Detection of Polyglutamine Protein Oligomers in Cells by Fluorescence Correlation Spectroscopy

Takahashi, Yasuo; Okamoto, Yuma; Popiel, H. Akiko; Fujikake, Nobuhiro; Toda, Tatsushi; Kinjo, Masataka; Nagai, Yoshitaka

doi:10.1074/jbc.m704789200

Cited by 89 publications

(87 citation statements)

References 50 publications

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“…3: C and D). Similar to our studies, other groups have demonstrated by different methods that Congo red inhibits oligomerization of mutant proteins with expanded polyglutamine using fluorescence resonance energy transfer and fluorescent correlation spectroscopy (28,39). These findings strengthen the hypothesis that improved mobility of mutant γPKC by Congo red is caused by inhibition of oligomerization.…”

Section: Discussionsupporting

confidence: 80%

Congo Red, an Amyloid-Inhibiting Compound, Alleviates Various Types of Cellular Dysfunction Triggered by Mutant Protein Kinase Cγ That Causes Spinocerebellar Ataxia Type 14 (SCA14) by Inhibiting Oligomerization and Aggregation

et al. 2010

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Abstract. Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation that induces apoptotic cell death. Congo red is widely used as a histological dye for amyloid detection. Recent evidence has revealed that Congo red has the property to inhibit amyloid oligomers and fibril formation of misfolded proteins. In the present study, we examine whether Congo red inhibits aggregate formation and cytotoxicity of mutant γPKC. Congo red likely inhibits aggregate formation of mutant γPKC -green fluorescent protein (GFP) without affecting its expression level in SH-SY5Y cells. Congo red counteracts the insolubilization of recombinant mutant γPKC, suggesting that the dye inhibits aggregation of mutant γPKC by a direct mechanism. Congo red also inhibits aggregation and oligomerization of mutant γPKC-GFP in primary cultured cerebellar Purkinje cells. Moreover, the dye reverses the improper development of dendrites and inhibits apoptotic cell death in Purkinje cells that express mutant γPKC-GFP. These results indicate that amyloid-inhibiting compounds like Congo red may be novel therapeutics for SCA14.

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Section: Discussionsupporting

confidence: 80%

Congo Red, an Amyloid-Inhibiting Compound, Alleviates Various Types of Cellular Dysfunction Triggered by Mutant Protein Kinase Cγ That Causes Spinocerebellar Ataxia Type 14 (SCA14) by Inhibiting Oligomerization and Aggregation

et al. 2010

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“…Small oligomers have been implicated as the causal agents in many proteinopathies. To monitor changes in the population of Htt103Q oligomers caused by our PrD enhancers and suppressors in yeast, we used fluorescence correlation spectroscopy (FCS) (25). To concentrate our analysis on the Htt103Q protein that was not localized to large aggregates, cell lysates were subjected to centrifugation to remove them.…”

Section: Resultsmentioning

confidence: 99%

“…For polyQ-containing proteins, oligomers form before IBs, their formation is polyQ length-dependent, and their presence is well correlated with cell death (25,37). Both chaperones that remodel oligomers and peptides that inhibit oligomer formation suppress toxicity (38,39).…”

Section: Discussionmentioning

confidence: 99%

Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1

Kayatekin

Matlack

Hesse

et al. 2014

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

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Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine-and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.protein misfolding | prions P rotein misfolding and aggregation underlie many neurodegenerative diseases. The causes of protein misfolding are numerous and include single amino acid changes and expansions of amino acid repeats. Polyglutamine (polyQ) expansions are an infamous example of the latter and are responsible for at least nine neurodegenerative conditions, including Huntington disease (HD). In most of these diseases the polyQ-expanded protein is expressed throughout the body, but tissue damage is restricted to the nervous system and, often, to a subset of cells depending on the causal protein. Thus, there are important cell type-specific determinants of toxicity, and these are specific to particular proteins. Despite these differences, disease severity is tightly linked to the number of glutamines. Progressively earlier onset occurs as the number of glutamines increases beyond a critical threshold of ∼35-45 residues (1). Moreover, in all cases, pathology is associated with misfolded forms of the polyQ-expanded protein.Thus, common features that arise from the expansions and result from shared aspects of polyQ-driven protein misfolding must contribute to pathology. Given the diverse nature of both the proteins bearing the polyQ tract and t...

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“…QBP1 decreased the amount of expanded polyQ protein oligomers, and the effect was greater for shorter length polyQ stretches [29,30] (Table 2). These results are consistent with our in vitro data, which show that QBP1 inhibits the monomeric conformational transition of the polyQ protein that occurs before oligomer and aggregate formation.…”

Section: Therapeutic Effects Of Qbp1 On Cell Culture Modelsmentioning

confidence: 99%

Inhibition of Protein Misfolding/Aggregation Using Polyglutamine Binding Peptide QBP1 as a Therapy for the Polyglutamine Diseases

et al. 2013

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Protein misfolding and aggregation in the brain have been recognized to be crucial in the pathogenesis of various neurodegenerative diseases, including Alzheimer's, Parkinson's, and the polyglutamine (polyQ) diseases, which are collectively called the "protein misfolding diseases". In the polyQ diseases, an abnormally expanded polyQ stretch in the responsible proteins causes the proteins to misfold and aggregate, eventually resulting in neurodegeneration. Hypothesizing that polyQ protein misfolding and aggregation could be inhibited by molecules specifically binding to the expanded polyQ stretch, we identified polyQ binding peptide 1 (QBP1). We show that QBP1 does, indeed, inhibit misfolding and aggregation of the expanded polyQ protein in vitro. Furthermore overexpression of QBP1 by the crossing of transgenic animals inhibits neurodegeneration in Drosophila models of the polyQ diseases. We also introduce our attempts to deliver QBP1 into the brain by administration using viral vectors and protein transduction domains. Interestingly, recent data suggest that QBP1 can also inhibit the misfolding/aggregation of proteins responsible for other protein misfolding diseases, highlighting the potential of QBP1 as a general therapeutic molecule for a wide range of neurodegenerative diseases. We hope that in the near future, aggregation inhibitor-based drugs will be developed and bring relief to patients suffering from these currently intractable protein misfolding diseases.

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Detection of Polyglutamine Protein Oligomers in Cells by Fluorescence Correlation Spectroscopy

Cited by 89 publications

References 50 publications

Congo Red, an Amyloid-Inhibiting Compound, Alleviates Various Types of Cellular Dysfunction Triggered by Mutant Protein Kinase Cγ That Causes Spinocerebellar Ataxia Type 14 (SCA14) by Inhibiting Oligomerization and Aggregation

Congo Red, an Amyloid-Inhibiting Compound, Alleviates Various Types of Cellular Dysfunction Triggered by Mutant Protein Kinase Cγ That Causes Spinocerebellar Ataxia Type 14 (SCA14) by Inhibiting Oligomerization and Aggregation

Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1

Inhibition of Protein Misfolding/Aggregation Using Polyglutamine Binding Peptide QBP1 as a Therapy for the Polyglutamine Diseases

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