-3 and Ub chains in vivo. Cells were transfected with the indicated plasmids encoding N-terminal (FLAG)₃-tagged Atx-3 proteins. Lysates were subjected to immunoprecipitation with Ab to the FLAG epitope. Immunoprecipitated proteins were blotted and reacted with Ab to Ub (Upper) or to the FLAG epitope (Lower). (B) Noncovalent interaction of Atx-3 and Ub chains in vivo. Cells were transfected with empty vector or vector encoding FLAG-tagged wild-type Atx-3. Lysates made in Nonidet P-40 or RIPA buffers, as indicated, were subjected to immunoprecipitation with Ab to the FLAG epitope. Immunoprecipitated proteins were blotted and reacted with Ab to Ub (Upper) or to the FLAG epitope (Lower).

Fig. 4.

PUBH motif-dependent recruitment of Atx-3 to polyQ aggregates. HEK293 cells were transfected with constructs encoding GFP-Atx-3 fusions and/or a CFP fusion with expanded polyQ (CFP-Q78), as indicated. The GFP-Atx-3 S236A/S256A/S359A mutant is in the context of the full-length protein (1–373). Imaging was done with filters for GFP (Left and Right) or CFP (Center).

Fig. 5.

Ub-binding-motif-dependent recruitment of p62/Sequestosome-1 to polyQ aggregates. HEK293 cells were transfected with constructs encoding GFP-p62 fusions and/or a CFP fusion with expanded polyQ (CFP-Q78), as indicated. Imaging was done with filters for GFP (Left and Right) or CFP (Center).