Evidence for Proteasome Involvement in Polyglutamine Disease: Localization to Nuclear Inclusions in SCA3/MJD and Suppression of Polyglutamine Aggregation in vitro

Chai, Yaohui; Koppenhafer, Stacia L.; Shoesmith, Sarah J.; Perez, Matthew K.; Paulson, Henry L.

doi:10.1093/hmg/8.4.673

Cited by 378 publications

(273 citation statements)

References 43 publications

Supporting

Mentioning

251

Contrasting

Order By: Relevance

“…First, we examined the role of the ubiquitin/proteasome pathway. Aggregates of several polyQ disease proteins in human neurons are ubiquitinated and contain proteasome components (26)(27)(28)40). We used three different mutant yeast strains that affect the ubiquitin/proteasome pathway at different steps.…”

Section: Discussionmentioning

confidence: 99%

“…Because aggregates of Ht (25) and other glutamine-repeat proteins associated with disease (26)(27)(28) are ubiquitinated in mammalian cells and are associated with components of the proteasome, it has been suggested that the ubiquitin/proteasome pathway might be involved in aggregate formation. We were unable to detect ubiquitinated Ht protein in yeast cells (data not shown).…”

Section: Mutant Ht Forms Cytoplasmic Aggregates In Yeastmentioning

confidence: 99%

See 1 more Smart Citation

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Krobitsch

Lindquist

2000

Proc. Natl. Acad. Sci. U.S.A.

514

438

View full text Add to dashboard Cite

P olyglutamine (polyQ) expansions in several unrelated proteins are responsible for at least eight inherited neurodegenerative diseases. These include Huntington's disease (HD), spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and spinocerebellar ataxia types 1, 2, 3, 6, and 7 (1-3). Perhaps the most baffling aspect of these diseases is that the proteins are expressed widely in brain and other tissues, yet each is toxic in a different, highly specific group of neurons and produces a distinct pathology (2).The major characteristic of HD is a selective loss of neurons in the striatum and cortex leading to movement disorders, dementia, and eventually, death (4, 5). The causative agent is a 350-kDa protein, huntingtin (Ht), with glutamine expansions in the N-terminal region (6). The toxicity of Ht in specific neurons correlates with the length of the glutamine expansion, but the mechanism of toxicity is unknown (7,8).A central event in HD is the production of an N-terminal fragment of Ht that aggregates in affected neurons during the natural progression of the disease in humans (9). In transgenic animal models an N-terminal fragment is sufficient to produce an HD-like phenotype, which also depends on the length of the Q repeat (10, 11). Aggregates are found in both the nucleus and/or the cytoplasm of affected neurons in human patients, transgenic animals, and cell lines (12). It is by no means clear, however, whether the aggregates are themselves pathogenic, simply benign byproducts (and thereby markers) of other pathogenic polyQ misfolding events, or a defense mechanism whose purpose is to reduce the interaction of toxic misfolded polyQ proteins with other proteins.Indeed, even the mechanisms underlying the aggregation of these fragments are unknown. A further complexity is that several other proteins interact with Ht. These include HAP1, HIP1, Hsp35, WW domain-containing proteins, the ubiquitin-conjugating enzyme hE2-25k, the SH3GL3 protein, cystathione ␤-synthase, and calmodulin (12)(13)(14).These problems are inherently difficult to study. Although several mammalian cell-line and transgenic-animal models exist for studying Ht, none is as readily amenable to genetic analysis as yeast. We demonstrate that the aggregation of N-terminal fragments of Ht in yeast cells depends on the length of its polyQ repeat and that this aggregation depends on the balance of chaperone protein activities in the cell. Furthermore, N-terminal fragments with up to 103 glutamines have little or no toxicity in either the aggregated or the soluble state. Thus, yeast cells should provide a valuable system for investigating general factors that affect aggregation and for determining what neuronal cell-type specific factors might inf luence toxicity. Materials and MethodsPlasmid Construction. Plasmids encoding fusions between the N-terminal region of Ht and green fluorescent protein (GFP) were the kind gift of G. Lawless (Univ. of California, Los Angeles).To create yeast expression plasmids for HtQ25 or HtQ103, DNAs were d...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Mutant Ht Forms Cytoplasmic Aggregates In Yeastmentioning

confidence: 99%

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Krobitsch

Lindquist

2000

Proc. Natl. Acad. Sci. U.S.A.

