Single-step detection of mutant huntingtin in animal and human tissues: A bioassay for Huntington’s disease

Weiss, Andreas; Abramowski, Dorothée; Bibel, Miriam; Bodner, Ruth; Chopra, Vanita; DiFiglia, Marian; Fox, Jonathan H.; Kegel, Kimberly B.; Klein, Corinna; Grueninger, Stephan; Hersch, Steven M.; Housman, David E.; Régulier, Etienne; Rosas, H. Diana; Stefani, Muriel; Zeitlin, Scott; Bilbe, Graeme; Paganetti, Paolo

doi:10.1016/j.ab.2009.08.001

Cited by 117 publications

(120 citation statements)

References 41 publications

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“…We conjugated several primary antibodies raised to epitopes known to fall between residues 1 and 117 of huntingtin. We chose to use both the 2B7 and the 4C9 monoclonal antibodies that target residues 8-13 of the N17 domain and residues 61-71 of the polyproline region of huntingtin, respectively (26). As an optimal pair for FLIM-FRET, we chose to conjugate the antibodies with either alexa488 or alexa546 dyes.…”

Section: Measuring the Conformation Of The Amino Terminus Within Thementioning

confidence: 99%

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Caron

Desmond

Xia

et al. 2013

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

130

150

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Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion within the huntingtin gene that encodes a polymorphic glutamine tract at the amino terminus of the huntingtin protein. HD is one of nine polyglutamine expansion diseases. The clinical threshold of polyglutamine expansion for HD is near 37 repeats, but the mechanism of this pathogenic length is poorly understood. Using Förster resonance energy transfer, we describe an intramolecular proximity between the N17 domain and the downstream polyproline region that flanks the polyglutamine tract of huntingtin. Our data support the hypothesis that the polyglutamine tract can act as a flexible domain, allowing the flanking domains to come into close spatial proximity. This flexibility is impaired with expanded polyglutamine tracts, and we can detect changes in huntingtin conformation at the pathogenic threshold for HD. Altering the structure of N17, either via phosphomimicry or with small molecules, also affects the proximity between the flanking domains. The structural capacity of N17 to fold back toward distal regions within huntingtin requires an interacting protein, protein kinase C and casein kinase 2 substrate in neurons 1 (PACSIN1). This protein has the ability to bind both N17 and the polyproline region, stabilizing the interaction between these two domains. We also developed an antibody-based FRET assay that can detect conformational changes within endogenous huntingtin in wild-type versus HD fibroblasts. Therefore, we hypothesize that wild-type length polyglutamine tracts within huntingtin can form a flexible domain that is essential for proper functional intramolecular proximity, conformations, and dynamics.polyglutamine diseases | conformational switching | FLIM-FRET | neurodegeneration | fluorescence lifetime imaging microscopy H untington disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene (1). The pathogenic threshold of CAG expansion is ∼37 repeats, with increased repeats leading to an earlier age-onset of HD (2-4). This CAG mutation results in an expanded polyglutamine tract in the amino terminus of the gene's protein product, huntingtin (1). To date, no phenotypes at the level of huntingtin molecular biology or animal models can be attributed to polyglutamine lengths near the human pathogenic disease threshold (5).Polyglutamine or glutamine-rich domains are also found in transcription factors such as the Sp-family and cAMP response element-binding protein (CREB) and are defined as proteinprotein interaction motifs used to scaffold and regulate transcription by RNA polymerase II (6, 7). In the transducin-like enhancer of split (TLE) corepressor proteins, the glutamine-rich domains are thought to allow dimerization (8). However, the role of polyglutamine in proteins such as huntingtin may be distinct from that of a protein-protein interaction or dimerization domain.The polyglutamine tract of huntingtin is flanked on...

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Section: Measuring the Conformation Of The Amino Terminus Within Thementioning

confidence: 99%

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Caron

Desmond

Xia

et al. 2013

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

130

150

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“…The N-terminal 1212 amino acids of HTT with 128Q and the 4C mutations (D513A, D530A D552A, and D589A) were transiently transfected and overexpressed together with WT CASP6 or mutant CASP6 C264/277S in COS7 cells for 24 h. 47 Cell pellets from COS7 cells or Q111 and Q7 cells were lysed with SDP+ lysis buffer 65 and diluted in SDP+ lysis buffer to a final protein concentration of 2 μg/μl. 66,67 The detection of the 586 HTT fragment was performed using a combination of the monoclonal BKP1 antibody raised against the HTT N-terminus and an in-house 586 neo-epitope antibody raised against the C-terminus of 586 cleaved HTT. 47 The level of 586 HTT fragment was normalized to total HTT measures with the combination of BKP1 and 2166 raised against exon 2.…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation of caspase-6 by HIP14 regulates its activation

et al. 2016

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Caspase-6 (CASP6) has an important role in axonal degeneration during neuronal apoptosis and in the neurodegenerative diseases Alzheimer and Huntington disease. Decreasing CASP6 activity may help to restore neuronal function in these and other diseases such as stroke and ischemia, where increased CASP6 activity has been implicated. The key to finding approaches to decrease CASP6 activity is a deeper understanding of the mechanisms regulating CASP6 activation. We show that CASP6 is posttranslationally palmitoylated by the palmitoyl acyltransferase HIP14 and that the palmitoylation of CASP6 inhibits its activation. Palmitoylation of CASP6 is decreased both in Hip14 −/− mice, where HIP14 is absent, and in YAC128 mice, a model of Huntington disease, where HIP14 is dysfunctional and where CASP6 activity is increased. Molecular modeling suggests that palmitoylation of CASP6 may inhibit its activation via steric blockage of the substrate-binding groove and inhibition of CASP6 dimerization, both essential for CASP6 function. Our studies identify palmitoylation as a novel CASP6 modification and as a key regulator of CASP6 activity.

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“…Until recently, the detection of soluble Htt was limited by the intrinsic insensitivity of assays, and a lack of suitable antibodies. Novel antibodies and a fluorescence-enhanced antibody assay using timeresolved Förster resonence energy transfer (TR-FRET) has now yielded a reliable, highly sensitive method of quantifying both mutant and total huntingtin in blood samples [19]. This assay is likely to empower future clinical trials.…”

Section: Biofluidsmentioning

confidence: 99%