- Email: [email protected]
Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins
Accepted Manuscript Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins Margherita Verani, Maria Bustamante, Paola Martufi, Manuel Daldin, Cristina Cariulo, Lucia Azzollini, Valentina Fodale, Francesca Puglisi, Andreas Weiss, Douglas Macdonald, Lara Petricca, Andrea Caricasole PII:
S0006-291X(16)31312-2
DOI:
10.1016/j.bbrc.2016.08.057
Reference:
YBBRC 36258
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 5 August 2016 Accepted Date: 8 August 2016
Please cite this article as: M. Verani, M. Bustamante, P. Martufi, M. Daldin, C. Cariulo, L. Azzollini, V. Fodale, F. Puglisi, A. Weiss, D. Macdonald, L. Petricca, A. Caricasole, Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.08.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins Margherita Verani1,2, Maria Bustamante2, Paola Martufi2, Manuel Daldin2, Cristina Cariulo2,
Lara Petricca*1,2 and Andrea Caricasole*1,2,$% 1. IRBM Promidis Via Pontina km 30.600 00071 Pomezia, Rome, Italy
SC
2. IRBM Science Park, Via Pontina km 30.600 00071 Pomezia, Rome, Italy
RI PT
Lucia Azzollini1,2, Valentina Fodale1,2, Francesca Puglisi2, Andreas Weiss1#, Douglas Macdonald3,
AC C
EP
TE D
M AN U
3. CHDI Management/CHDI Foundation, Los Angeles, CA 90045, USA
*These authors contributed equally to the present work #: Current address: Evotec AG, Manfred Eigen Campus, Hamburg, Germany %: Current address: Alzheimer’s Research UK Drug Discovery Institute, University of Cambridge, Cambridge, UK
$: To whom correspondence should be addressed
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
Abstract We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical
RI PT
methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized,
SC
unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based
M AN U
conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-
AC C
EP
TE D
mediated conformational change in different proteins associated with CAG triplet repeat disorders.
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
Introduction The human genome contains several loci encoding polyglutamine domains, encoded by CAG repeats [1]. CAG triplet repeat disorders represent a class of rare genetic neurodegenerative conditions
RI PT
associated with the expansion of a polyglutamine (polyQ; Q being encoded by CAG or CAA) repeat domain [2,3]. Although different neuronal subtypes and brain regions are characteristically affected in each disease, a unifying feature is the production of intracellular/neuronal protein aggregates, typically enriched for the mutant (polyQ-expanded) protein or its expanded polyQ-containing fragments [2,4,5]. This invariably leads
SC
to progressive neurodegeneration that cannot presently be addressed therapeutically [6]. Although loss of normal protein function may play a role in at least some cases [7–9], pathology is thought to be driven largely through a gain of function correlated with polyQ expansion [10,11] and associated with
M AN U
conformational change [12–15] that leads to one or more dysfunctions affecting intra- and/or intermolecular interactions, subcellular localization and compartmentalized activity, proteolysis, protein clearance and oligomerization/aggregation. This is exemplified by the prototypical CAG triplet repeat disorder, Huntington’s disease (HD) [16–22], a progressive, autosomal dominant neurodegenerative disease caused by expansion of a polyQ encoding repeat within exon 1 of the huntingtin (HTT) gene [22]. The disease is associated with the generation of N-terminal fragments of mutant HTT (mHTT) protein
TE D
comprising the expanded polyQ domain and likely produced through mechanisms such as aberrant splicing and proteolysis [23–25]. Misfolding and self-association of these fragments give rise to oligomers and the distinctive aggregates observed in the HD brain, which can give rise to HD-like phenotype in animal models [29,25]. Sensitive, accurate measurements of soluble wild type and mHTT conformers in different
EP
preclinical and clinical samples is possible using time-resolved Förster Energy Transfer (TR-FRET) based immunoassays [26,27]; measurement is based on the labeling of an antibody pair with a rare earth ion
AC C
fluorophore donor and an acceptor fluorophore, which generate a specific TR-FRET signal when the donor and acceptor labeled antibodies bind to their antigen simultaneously. Several studies have addressed the conformational changes associated with polyQ expansion, particularly in HTT [15]. The recent development of conformational, TR-FRET based immunoassays [15,28] has confirmed the apparent structural rigidity imparted on HTT by polyQ expansion [29] and has opened the possibility of developing approaches to modulate mHTT conformation with the aims to alter its toxic properties. The exquisite polyQ dependence of the conformational immunoassays, requiring interrogation of the polyQ domain with specific antibodies [15,28], suggests that a similar approach may be used to interrogate the conformation of other polyQ repeat disease-associated proteins and supports an intrinsic structural property of polyQ repeats in imparting conformational rigidity. Here, we demonstrate the general applicability of the conformational immunoassay to detect conformational rigidity imparted by polyQ expansion in other polyQ repeat IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT proteins, exemplified by the androgen receptor (whose polyQ domain is expanded in spinal bulbar muscular atrophy, SBMA [30–32]) and TATA-binding protein (TBP, whose polyQ domain is expanded in spinocerebellar ataxia 17, SCA17 [33,5,34]). Further, we provide additional evidence supporting the notion that conformational rigidity imparted on relevant proteins by polyQ expansion is due to an intrinsic structural property of the polyQ domain.
