Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation

Monsellier, Élodie; Redeker, Virginie; Ruiz-Arlandis, Gemma; Bousset, Luc; Melki, Ronald

doi:10.1074/jbc.m114.603332

Cited by 77 publications

(118 citation statements)

References 68 publications

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“…These findings suggest that delays in enzymatic cleavage, sequence modifications, and/or sample heterogeneity influence greatly the aggregation kinetics and morphology of Httex1 fibrils. Consistent with these observations, recent studies showed that removal of the cleaved fusion proteins before initiating Httex1 aggregation resulted in an increased aggregation propensity of Httex1 (44). This further underscores the critical importance of using homogeneous preparations of native Httex1 to ensure accurate assessment of changes in the aggregation kinetics and structural properties of Httex1.…”

Section: Discussionsupporting

confidence: 64%

“…4). Interestingly, including an additional purification step after the enzymatic cleavage also resulted in a fast fibrillization of recombinant Httex1 with a polyQ-length of 17Q at 20 M generated from a MBP-Httex1-17Q-His 6 fusion protein (44). However, in our hands no abundant fibril formation was observed for Httex1-7Q/15Q even after weeks of incubation at 60 M (Fig.…”

Section: Discussioncontrasting

confidence: 44%

“…5). This discrepancy in the aggregation behavior might be caused by the additional non-native amino acids at the N (Gly-Ala dipeptide) and C termini (His 6 tag) of the Httex1-17Q protein used by Monsellier et al (44) and the fact that other polyQ model systems lack significant domains of Httex1 and/or incorporate non-native sequences (20 -22, 55, 60, 62). These findings indicate that differences in the primary sequence can have a strong effect on the aggregation properties of Httex1, which could make it difficult to compare aggregation data from different laboratories.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies using model polyQ peptide systems or partial Httex1 sequences demonstrated that non-pathogenic polyQ repeats even as short as 15Q can aggregate in vitro (22,55,60,61), whereas Httex1 proteins with non-pathogenic polyQ repeats produced from fusion proteins by in situ enzymatic cleavage often failed to form fibrils in vitro (26 -28, 37, 38). When Httex1 with wild type polyQ repeats (25Q) is purified after the enzymatic cleavage (44) or is produced as a tagfree protein by the intein strategy (23Q), it aggregates and forms amyloid-like fibrils in a concentration-dependent manner (Fig. 4).…”

Section: Discussionmentioning

confidence: 99%

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An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation

Vieweg

Ansaloni

Wang

et al. 2016

Journal of Biological Chemistry

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The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltosebinding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an inteinbased strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1-7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties. Huntington disease (HD)2 is a fatal neurodegenerative disorder caused by a CAG expansion within the first exon (Exon 1) of the huntingtin gene, IT15 (1). The CAG repeat length ranges between 6 and 35 in healthy subjects, whereas patients with HD exhibit lengths of 36 or greater resulting in the synthesis of a mutant Huntingtin protein (Htt) with an expanded polyglutamine (polyQ) domain (2). The length of the polyQ tract directly correlates with disease severity and is inversely correlated with disease age of onset (3). HD patients suffer from motor impairments, cognitive decline, and depression due to neurodegeneration in the striatum and cortex (4 -8).The accumulation of N-terminal fragments of mutant Htt in the nucleus and cytoplasm of striatal neurons led to the hypothesis that these fragments play causative roles in the neurodegeneration and pathology observed in HD (9 -13). This hypothesis is supported by the observation that the overexpression of the expanded Exon 1 in R6/2 transgenic mice recapitulates several key symptoms of HD (14). Subsequent studies demonstrated that aberrant splicing in HttQ150 knock-in mice gave rise to a short mRNA, which translates into the Huntingtin Exon 1 protein (Httex1), thus linking the genetic cause of HD to the generation of a highly toxic N-terminal Htt fragment (15). Together, these findings highlight the importance of this region in HD pathogenesis and underscore the critical importance of investigating the structure, aggregation, a...

