One‐Pot Semisynthesis of Exon 1 of the Huntingtin Protein: New Tools for Elucidating the Role of Posttranslational Modifications in the Pathogenesis of Huntington’s Disease

Ansaloni, Annalisa; Wang, Zheming; Jeong, Jae Sun; Ruggeri, Francesco Simone; Dietler, Giovanni; Lashuel, Hilal A.

doi:10.1002/ange.201307510

Cited by 14 publications

(21 citation statements)

References 41 publications

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“…We and others have previously shown that the polyQ repeat length strongly influences the conformation and aggregation properties of Httex1, with higher polyQ favoring the formation of a more compact polyQ domain and accelerating Htt aggregation in vitro and in vivo 30,45,46 .…”

Section: The Length Of the Polyq Domain Is Another Key Determinant Ofmentioning

confidence: 88%

Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

Riguet

Mahul‐Mellier

Maharjan

et al. 2020

Preprint

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Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington’s disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt inclusions in cells overexpressing mutant exon1 of the Htt protein. Our findings provide novel insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve 1) polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; 2) recruitment and accumulation of remodeled or dysfunctional membranous organelles; and 3) impairement of the protein quality control and degradation machineries. Interestingly, nuclear and cytoplasmic Htt inclusions exhibited distinct biochemical composition and ultrastructural properties, suggesting different formation mechanisms and toxicity.Graphical AbstractSchematic depictions and original electron micrographs of cytoplasmic inclusions formed by native (tag-free) mutant Huntington exon1 proteins (Httex1 72Q, left) and the corresponding GFP fusion protein (Httex1 72Q-GFP).

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Section: The Length Of the Polyq Domain Is Another Key Determinant Ofmentioning

confidence: 88%

Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

Riguet

Mahul‐Mellier

Maharjan

et al. 2020

Preprint

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“…Second, fusing these proteins to mutant Httex1 proteins has been shown to alter their aggregation properties and favors the formation of round particles or amorphous aggregates (27,39,41,59). Third, the use of enzymatic cleavage to remove these proteins or other solubilizing motifs often leaves behind additional amino acids in the protein sequence (Table 1) and/or occasionally results in the generation of undesired cleavage products (19,28,50).…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, Httex1 proteins generated by enzymatic cleavage often form heterogeneous fibrils that exhibit the tendency to associate into bundles (26 -28, 39, 40, 45), unlike the tag-free Httex1 proteins produced by the intein strategy herein (Figs. 3-6), semisynthesis, or chemical synthesis, which form very uniform and smooth amyloid-like fibrils (50,52). Moreover, several studies based on in situ cleavage of Httex1 fusion proteins have reported that Httex1 proteins with non-pathogenic polyQ repeats do not form fibrils (26 -28, 37, 38), whereas studies using synthetic (52), semisynthetic (50), or recombinant tagfree Httex1 proteins have shown that Httex1 with 23Q at concentrations ranging from 15 to 120 M can form fibrils in vitro (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…This could alter the biophysical and biochemical properties of Httex1 because of the important role of the Nt17 domain and prolinerich domain in regulating the conformational and aggregation properties of the protein (18,20,22,32,47,49). Moreover, it has been shown that commonly used enzymes such as trypsin and thrombin may lead to cleavages within the Nt17 domain and result in the generation of undesired Httex1 fragments (19,28,50). The net effect of incomplete and/or unspecific enzymatic cleavage of Httex1 fusion proteins is the generation of hetero- The cited fusion constructs were chosen from studies that focused on the characterization of the aggregation propensity and aggregate structure of Httex 1 in vitro.…”

Section: Huntington Disease (Hd)mentioning

confidence: 99%

“…This intein-based strategy eliminates the need for proteolytic enzymes and has been successfully used to produce Httex1 proteins with polyQ repeats ranging from 7Q to at least 49Q. Biophysical characterization of these proteins revealed that they exhibit the expected aggregation properties of tag-free Httex1 proteins produced by chemical or semisynthetic strategies (50,52). The use of tagfree Httex1 proteins enabled us to decipher subtle effects of concentration and polyQ-repeat length on Httex1 oligomerization and fibril formation, which were not observed previously using Httex1 fusion constructs or Httex1-based peptide model systems containing non-native sequences.…”

Section: Huntington Disease (Hd)mentioning

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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Peptide o‐Aminoanilides as Crypto‐Thioesters for Protein Chemical Synthesis

Wang¹,

Fang²,

He³

et al. 2014

Angewandte Chemie

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Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO 2 in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.

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One‐Pot Semisynthesis of Exon 1 of the Huntingtin Protein: New Tools for Elucidating the Role of Posttranslational Modifications in the Pathogenesis of Huntington’s Disease

Cited by 14 publications

References 41 publications

Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

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

Peptide o‐Aminoanilides as Crypto‐Thioesters for Protein Chemical Synthesis

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