RI PT
Materials and Methods Plasmids
For expression of proteins of interest, cDNA inserts were designed as follows. HTT exon 1
SC
sequences with Q16 and Q72 were essentially as described previously [15], with a FLAG epitope Cterminally fused to the end of exon 1. Art polyQ sequences were designed to encode proteins of similar
M AN U
molecular weight and identical polyQ lengths to HTT exon 1 proteins, and to include N-terminal and Cterminal epitopes for easy TR-FRET detection (MYC and FLAG). In Art polyQ proteins, the methionine codon is
sequentially
followed (as
a translational fusion)
by
a MYC epitope,
a linker region
(ANSSIDLISVPVEYPYDVPDYASR; [35]), a polyQ encoding domain (Q16 or Q72), and a second linker region (RPACKIPNDLKQKVMNH; [35]) followed by a C-terminal FLAG. Art polyMYC sequences were designed to encode proteins comparable to Art polyQ proteins except for the polyQ domains, which were replaced by
TE D
sequences encoding either 2 MYC epitopes (to mimick the 8-10 residue epitope of anti-polyQ antibodies such as MW1) or 8 MYC epitopes (to mimick the multimerized MW1 epitope present in a Q72 expansion). In Art polyMYC proteins the methionine codon is sequentially followed (as a translational fusion) by the ANSSIDLISVPVEYPYDVPDYASR linker region, the polyMYC encoding domain (2xMYC or 8xMYC), the second
EP
linker region RPACKIPNDLKQKVMNH and a C-terminal FLAG. Relevant protein sequences comprising the polyQ domains of androgen receptor (AR) and TATA binding protein (TBP) were selected based on
AC C
previously published constructs ([36,37] for AR and [38] for TBP). cDNAs encoding these protein fragments were designed to include N-terminal MYC and C-terminal FLAG epitopes. HEK293T cell culture and transfection HEK293T cells were cultured, transiently transfected and whole cell lysates were prepared at the indicated times after transfection, exactly as previously reported in [15,39].
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
SDS-PAGE and immunoblotting SDS-PAGE and immunoblotting were performed as described in [15]. The anti-MYC (cat. M4439), anti-FLAG (cat. F1804) and anti-GAPDH (cat. G9545) antibodies were obtained from Sigma-Aldrich. The
RI PT
MW1 antibody [40] was obtained from the Developmental Studies Hybridoma Bank, The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Antibody 4C9 was described previously [27,41] and obtained from the CHDI Foundation (New York, NY). Custom terbium cryptate and D2fluorophore antibody labeling was performed by CisBio (Bagnols, France) as reported in [15].
SC
TR-FRET assays
TR-FRET assays were performed essentially as described previously [15]. The specificity of the FLAG-
M AN U
MW1, FLAG-4C9 and FLAG-MYC TR-FRET immunoassays was tested by a) omitting one of the antibodies of the TR-FRET-pair in each assay and b) performing the measurements on lysates overexpressing unrelated proteins (data not shown).
Results and discussion
TE D
Previous studies by us [15], and others [28] reported TR-FRET immunoassay detection of a conformational change imparted by mutant polyQ expansion on soluble HTT. These studies identified an absolute requirement on polyQ interrogation (with one of the TR-FRET antibodies in the antibody pair) for conformational change detection by TR-FRET immunoassay methods. Therefore, we decided to further
EP
investigate the exquisite polyQ dependence of the structural change detected by the conformational immunoassay and to determine its relevance to other polyQ disease proteins associated with
AC C
neurodegeneration, by studying proteins expressed in a biological context (transfected human cells). In order to do this, we designed different expression constructs (Fig. 1Ai) encoding either HTT with Q16 or Q72, an artificial protein encoding polyQ domains identical to those present in the HTT constructs employed (Q16 or Q72), or a comparable artificial construct where the polyQ domain is replaced by a multimerized MYC epitope (2xMYC or 8xMYC) to mimic theoretical epitope availability present for antipolyQ antibody MW1 on HTT Q16 and Q72, respectively. Correct expression of these constructs following transient transfection of HEK293T cells was verified by SDS-PAGE followed by immunoblotting (Fig. 1Aii). In these blots, detection with antibody MW1 predominantly detected polyQ expansion (Q72) in mutant HTT and mutant Art polyQ (Q72) over the wildtype (Q16) proteins, while detection with anti-MYC detected the presence of the MYC epitope in Art polyQ and Art polyMYC encoding constructs (Fig. 1Aii). Anti-FLAG immunoblotting detected expression of all constructs at the expected molecular weight (Fig. 1Aii). IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT Additional constructs (Fig. 1Bi) were designed, encoding androgen receptor (AR) and TATA binding protein (TBP) fragments comprising their wild type (Q19 for AR and Q36 for TBP) or mutant counterparts (Q65 and Q80, respectively). The AR and TBP protein regions encoded by the cDNAs were selected based on previous studies using cDNAs encoding protein fragments of the respective proteins ([36,37] for AR and [38] for TBP). Again, correct expression of these constructs following transient transfection of HEK293T cells was
RI PT
verified by SDS-PAGE followed by immunoblotting (Fig. 1Bii). MYC and FLAG epitopes at the N-terminus and C-terminus of these constructs were introduced in order to enable TR-FRET measurements independent of anti-HTT specific antibodies (including a FLAG epitope at the C-terminus of HTT exon 1-encoding constructs). These constructs were first used to investigate the role of the protein context on the structural rigidity imparted by polyQ expansion, as detected by the conformational immunoassay. Second, as polyQ
SC
expansions likely result in an increase in the number of epitopes for anti-polyQ antibodies we asked if increasing epitope number per se is sufficient to alter protein conformation as detected by TR-FRET based
M AN U
immunoassays, or if this is based on an intrinsic property of polyQ repeats.
