The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity

Barrington Burnett 0 Fusheng Li 0 Randall N. Pittman 0 0 Department of Pharmacology, University of Pennsylvania School of Medicine , Philadelphia, PA 19104-6084 , USA The ubiquitin-proteasome pathway is critically involved in the pathology of neurodegenerative diseases characterized by protein misfolding and aggregation. Data in the present study suggest that the polyglutamine neurodegenerative disease protein, ataxin-3 (AT3), functions in the ubiquitin-proteasome pathway. AT3 contains an ubiquitin interaction motif (UIM) domain that binds polyubiquitylated proteins with a strong preference for chains containing four or more ubiquitins. Mutating the conserved leucine in the first UIM (L229A) almost totally eliminates binding to polyubiquitin chains while a similar mutation in the second UIM (L249A) also inhibits binding to polyubiquitin chains but to a lesser extent. Both wild-type and pathological AT3 increase cellular levels of a short-lived GFP that is degraded by the ubiquitin-proteasome pathway. AT3 has several properties characteristic of ubiquitin proteases including decreasing polyubiquitylation of 125I-lysozyme by removing ubiquitin from polyubiquitin chains, cleaving a ubiquitin protease substrate, and binding the specific ubiquitin protease inhibitor, ubiquitin-aldehyde. Mutating the predicted catalytic cysteine in AT3 inhibits each of these ubiquitin protease activities. The ability to bind and cleave ubiquitylated proteins is consistent with AT3 playing a role in the ubiquitin-proteasome system. This raises the possibility that pathological AT3, which tends to misfold and aggregate, may be exposed to aggregateprone misfolded/denatured proteins as part of its normal function. - INTRODUCTION Spinocerebellar ataxia type-3 (SCA3) is the most common dominantly inherited cerebellar ataxia and a member of the polyglutamine neurodegenerative disease family (14). As is the case with other members of this family, the protein mutated in SCA3, ataxin-3 (AT3), causes pathology through an apparent gain of function associated with expansion of a CAG repeat that codes for a polyglutamine domain within the protein. Other diseases in this family include Huntingtons, dentatorubral pallidoluysian atrophy, spinal and bulbar muscular atrophy, and SCA1, 2, 6 and 7. A prominent feature in these diseases is the presence of nuclear, and in some cases cytoplasmic inclusions of aggregated pathological protein (58). Typically, inclusions are highly ubiquitylated and in many cases proteasomes are associated with inclusions which suggests an attempt to degrade the aggregated proteins (5,6,911). Targeting a protein for proteasome degradation is a multistep iterative process involving an E1 activating enzyme, E2 ubiquitin carrier/conjugating enzymes and E3 ubiquitin ligases which generate polyubiquitin chains linked to the e-amino group of lysine(s) in the protein targeted for degradation (12). Editing and disassembly of polyubiquitin chains as well as recycling ubiquitin is critical for cellular homeostasis. These functions are carried out by cysteine proteases known as deubiquitinating enzymes (DUBs) (13,14). The two major families of DUBs are ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (USPs). UCHs are wellconserved proteases that prefer cleaving small leaving groups from the C-terminus of ubiquitin and primarily function in maintaining high levels of free ubiquitin in cells. USPs are a large highly divergent family of proteases with substrate specificities ranging from very general deubiquitinating properties to highly specific cleavage of ubiquitin from a single or small number of protein targets; USPs primarily function in ubiquitin chain editing and disassembly (13,14). DUBs as well as other regulators of the ubiquitin proteasome pathway (UPP) have been linked to neurodegenerative diseases that are characterized by protein misfolding and aggregation. In some cases mutations or polymorphisms in regulators of the UPP are responsible for inherited forms of the disease or modulate the disease phenotype. For instance, mutation of Parkin, an E3 ubiquitin ligase, results in early onset Parkinsons disease, and a polymorphism resulting in reduced ligase activity of the bifunctional DUB, UCH-L1 (15), is associated with decreased susceptibility to Parkinsons disease (16,17). This same polymorphism in UCH-L1 has also been linked to the age of onset in Huntingtons disease (18). Mutations in UCH-L3, as well as mutations in ubiquitin and ubiquitin ligases enhance degeneration in a Drosophila model of SCA1 (19). These and other studies support the hypothesis that the UPP is intimately linked to neurodegenerative diseases characterized by protein misfolding and aggregation (20,21). Although misfolded polyglutamine proteins may be exposed to the UPP as part of normal protein turnover there is little data to suggest that polyglutamine proteins are exposed to the UPP as part of their normal cellular functions. If a polyglutamine protein were part of this pathway it would create a particularly dangerous situation in which the pathological protein with its destabilizing expanded polyglutamine domain (22) and tendency to aggregate (23,24) is exposed to misfolded proteins which could increase the probability of protein aggregation. Based on its sequence, AT3 may function in the UPP. Depending on the splice variant, AT3 has either two or three potential ubiquitin interaction motifs (UIMs) (25) that have recently been shown to bind mono and/or polyubiquitylated proteins (26,27). During the course of our studies to determine if AT3 binds ubiquitylated species, we found that AT3 not only binds polyubiquitylated chains and proteins but it also exhibits ubiquitin protease activity that is inhibited by mutating the predicted active site cysteine. Results AT3 binds ubiquitin chains containing four or more ubiquitins through its UIM domain The UIM is a recently identified protein motif that binds mono and/or polyubiquitylated proteins (2527). The major splice form of AT3 has two predicted UIMs located between its conserved N terminus and the polyglutamine domain (Fig. 1A). To determine if AT3 binds ubiquitylated proteins, cell lysates were incubated with MBP-AT3 and associated proteins pulled down with amylose beads. Both wild-type and pathological AT3 bind ubiquitylated cellular proteins equally whereas MBP does not bind ubiquitylated proteins (Fig. 1B). Monoubiquitylated as well as polyubiquitylated proteins are present in cell lysates; therefore, the preference of AT3 for ubiquitylated species was determined by the ability of AT3 to bind free ubiquitin, a monoubiquitin fusion protein, or polyubiquitin chains of varying lengths. Under assay conditions used, AT3 does not bind free ubiquitin, a monoubiquitin fusion protein or ubiquitin chains containing less than four ubiquitins (Fig. 1C). However, AT3 binds polyubiquitin chains containing four or more ubiquitins which is the (...truncated)