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Section: Discussionsupporting

confidence: 64%

Section: Discussioncontrasting

confidence: 44%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation

Vieweg

Ansaloni

Wang

et al. 2016

Journal of Biological Chemistry

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“…For example, polyQ is much more aggregation-prone on its own than when it is embedded within the context of a disease-associated protein [5, 6]. Furthermore, short fragments of the HD-associated htt protein are less susceptible to protein turnover than longer fragments [7], and the immediate N-terminus of htt has been shown to interact with certain molecular chaperones [8, 9]. …”

Section: Introductionmentioning

confidence: 99%

The struggle by Caenorhabditis elegans to maintain proteostasis during aging and disease

Kikis

2016

Biol Direct

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The presence of only small amounts of misfolded protein is an indication of a healthy proteome. Maintaining proteome health, or more specifically, “proteostasis,” is the purview of the “proteostasis network.” This network must respond to constant fluctuations in the amount of destabilized proteins caused by errors in protein synthesis and exposure to acute proteotoxic conditions. Aging is associated with a gradual increase in damaged and misfolded protein, which places additional stress on the machinery of the proteostasis network. In fact, despite the ability of the proteostasis machinery to readjust its stoichiometry in an attempt to maintain homeostasis, the capacity of cells to buffer against misfolding is strikingly limited. Therefore, subtle changes in the folding environment that occur during aging can significantly impact the health of the proteome. This decline and eventual collapse in proteostasis is most pronounced in individuals with neurodegenerative disorders such as Alzheimer’s Disease, Parkinson’s Disease, and Huntington’s Disease that are caused by the misfolding, aggregation, and toxicity of certain proteins. This review discusses how C. elegans models of protein misfolding have contributed to our current understanding of the proteostasis network, its buffering capacity, and its regulation. Reviewers: This article was reviewed by Luigi Bubacco, Patrick Lewis and Xavier Roucou.

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Recent Advances and Applications of Nanoscale Infrared Spectroscopy and Imaging

Unger

Marcott²

2017

Encyclopedia of Analytical Chemistry

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In conventional infrared (IR) microspectroscopy, the rule of thumb is that you cannot obtain IR spectra of objects smaller than 5–10 m in size. This is based on the diffraction of radiation, and therefore, the spatial resolution is limited to be on the order of the wavelength of light used to make the measurement. Recently, researchers have developed new techniques that can overcome this limit, where the spatial resolution is not limited by diffraction. In this context, this article will focus on the combination of atomic force microscopy and infrared spectroscopy (AFM‐IR) as well as scattering scanning nearfield optical microscopy (s‐SNOM). Both techniques, AFM‐IR and s‐SNOM, are complementary with different strengths and weaknesses. AFM‐IR directly detects IR radiation absorbed by the sample using the AFM (atomic force microscope) probe tip to sense thermal expansion. This thermal expansion depends primarily on the absorption coefficient of the sample and is largely independent of other optical properties of the AFM tip and the sample. Thus, the AFM‐IR technique is preferred to measure absorption spectra with correlation to conventional IR spectroscopy and, furthermore, can be widely used for studies of soft materials such as polymers and life science samples. On the other hand, s‐SNOM detects radiation scattered by nanometer‐scale regions directly under the AFM probe tip. The scattered field depends on the complex optical constants of both the tip and the sample and contains rich information about nano‐optical phenomena. s‐SNOM is an advanced technique for imaging nanoscale contrast based on optical properties, with diverse applications in advanced materials, devices, and fundamental light/matter interactions. In this article, both the AFM‐IR and s‐SNOM approaches will be described with example applications of how they can be used to chemically characterize materials at the nanoscale.

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Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation

Cited by 77 publications

References 68 publications

An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation

An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation

The struggle by Caenorhabditis elegans to maintain proteostasis during aging and disease

Recent Advances and Applications of Nanoscale Infrared Spectroscopy and Imaging

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