A conformational constraint is imparted on proteins by repeat-length expansion of the polyQ domain We therefore asked if a polyQ domain can determine conformational changes by itself, irrespective of protein context, by interrogating wholly artificial constructs bearing a short (Q16) or mutant-like (Q72) polyQ expansion, as well as AR and TBP protein fragments, comprising their respective polyQ domains (wild
TE D
type or mutant), as their non-polyQ sequences are unrelated to each other and HTT. To do this, we individually transfected each plasmid of the four pairs of constructs into HEK293T cells (Fig. 2A). The first pair of constructs (HTT Ex1 Q16 or Q72) encoded HTT exon 1 with a Q16 or a Q72 repeat (Fig. 2Ai). This pair served as a reference, control pair, and yielded comparable signals for lysates expressing the Q16 and
EP
Q72 HTT exon 1 proteins with the FLAG-4C9 TR-FRET antibody pair, indicative of comparable expression levels of the two constructs in HEK293T cells (Fig. 2Aii). As expected, the FLAG-MW1 antibody pair
AC C
produced a TR-FRET signal which was polyQ-dependent (Fig. 2Aiii), as previously reported for a HTT-specific polyQ-dependent TR-FRET antibody pair (2B7-MW1; [15,28]). The second pair of constructs encoded a Q16 or Q72 polyQ expansion flanked at the N-terminus and C-terminus by artificial sequences, and bearing an N-terminus MYC epitope and a C-terminal FLAG epitope (Fig. 2Bi). This pair of constructs (Art Q16 and Art Q72) was designed to determine if HTT sequences N-terminal or C-terminal of the polyQ are absolutely required for HTT’s conformational plasticity detected by the TR-FRET 2B7/MW1 immunoassay. Again comparable signals for lysates expressing the Q16 and Q72 Art proteins were obtained with the FLAG-4C9 TR-FRET antibody pair (Fig. 2Bii), while a polyQ-dependent TR-FRET signal was obtained with the FLAGMW1 antibody pair (Fig. 2Biii). The third and fourth pairs of constructs encoded wild type or mutant AR and TBP proteins, respectively (Fig. 2Ci and 2Di). As observed for HTT proteins and for the Art polyQ proteins, the FLAG-4C9 TR-FRET antibody pair did not result in increased signal in the expanded (mutant) proteins IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT (Fig. 2Cii and 2Dii). Significantly, however, the FLAG-MW1 TR-FRET pair showed a strong increase in lysates expressing the mutant AR protein (Fig. 2Ciii), and the polyQ-dependent increase was evident even in lysates expressing mutant TBP, although a significant polyQ expansion is present in wild type TBP already (Fig. 2Diii). Collectively, these data are consistent with the capacity of MW1-based TR-FRET immunoassays to detect repeat length-dependent changes in polyQ epitopes in different proteins, including HTT. We next
RI PT
turned to determining if the FLAG-MW1 TR-FRET immunoassay can detect the temperature- and polyQdependent conformational change previously reported in HTT for the 2B7-MW1 TR-FRET immunoassay [15,28] and if this is detectable in artificial proteins (Art polyQ) and in polyQ disease-associated proteins other than HTT (AR and TBP). As shown in Fig. 3Aii, a control (FLAG-4C9) TR-FRET immunoassay (which does not interrogate the polyQ domain) yields an essentially identical and polyQ-independent signal in Q16 and
SC
Q72 HTT proteins (Fig. 3A), as previously reported for the 4C9-MW1 TR-FRET immunoassay [15,28], while the FLAG-MW1 TR-FRET immunoassay easily detects the conformational change imparted by polyQ
M AN U
expansion on HTT exon 1 protein, as previously reported for the 2B7-MW1 TR-FRET immunoassay [15,28]. Similar results were obtained for lysates expressing Art polyQ (Fig. 3B), AR (Fig. 3C) and TBP (Fig. 3D), where the control (FLAG-4C9) TR-FRET immunoassay yields a polyQ-independent signal in wild type and mutant proteins (Fig. 3Bii, 3Cii and 3Dii), while the FLAG-MW1 TR-FRET immunoassay easily detects the conformational change imparted by polyQ expansion on all proteins. Together with the previously reported requirement of the conformational TR-FRET immunoassays for polyQ interrogation and its independence
TE D
on HTT protein fragment length [15,28], these data are strongly supportive of the notion that the polyQ domain is necessary and sufficient to produce a repeat-length conformational constraint on proteins. Importantly, the data demonstrate that polyQ expansion can alter the conformational properties of other
EP
proteins associated with CAG repeat-disorders in a manner similar to that observed for HTT. Intrinsic nature of the conformational constraint imparted on proteins by repeat-length expansion of the
AC C
polyQ domain
