The expansion of trinucleotide CAG repeats encoding glutamines within specific cellular proteins is the cause of inherited neurodegenerative diseases termed polyglutamine disorders. Huntington’s disease, spinobulbar muscular atrophy (SBMA), dentatorubral–pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias (SCA) 1, 2, 3, 6, 7, and 17 are all caused by an abnormally expanded polyQ domain (reviewed in Cummings and Zoghbi, 2000
; McCampbell et al., 2001
; Orr and Zoghbi, 2007
). A striking neuropathological hallmark of these polyQ diseases is the presence of neuronal insoluble cytoplasmic aggregates and nuclear insoluble inclusions (NIIs) formed by mutant polyQ proteins (reviewed in Ross, 2002
). The role NIIs and cytoplasmic aggregates in the pathological processes of polyQ diseases remains contentious. While some consider the NIIs/cytoplasmic aggregates as direct toxic intermediates, some have proposed that NIIs/cytoplasmic aggregates are a point of sequestration of a toxic product and thereby beneficial to a neuron (Arrasate et al., 2004
; Ross et al., 1999
Multiple lines of evidence support the view that protein aggregation is a complex process that is initiated by the accumulation of misfolded polyQ-containing proteins into a variety of higher-order intermediate conformational assemblies that ultimately form insoluble inclusion bodies. Conformational rearrangements of these mutated proteins likely change their biological activities and contribute to their toxicities. Importantly, recent reports suggest that the toxicity of mutant polyQ-containing proteins might be not related to the insoluble inclusion bodies but rather to soluble oligomeric or other intermediate conformations (Kayed et al., 2003
). In support of this idea, polyQ-induced neurodegeneration can be prevented by pharmacological and molecular manipulations that target processes leading to oligomerization and aggregation. For instance, inhibition of polyglutamine oligomerization in a transgenic mouse model of HD has a marked protective effect on survival, weight loss, and motor function (Sanchez et al., 2003
), supporting the idea that oligomerization of expanded polyglutamine may play a pivotal role in the protein’s toxicity. Similarly, the green tea polyphenol epi-gallocatechin-3-gallate (EGCG) inhibits aggregation of polyQ Httex1 in vitro and reduces polyQ-induced cytotoxicity in yeast cells and Drosophila models of the disease (Ehrnhoefer et al., 2006
In addition to pharmacological approaches, modulations of cellular pathways that prevent aggregation also reverse neurotoxicity. Molecular chaperones are the first line of defense against protein aggregation, and numerous studies have shown that overexpression of chaperones prevents polyQ aggregation and reduces the pathology of neurodegenerative diseases (Muchowski and Wacker, 2005
). In cultured cells, the chaperones heat shock protein 40 kDa (Hsp40) and 70 kDa (Hsp70) have been found to prevent the formation of spherical and annular oligomeric structures by a polyQ Httex1 (Wacker et al., 2004
). Significantly, overexpression of Hsp70 in SCA1 transgenic mice decreases neurodegeneration and improves motor coordination (Cummings et al., 2001
). Similarly, inhibiting polyQ aggregation by overexpressing Hsp70 (Warrick et al., 1999
) and Hsp40 (Chan et al., 2000
) rescues neurodegeneration in Drosophila models of SCA3. Altogether, these findings strongly suggest that specific polyQ conformational structures confer the cellular toxicity of the mutated proteins and that the identification of novel pharmacological, molecular, or genetic means to prevent polyQ aggregation is likely to have direct impact on developing therapeutic strategies for neurodegenerative diseases such as HD and SCA3.
We have recently documented that the UL97 kinase of HCMV prevents the aggregation of viral components during infection (Prichard et al., 2008
). UL97 is a ~80-kDa tegument protein composed of 707 amino acids that is expressed during HCMV infection (Michel et al., 1998
). UL97 contains a nuclear localization signal within its N-terminal domain and is targeted to the nucleus in infected or transfected cells (Prichard et al., 2005
). UL97 is a kinase and shows homology to cellular serine/threonine kinases within conserved subdomains involved in substrate and ATP binding (Michel et al., 1998
). The kinase activity of UL97 requires the invariant lysine at position 355, and the K355M mutant is inactive (Marschall et al., 2001
). UL97 is important for viral replication, and a recombinant virus with a large deletion in UL97 replicates poorly and contains abnormal aggregates of viral proteins within the nuclei (Prichard et al., 1999
). One of the aggregated proteins is the pp65 viral tegument protein, which also forms nuclear aggregates when expressed in transfected mammalian cells. Importantly, UL97, but not the catalytically inactive UL97 K355M, prevents pp65 aggregation (Prichard et al., 2005
), suggesting that UL97 has antiaggregation activity and that the antiaggregation effect of UL97 is dependent on its kinase activity.
In addition to viral proteins, UL97 also prevents aggregation of GFP170*, a protein chimera formed by fusing an internal segment (amino acids 566–1375) of the Golgi protein GCP-170 to the C-terminus of GFP (Misumi et al., 2001
; Hicks and Machamer, 2002
; Prichard et al., 2008
). We have shown previously that GFP170* forms nuclear aggregates similar in structure to those formed by the viral pp65 and also deposits in large ribbon-like aggregates within the cytoplasm (Fu et al., 2005a
). UL97 prevents the formation of both the nuclear and the cytoplasmic GFP170* aggregates (Prichard et al., 2008
). As with pp65, the catalytically inactive UL97/K355M mutant is unable to prevent GFP170* aggregation. Thus, UL97 prevents the aggregation of both a viral and a cellular protein. Herein, we examined the possibility that UL97 may possess a general antiaggregation activity and may serve as a tool for understanding and inhibiting the mechanisms that contribute to aggregation in polyQ diseases.
We report that UL97 has a strong antiaggregation effect on non-polyQ proteins as well as polyQ-expanded proteins associated with HD and SCA3. We show that UL97 prevents the deposition of aggregates of the non-polyQ Werner protein (WRN) that causes the premature aging disease Werner syndrome. We also show that UL97 prevents aggregation of a pathogenic construct that encodes the full-length ataxin-3 containing a 72-glutamine expansion (AT3-72Q), and of a pathogenic N-terminal huntingtin domain corresponding to the exon1 of this protein and containing an expanded track of 82 glutamine residues (HttExon1-82Q). In all cases, the catalytically inactive UL97/ K355M mutant does not prevent aggregation. The similarity of the UL97 effect on the viral pp65 protein, the non-polyQ GFP170* and WRN proteins, and the polyQ AT3-72Q and HttExon1-82Q proteins suggests that UL97 has general antiaggregation effect. This similarity is also consistent with the hypothesis that aggregation of diverse proteins may occur through a common mechanism that is targeted by the UL97 kinase. In agreement, we show that UL97 disperses nuclear PML bodies and causes a decrease in p53-mediated transcription.
Our results identify UL97 as a novel means to inhibit the aggregation of polyQ proteins. They also designate UL97 as a new molecular tool to further examine the cellular mechanisms that lead to polyQ aggregation and neurodegeneration in HD and SCA3.