514

438

View full text Add to dashboard Cite

show abstract

“…Methods for cell culture, transfection, and IF have been described (3,5,(27)(28). In cotransfections, constructs encoding polyQ proteins or control vector were used at a molar ratio of 1:1 with pEGFP-CBP construct.…”

Section: Methodsmentioning

confidence: 99%

Live-cell imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model of pathogenesis

Chai

Shao

Miller

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

189

136

View full text Add to dashboard Cite

Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent proteintagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders.A t least nine inherited neurodegenerative diseases are caused by CAG triplet repeat expansions that encode expanded polyglutamine (polyQ) in the disease proteins (1). A unifying feature of polyQ diseases is the formation of neuronal intracellular inclusions by the disease protein, most commonly in the nuclear inclusions (NIs). Growing evidence suggests that polyQ expansion promotes protein misfolding, resulting in neuronal dysfunction and degeneration by still unknown mechanisms.Two leading, yet compatible, theories of polyQ pathogenesis are failures in protein homeostasis and perturbations in transcriptional regulation. Evidence implicating problems in protein homeostasis includes the formation of ubiquitin-positive inclusions in disease tissue, the recruitment of molecular chaperones and proteasome components to NI, and the ability of molecular chaperones to suppress toxicity in disease models (2-11). Evidence supporting transcriptional dysregulation includes expanded polyQ-induced changes in gene expression, the inhibition of histone acetyltransferases by polyQ proteins, and the fact that at least two disease proteins are transcription factors (1, 12-13). In addition, the nucleus is recognized to be a critical site for polyQ toxicity (14-16), suggesting that one or more nuclearspecific functions are perturbed in disease.A compelling link between these two leading theories is the fact that many proteins implicated in transcriptional control become redistributed into NI (17-27). One intriguing example is the transcription coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), which colocalizes to the NI formed by several expanded polyQ proteins (18-20, 25, 27). Moreover, overexpressed CBP suppresses polyQ toxicity in cellular models of polyQ disease. These results suggest a model of pathogenesis (25) in which sequestration of CBP by polyQ proteins inhib...

show abstract

“…The formation of nuclear inclusions depends on the length of poly-Q repeats and on as yet unidentified factors in the host cells. Studies in cell culture systems and in transgenic mice show that the nuclear inclusions recruit molecular chaperones, ubiquitin, and proteasomal subunits (Cummings et al, 1998;Chai et al, 1999b;Kim et al, 2002). The association of the degradative machineries suggests that nuclear inclusions may be involved in the proteolytic clearing of poly-Q aggregated substrates.…”

Section: Introductionmentioning

confidence: 99%

Nuclear Aggresomes Form by Fusion of PML-associated Aggregates

Gao

Tousson

et al. 2005

MBoC

View full text Add to dashboard Cite

Nuclear aggregates formed by proteins containing expanded poly-glutamine (poly-Q) tracts have been linked to the pathogenesis of poly-Q neurodegenerative diseases. Here, we show that a protein (GFP170*) lacking poly-Q tracts forms nuclear aggregates that share characteristics of poly-Q aggregates. GFP170* aggregates recruit cellular chaperones and proteasomes, and alter the organization of nuclear domains containing the promyelocytic leukemia (PML) protein. These results suggest that the formation of nuclear aggregates and their effects on nuclear architecture are not specific to poly-Q proteins. Using GFP170* as a model substrate, we explored the mechanistic details of nuclear aggregate formation. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses show that GFP170* molecules exchange rapidly between aggregates and a soluble pool of GFP170*, indicating that the aggregates are dynamic accumulations of GFP170*. The formation of cytoplasmic and nuclear GFP170* aggregates is microtubule-dependent. We show that within the nucleus, GFP170* initially deposits in small aggregates at or adjacent to PML bodies. Time-lapse imaging of live cells shows that small aggregates move toward each other and fuse to form larger aggregates. The coalescence of the aggregates is accompanied by spatial rearrangements of the PML bodies. Significantly, we find that the larger nuclear aggregates have complex internal substructures that reposition extensively during fusion of the aggregates. These studies suggest that nuclear aggregates may be viewed as dynamic multidomain inclusions that continuously remodel their components. INTRODUCTIONNewly synthesized proteins must be properly folded and modified to function correctly. Eukaryotic cells have developed extensive folding machineries to ensure the fidelity of protein processing. Nevertheless, misfolding can occur due to mutations within a protein, outside stresses, or the overexpression of proteins. Misfolded proteins often expose their hydrophobic domains, which leads to nonproductive protein associations and results in aggregation. Aggregated proteins tend to coalesce and form large deposits termed inclusion bodies, Russell bodies, or aggresomes, depending on their composition and location. Formation of such inclusions underlies a number of aggresomal diseases, including Alzheimer's disease, Parkinson's disease, familial amyotrophic lateral sclerosis, and the poly-glutamine (poly-Q) neuropathologies (reviewed in Zoghbi and Orr, 2000;Garcia-Mata et al., 2002).The biological processes leading to protein aggregation have been actively investigated (reviewed in Kopito, 2000;Garcia-Mata et al., 2002;Goldberg, 2003;Selkoe, 2003). Aggregation of proteins most likely occurs cotranslationally, while nascent peptide chains are synthesized on polyribosomes. If the nascent peptides cannot fold correctly, they will aggregate to form aggresomal particles. Small aggresomal particles form throughout the cell and are quickly transported toward the microtubule (MT)...

show abstract

Evidence for Proteasome Involvement in Polyglutamine Disease: Localization to Nuclear Inclusions in SCA3/MJD and Suppression of Polyglutamine Aggregation in vitro

Cited by 378 publications

References 43 publications

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Live-cell imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model of pathogenesis

Nuclear Aggresomes Form by Fusion of PML-associated Aggregates

Contact Info

Product

Resources

About