We next asked if a multimerized artificial epitope (MYC), designed to mimic epitope recognition by MW1 of a Q16 or a Q72 repeat can impart a conformational constraint detectable by a relevant TR-FRET immunoassay. The multimerized MYC epitope presents either 2 or 8 epitopes for its specific (anti-MYC) antibody, in order to mimic MW1 recognition of a Q16 and Q72 polyQ region (which appears to involve a linear epitope of 8-10 glutamines [42,43]), and is flanked at the N-terminus and C-terminus by artificial sequences (the same as in Art polyQ). This pair of constructs (Art 2xMYC and 8xMYC; Fig. 4Ai) was then used to determine the dependence of the polyQ- and temperature-dependent conformational changes on epitope multimerization rather than intrinsic polyQ properties. If interrogation of a multimerized, nonpolyQ epitope with a specific antibody could substitute for the polyQ-anti polyQ antibody interaction, then epitope multimerization rather than polyQ conformation would be relevant for the observed effect. IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT Analysis of the HEK293T cells lysates with the FLAG-MYC pair detected a relatively higher signal in the sample expressing the Art polyMYC 8xMYC construct, as might be expected given the presence of a multimerized MYC epitope. Interestingly, when the lysates where interrogated using this TR-FRET immunoassay at the two temperatures (20°C and 4°C, as done for the 2B7-MW1 and FLAG-MW1 TR-FRET immunoassays to detect the repeat-length conformational constraint imposed by polyQ on HTT and other
RI PT
polyQ proteins), no MYC epitope-length dependent effect was observed, unlike results obtained when polyQ repeat proteins are interrogated with relevant TR-FRET immunoassays (where one antibody in the TR-FRET pair interrogates the polyQ domain). This discordance in behaviour between multimerized MYC and polyQ epitopes indicates that conformational differences detected by TR-FRET immunoassays at the two temperatures are due to some inherent properties of the polyQ region rather than simple
SC
multimerization of an epitope.
In conclusion, we have provided evidence that polyQ expansion can also influence the
M AN U
conformation of other polyQ repeat-associated proteins. Consistent with previous reports, our data also show that an intrinsic property of polyQ repeats, rather than simple epitope multimerization, is associated with this effect. The conformational, TR-FRET based immunoassays may prove useful, practical and scalable tools to identify and further develop modifiers of conformation for a range of CAG repeat-associated proteins, which could constitute the basis for novel therapeutic development in different CAG repeat
Figure legends
TE D
expansion diseases.
EP
Figure 1. Schemes of the different constructs, and confirmation of their correct expression in transiently transfected HEK293T cells. A. Details and characterization of constructs encoding HTT exon 1 (HTT Ex1; Q16
AC C
and Q72) and similarly sized artificial proteins encoding multimeric MW1 (Art polyQ; Q16 and Q72) or antiMYC epitopes (Art polyMYC; 2x and 8x). i) Schemes of constructs ii). Representative immunoblots of whole cell lysates from HEK293T cells transiently transfected with the constructs in Ai and probed for different antibodies to confirm presence of polyQ expansions (MW1), MYC epitopes (anti-MYC), expression of all constructs (anti-FLAG) and GAPDH (anti-GAPDH) to control for total protein content. B. Details and characterization of constructs encoding androgen receptor (AR) and TATA binding protein (TBP) protein fragments. i) Schemes of constructs encoding wild type and mutant AR (Q19 and Q65) and TATA binding protein (TBP; Q36 and Q80). ii) Representative immunoblots of whole cell lysates from HEK293T cells transiently transfected with the constructs in Ai and probed for different antibodies to confirm presence of polyQ expansions (MW1), MYC epitopes (anti-MYC), expression of all constructs (anti-FLAG) and GAPDH (anti-GAPDH) to control for total protein content. Numbers indicate MW (in kDa). IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT Figure 2. TR-FRET analysis of HEK293T cell lysates expressing the different constructs (HTT Ex1, Art polyQ, AR and TBP; Ai, Bi, Ci, Di, respectively) with wild type or mutant polyQ repeats, using the FLAG-4C9 (HTT ex1; Ai) or FLAG-MYC antibody pairs (Bii, Cii, Dii, respectively), which do not interrogate the polyQ repeat. TR-FRET analysis of HEK293T cell lysates expressing the different constructs with the FLAG-MW1 antibody pair, which interrogates the polyQ repeat, is shown in Aiii, Biii, Ciii, Diii. Data obtained from a
RI PT
representative experiment are shown. At least three independent biological replicates were performed. Figure 3. Conformational immunoassay analysis of cell lysates expressing the different constructs (HTT Ex1, Art polyQ, AR and TBP. Data represent ratiometric values of TR-FRET signals obtained for each construct at the two temperatures (4°C and 20°C). Ai, Bi, Ci, Di, respectively) with wild type or mutant polyQ repeats.
