A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70

Ormsby, Angelique R.; Ramdzan, Yasmin M.; Mok, Yee-Foong; Jovanoski, Kristijan D.; Hatters, Danny M.

doi:10.1074/jbc.m113.486944

Cited by 29 publications

(35 citation statements)

References 62 publications

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“…Data show transfection efficiency as standardized to a gate (FITC channel) defining 98% background fluorescence using untransfected cells as a reference. The percentage of transfected cells with inclusions was defined using PulSA as described previously (11,12), with the nonaggregating mutants to set the reference gate positions (n ϭ 3; mean Ϯ S.D. are shown).…”

Section: Resultsmentioning

confidence: 99%

“…SVA is a benchmark method for investigating mass heterogeneity of purified proteins (13), but to date has had limited application in cell lysates using fluorescently tagged proteins. We applied this method successfully to cell lysates to decipher the aggregation patterns of Httex1 and other proteins (10,12,14). We have also developed a strategy to separate cells expressing mutant protein enriched in inclusions from those lacking inclusions using Pulse Shape Analysis (PulSA) by flow cytometry, which enables a second layer of control over the study of protein oligomerization in cells with visible inclusions or not (11).…”

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confidence: 99%

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Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

Polling

Mok

Ramdzan

et al. 2014

Journal of Biological Chemistry

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Background: Protein aggregation is associated with neurodegenerative diseases. Results: We defined how the oligomeric state of disease-relevant mutant protein and homopolypeptides relate to clustering into inclusion subtypes IPOD and JUNQ. Conclusion: JUNQ protein and homopolypeptides relate to constitutively disrupted oligomeric states irrespective of inclusion formation. Significance: JUNQ inclusions may arise by cellular failure in degradation of abnormal oligomeric states.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

Polling

Mok

Ramdzan

et al. 2014

Journal of Biological Chemistry

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“…Development of techniques that enable the aggregation state of α-syn to be tracked in cells, such that this can be linked to specific biological outcomes, would greatly assist future work in this area. For example, the development of conformational sensors of α-syn capable of distinguishing monomeric, oligomeric and fibrillar species, such as those developed for Htt (Ormsby et al 2013), would facilitate investigation of the precise mechanism(s) by which sHsps act to prevent α-syn aggregation in cells.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, N2a cells represent a well-established neuronal model used for characterising intracellular protein aggregation (Krishnan et al 2006;Ojha et al 2011;Ormsby et al 2013;Chai et al 2016). Cells were cultured in Dulbecco's modified Eagle's medium/Ham's Nutrient Mixture F12 (DMEM/F12; ThermoFischer Scientific, Waltham, USA) supplemented with 10% (v/v) foetal bovine serum (FBS; Bovagen Biologicals, East Keilor, Australia) and 2.5 mM L-glutamine (ThermoFischer Scientific, Waltham, USA).…”

Section: Culture and Transfection Of Mammalian Cell Linesmentioning

confidence: 99%

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

Cox

Ecroyd

2017

Cell Stress and Chaperones

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Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cellbased model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. As such, this work sought to establish a robust model of intracellular α-syn aggregation using Neuro-2a cells. Aggregation of α-syn was found to be sensitive to inhibition of autophagy and the proteasome, resulting in a significant increase in the proportion of cells containing α-syn inclusions. This model was then used to evaluate the capacity of the sHsps, αB-c and Hsp27, to prevent α-syn aggregation in cells. To do so, we used bicistronic expression plasmids to express the sHsps. Unlike traditional fluorescent fusion constructs, these bicistronic expression plasmids enable only individual transfected cells expressing the sHsps (via expression of the fluorescent reporter) to be analysed, but without the need to tag the sHsp, which can affect its oligomeric structure and chaperone activity. Overexpression of both αB-c and Hsp27 significantly reduced the intracellular aggregation of α-syn. Thus, these findings suggest that overexpressing or boosting the activity of sHsps may be a way of preventing amyloid fibrillar aggregation of α-syn in the context of neurodegenerative disease.

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“…Furthermore, this interaction may actually help mediate the coalescing of the aggregates into larger protein deposits. There is evidence that protein inclusions are protective in the cell; studies have reported a lack of correlation between amyloid deposition in the brain and disease progression (Schmitz et al 2004 ;Rabinovici and Jagust 2009 ), and in cell culture models of protein aggregation multiple inclusions can be present in otherwise 'healthy' cells (Arrasate et al 2004 ;Gong et al 2008 ;Ormsby et al 2013 ). Thus, by promoting the formation of inclusions sHsps may assist in shielding the cell from toxic oligomers.…”

Section: Interactions With Amyloid Fibrilsmentioning

confidence: 94%

Redefining the Chaperone Mechanism of sHsps: Not Just Holdase Chaperones

Ecroyd

2015

Heat Shock Proteins

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The small heat-shock proteins (sHsps) are molecular chaperones that play a fundamental role in maintaining cellular protein homeostasis (proteostasis) by preventing the aggregation of destabilised proteins. They are generally described as 'holdase' type chaperones since they have the ability to bind partially folded intermediate states of target proteins, in an ATP-independent manner, and, in doing so, they can form high molecular weight complexes with some of them. However, recent work has shown that the ability of sHsps to interact with target proteins is multi-faceted. This review highlights the mechanisms by which sHsps can interact with aggregation-prone target proteins and proposes that they should be considered as protein 'stabilisers' rather than 'holdase' chaperones.

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A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70

Cited by 29 publications

References 62 publications

Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

Redefining the Chaperone Mechanism of sHsps: Not Just Holdase Chaperones

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