SC
Data obtained using the FLAG-4C9 (HTT ex1; Ai) or FLAG-MYC antibody pairs (Bii, Cii, Dii, respectively), which do not interrogate the polyQ repeat. In Aiii, Biii, Ciii, Diii the data obtained by TR-FRET analysis of HEK293T cell lysates expressing the different constructs with the FLAG-MW1 antibody pair, which
M AN U
interrogates the polyQ repeat, are shown. Data represent the means and standard deviations of the means of at least three independent experiments (one-way ANOVA Tukey's Multiple Comparison Test, degrees of significance are indicated).
Figure 4. A. Standard and conformational TR-FRET analysis of the Art-polyMYC construct.. A i) Details of the Art polyMYC constructs, designed to mimick the presence of a small (2xMYC) or larger (8xMYC) number of
TE D
epitopes for the interrogating antibody (as in the case of Q16 and Q72 for MW1). ii) Data obtained by TRFRET analysis of HEK293T cell lysates expressing the two constructs with the FLAG-MYC antibody pair, which interrogates the MYC repeat from one representative experiment are shown. iii) ratio of TR-FRET signals obtained at the two temperatures (4°C/20°C) using the FLAG-MYC antibody pair on these constructs
EP
(similar to FLAG-MW1 conformational TR-FRET analysis of Art polyQ). Data represent the means and standard deviations of the means of at least three independent experiments ((one-way ANOVA Tukey's degrees of significance are indicated). B. Schematic interpretation of the lack of
AC C
Multiple Comparison Test,
conformational effects of temperature and epitope multimerization on TR-FRET signals obtained with the FLAG-MYC antibody pair on Art polyMYC constructs, compared to the effects observed on TR-FRET signals with the FLAG-MW1 antibody pair on Art polyQ constructs with i) short (Q16) and ii) long (Q72) polyQ repeats.
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[5]
[6]
[7]
[8]
[9]
[10]
SC
M AN U
[4]
TE D
[3]
EP
[2]
S.L. Butland, R.S. Devon, Y. Huang, C.L. Mead, A.M. Meynert, S.J. Neal, et al., CAG-encoded polyglutamine length polymorphism in the human genome, BMC Genomics. 8 (2007) 126. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17519034. C.M. Everett, N.W. Wood, Trinucleotide repeats and neurodegenerative disease, Brain. 127 (2004) 2385–2405. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=15329351. J.R. Gatchel, H.Y. Zoghbi, Diseases of unstable repeat expansion: mechanisms and common principles, Nat Rev Genet. 6 (2005) 743–755. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16205714. S.W. Davies, K. Beardsall, M. Turmaine, M. DiFiglia, N. Aronin, G.P. Bates, Are neuronal intranuclear inclusions the common neuropathology of triplet-repeat disorders with polyglutamine-repeat expansions?, Lancet. 351 (1998) 131–133. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=9439509. M.J. Friedman, A.G. Shah, Z.H. Fang, E.G. Ward, S.T. Warren, S. Li, et al., Polyglutamine domain modulates the TBP-TFIIB interaction: implications for its normal function and neurodegeneration, Nat Neurosci. 10 (2007) 1519–1528. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17994014. N.A. Di Prospero, K.H. Fischbeck, Therapeutics development for triplet repeat expansion diseases, Nat Rev Genet. 6 (2005) 756–765. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16205715. E. Cattaneo, D. Rigamonti, D. Goffredo, C. Zuccato, F. Squitieri, S. Sipione, Loss of normal huntingtin function: new developments in Huntington’s disease research, Trends Neurosci. 24 (2001) 182–188. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=11182459. P.S. Thomas Jr., G.S. Fraley, V. Damian, L.B. Woodke, F. Zapata, B.L. Sopher, et al., Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy, Hum Mol Genet. 15 (2006) 2225–2238. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16772330. C. Zuccato, M. Valenza, E. Cattaneo, Molecular mechanisms and potential therapeutical targets in Huntington’s disease, Physiol Rev. 90 (2010) 905–981. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=20664076. H.T. Orr, Polyglutamine neurodegeneration: expanded glutamines enhance native functions, Curr Opin Genet Dev. 22 (2012) 251–255. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=22284692.
AC C
[1]
RI PT
Reference list
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[16]
[17]
[18]
[19]
[20]
[21]
[22] [23]
RI PT
SC
[15]
M AN U
[14]
TE D
[13]
EP
[12]
J.C. Jacobsen, G.C. Gregory, J.M. Woda, M.N. Thompson, K.R. Coser, V. Murthy, et al., HD CAGcorrelated gene expression changes support a simple dominant gain of function, Hum Mol Genet. 20 (2011) 2846–2860. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=21536587. J. Shao, M.I. Diamond, Polyglutamine diseases: emerging concepts in pathogenesis and therapy, Hum Mol Genet. 16 Spec No (2007) R115–23. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17911155. Y. Nagai, H.A. Popiel, Conformational changes and aggregation of expanded polyglutamine proteins as therapeutic targets of the polyglutamine diseases: exposed beta-sheet hypothesis, Curr Pharm Des. 14 (2008) 3267–3279. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19075705. B. Almeida, S. Fernandes, I.A. Abreu, S. Macedo-Ribeiro, Trinucleotide repeats: a structural perspective, Front Neurol. 4 (2013) 76. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=23801983. V. Fodale, N.C. Kegulian, M. Verani, C. Cariulo, L. Azzollini, L. Petricca, et al., Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy, PLoS One. 9 (2014) e112262. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=25464275. N. Hazeki, K. Nakamura, J. Goto, I. Kanazawa, Rapid aggregate formation of the huntingtin Nterminal fragment carrying an expanded polyglutamine tract, Biochem Biophys Res Commun. 256 (1999) 361–366. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=10079189. A. Lunkes, K.S. Lindenberg, L. Ben-Haiem, C. Weber, D. Devys, G.B. Landwehrmeyer, et al., Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions, Mol Cell. 10 (2002) 259–269. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=12191472. E. Roze, F. Saudou, J. Caboche, Pathophysiology of Huntington’s disease: from huntingtin functions to potential treatments, Curr Opin Neurol. 21 (2008) 497–503. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=18607213. C. Landles, K. Sathasivam, A. Weiss, B. Woodman, H. Moffitt, S. Finkbeiner, et al., Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease, J Biol Chem. 285 (2010) 8808–8823. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=20086007. D.E. Ehrnhoefer, L. Sutton, M.R. Hayden, Small changes, big impact: posttranslational modifications and function of huntingtin in Huntington disease, Neuroscientist. 17 (2011) 475–492. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=21311053. C.A. Ross, S.J. Tabrizi, Huntington’s disease: from molecular pathogenesis to clinical treatment, Lancet Neurol. 10 (2011) 83–98. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=21163446. G.P. Bates, R. Dorsey, J.F. Gusella, M.R. Hayden, C. Kay, B.R. Leavitt, et al., Huntington disease, Nat. Rev. Dis. Prim. (2015) 15005. http://dx.doi.org/10.1038/nrdp.2015.5. K. Sathasivam, A. Neueder, T.A. Gipson, C. Landles, A.C. Benjamin, M.K. Bondulich, et al., Aberrant
AC C
[11]
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
RI PT
SC
[28]
M AN U
[27]
TE D
[26]
EP
[25]
AC C
[24]
splicing of HTT generates the pathogenic exon 1 protein in Huntington disease., Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 2366–70. doi:10.1073/pnas.1221891110. T.A. Gipson, A. Neueder, N.S. Wexler, G.P. Bates, D. Housman, Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis, RNA Biol. 10 (2013) 1647–1652. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=24256709. B.A. Barbaro, T. Lukacsovich, N. Agrawal, J. Burke, D.J. Bornemann, J.M. Purcell, et al., Comparative study of naturally occurring huntingtin fragments in Drosophila points to exon 1 as the most pathogenic species in Huntington’s disease, Hum Mol Genet. 24 (2015) 913–925. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=25305076. B. Baldo, P. Paganetti, S. Grueninger, D. Marcellin, L.S. Kaltenbach, D.C. Lo, et al., TR-FRET-based duplex immunoassay reveals an inverse correlation of soluble and aggregated mutant huntingtin in Huntington’s disease, Chem. Biol. 19 (2012) 264–275. doi:10.1016/j.chembiol.2011.12.020. A. Weiss, D. Abramowski, M. Bibel, R. Bodner, V. Chopra, M. DiFiglia, et al., Single-step detection of mutant huntingtin in animal and human tissues: a bioassay for Huntington’s disease, Anal Biochem. 395 (2009) 8–15. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19664996. X. Cui, Q. Liang, Y. Liang, M. Lu, Y. Ding, B. Lu, TR-FRET assays of Huntingtin protein fragments reveal temperature and polyQ length-dependent conformational changes, Sci Rep. 4 (2014) 5601. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=24998512. N.S. Caron, C.R. Desmond, J. Xia, R. Truant, Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin., Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 14610–5. doi:10.1073/pnas.1301342110. A. Poletti, The polyglutamine tract of androgen receptor: from functions to dysfunctions in motor neurons, Front Neuroendocr. 25 (2004) 1–26. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=15183036. I. Palazzolo, A. Gliozzi, P. Rusmini, D. Sau, V. Crippa, F. Simonini, et al., The role of the polyglutamine tract in androgen receptor, J Steroid Biochem Mol Biol. 108 (2008) 245–253. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17945479. J.P. Chua, A.P. Lieberman, Pathogenic mechanisms and therapeutic strategies in spinobulbar muscular atrophy, CNS Neurol Disord Drug Targets. 12 (2013) 1146–1156. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=24040817. W.M. van Roon-Mom, S.J. Reid, R.L. Faull, R.G. Snell, TATA-binding protein in neurodegenerative disease, Neuroscience. 133 (2005) 863–872. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=15916858. R. Gao, T. Matsuura, M. Coolbaugh, C. Zuhlke, K. Nakamura, A. Rasmussen, et al., Instability of expanded CAG/CAA repeats in spinocerebellar ataxia type 17, Eur J Hum Genet. 16 (2008) 215–222. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=18043721. T.K. Kerppola, Bimolecular fluorescence complementation: visualization of molecular interactions in living cells, Methods Cell Biol. 85 (2008) 431–470. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=18155474. Y. Takeshita, R. Fujinaga, C. Zhao, A. Yanai, K. Shinoda, Huntingtin-associated protein 1 (HAP1) interacts with androgen receptor (AR) and suppresses SBMA-mutant-AR-induced apoptosis, Hum Mol Genet. 15 (2006) 2298–2312. IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[41]
[42]
[43]
RI PT
SC
M AN U
[40]
TE D
[39]
EP
[38]
AC C
[37]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16782802. J.E. Young, G.A. Garden, R.A. Martinez, F. Tanaka, C.M. Sandoval, A.C. Smith, et al., Polyglutamineexpanded androgen receptor truncation fragments activate a Bax-dependent apoptotic cascade mediated by DP5/Hrk, J Neurosci. 29 (2009) 1987–1997. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19228953. P.J. Kung, Y.C. Tao, H.C. Hsu, W.L. Chen, T.H. Lin, D. Janreddy, et al., Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models, Drug Des Devel Ther. 8 (2014) 1929–1939. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=25342886. M.B. Bustamante, A. Ansaloni, J.F. Pedersen, L. Azzollini, C. Cariulo, Z.M. Wang, et al., Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKK??, Biochem. Biophys. Res. Commun. 463 (2015) 1317–1322. doi:10.1016/j.bbrc.2015.06.116. J. Ko, S. Ou, P.H. Patterson, New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins, Brain Res Bull. 56 (2001) 319–329. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=11719267. P. Paganetti, A. Weiss, M. Trapp, I. Hammerl, D. Bleckmann, R.A. Bodner, et al., Development of a method for the high-throughput quantification of cellular proteins, Chembiochem. 10 (2009) 1678– 1688. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19492395. P. Li, K.E. Huey-Tubman, T. Gao, X. Li, A.P. West Jr., M.J. Bennett, et al., The structure of a polyQanti-polyQ complex reveals binding according to a linear lattice model, Nat Struct Mol Biol. 14 (2007) 381–387. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17450152. G.E. Owens, D.M. New, A.P. West, P.J. Bjorkman, Anti-PolyQ Antibodies Recognize a Short PolyQ Stretch in Both Normal and Mutant Huntingtin Exon 1., J. Mol. Biol. 427 (2015) 2507–19. doi:10.1016/j.jmb.2015.05.023.
IRBM-CONFIDENTIAL
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0006-291X(16)31312-2
DOI:
10.1016/j.bbrc.2016.08.057
Reference:
YBBRC 36258
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 5 August 2016 Accepted Date: 8 August 2016
Please cite this article as: M. Verani, M. Bustamante, P. Martufi, M. Daldin, C. Cariulo, L. Azzollini, V. Fodale, F. Puglisi, A. Weiss, D. Macdonald, L. Petricca, A. Caricasole, Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.08.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins Margherita Verani1,2, Maria Bustamante2, Paola Martufi2, Manuel Daldin2, Cristina Cariulo2,
Lara Petricca*1,2 and Andrea Caricasole*1,2,$% 1. IRBM Promidis Via Pontina km 30.600 00071 Pomezia, Rome, Italy
SC
2. IRBM Science Park, Via Pontina km 30.600 00071 Pomezia, Rome, Italy
RI PT
Lucia Azzollini1,2, Valentina Fodale1,2, Francesca Puglisi2, Andreas Weiss1#, Douglas Macdonald3,
AC C
EP
TE D
M AN U
3. CHDI Management/CHDI Foundation, Los Angeles, CA 90045, USA
*These authors contributed equally to the present work #: Current address: Evotec AG, Manfred Eigen Campus, Hamburg, Germany %: Current address: Alzheimer’s Research UK Drug Discovery Institute, University of Cambridge, Cambridge, UK
$: To whom correspondence should be addressed
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
Abstract We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical
RI PT
methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized,
SC
unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based
M AN U
conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-
AC C
EP
TE D
mediated conformational change in different proteins associated with CAG triplet repeat disorders.
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
Introduction The human genome contains several loci encoding polyglutamine domains, encoded by CAG repeats [1]. CAG triplet repeat disorders represent a class of rare genetic neurodegenerative conditions
RI PT
associated with the expansion of a polyglutamine (polyQ; Q being encoded by CAG or CAA) repeat domain [2,3]. Although different neuronal subtypes and brain regions are characteristically affected in each disease, a unifying feature is the production of intracellular/neuronal protein aggregates, typically enriched for the mutant (polyQ-expanded) protein or its expanded polyQ-containing fragments [2,4,5]. This invariably leads
SC
to progressive neurodegeneration that cannot presently be addressed therapeutically [6]. Although loss of normal protein function may play a role in at least some cases [7–9], pathology is thought to be driven largely through a gain of function correlated with polyQ expansion [10,11] and associated with
M AN U
conformational change [12–15] that leads to one or more dysfunctions affecting intra- and/or intermolecular interactions, subcellular localization and compartmentalized activity, proteolysis, protein clearance and oligomerization/aggregation. This is exemplified by the prototypical CAG triplet repeat disorder, Huntington’s disease (HD) [16–22], a progressive, autosomal dominant neurodegenerative disease caused by expansion of a polyQ encoding repeat within exon 1 of the huntingtin (HTT) gene [22]. The disease is associated with the generation of N-terminal fragments of mutant HTT (mHTT) protein
TE D
comprising the expanded polyQ domain and likely produced through mechanisms such as aberrant splicing and proteolysis [23–25]. Misfolding and self-association of these fragments give rise to oligomers and the distinctive aggregates observed in the HD brain, which can give rise to HD-like phenotype in animal models [29,25]. Sensitive, accurate measurements of soluble wild type and mHTT conformers in different
EP
preclinical and clinical samples is possible using time-resolved Förster Energy Transfer (TR-FRET) based immunoassays [26,27]; measurement is based on the labeling of an antibody pair with a rare earth ion
AC C
fluorophore donor and an acceptor fluorophore, which generate a specific TR-FRET signal when the donor and acceptor labeled antibodies bind to their antigen simultaneously. Several studies have addressed the conformational changes associated with polyQ expansion, particularly in HTT [15]. The recent development of conformational, TR-FRET based immunoassays [15,28] has confirmed the apparent structural rigidity imparted on HTT by polyQ expansion [29] and has opened the possibility of developing approaches to modulate mHTT conformation with the aims to alter its toxic properties. The exquisite polyQ dependence of the conformational immunoassays, requiring interrogation of the polyQ domain with specific antibodies [15,28], suggests that a similar approach may be used to interrogate the conformation of other polyQ repeat disease-associated proteins and supports an intrinsic structural property of polyQ repeats in imparting conformational rigidity. Here, we demonstrate the general applicability of the conformational immunoassay to detect conformational rigidity imparted by polyQ expansion in other polyQ repeat IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT proteins, exemplified by the androgen receptor (whose polyQ domain is expanded in spinal bulbar muscular atrophy, SBMA [30–32]) and TATA-binding protein (TBP, whose polyQ domain is expanded in spinocerebellar ataxia 17, SCA17 [33,5,34]). Further, we provide additional evidence supporting the notion that conformational rigidity imparted on relevant proteins by polyQ expansion is due to an intrinsic structural property of the polyQ domain.
RI PT
Materials and Methods Plasmids
For expression of proteins of interest, cDNA inserts were designed as follows. HTT exon 1
SC
sequences with Q16 and Q72 were essentially as described previously [15], with a FLAG epitope Cterminally fused to the end of exon 1. Art polyQ sequences were designed to encode proteins of similar
M AN U
molecular weight and identical polyQ lengths to HTT exon 1 proteins, and to include N-terminal and Cterminal epitopes for easy TR-FRET detection (MYC and FLAG). In Art polyQ proteins, the methionine codon is
sequentially
followed (as
a translational fusion)
by
a MYC epitope,
a linker region
(ANSSIDLISVPVEYPYDVPDYASR; [35]), a polyQ encoding domain (Q16 or Q72), and a second linker region (RPACKIPNDLKQKVMNH; [35]) followed by a C-terminal FLAG. Art polyMYC sequences were designed to encode proteins comparable to Art polyQ proteins except for the polyQ domains, which were replaced by
TE D
sequences encoding either 2 MYC epitopes (to mimick the 8-10 residue epitope of anti-polyQ antibodies such as MW1) or 8 MYC epitopes (to mimick the multimerized MW1 epitope present in a Q72 expansion). In Art polyMYC proteins the methionine codon is sequentially followed (as a translational fusion) by the ANSSIDLISVPVEYPYDVPDYASR linker region, the polyMYC encoding domain (2xMYC or 8xMYC), the second
EP
linker region RPACKIPNDLKQKVMNH and a C-terminal FLAG. Relevant protein sequences comprising the polyQ domains of androgen receptor (AR) and TATA binding protein (TBP) were selected based on
AC C
previously published constructs ([36,37] for AR and [38] for TBP). cDNAs encoding these protein fragments were designed to include N-terminal MYC and C-terminal FLAG epitopes. HEK293T cell culture and transfection HEK293T cells were cultured, transiently transfected and whole cell lysates were prepared at the indicated times after transfection, exactly as previously reported in [15,39].
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
SDS-PAGE and immunoblotting SDS-PAGE and immunoblotting were performed as described in [15]. The anti-MYC (cat. M4439), anti-FLAG (cat. F1804) and anti-GAPDH (cat. G9545) antibodies were obtained from Sigma-Aldrich. The
RI PT
MW1 antibody [40] was obtained from the Developmental Studies Hybridoma Bank, The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Antibody 4C9 was described previously [27,41] and obtained from the CHDI Foundation (New York, NY). Custom terbium cryptate and D2fluorophore antibody labeling was performed by CisBio (Bagnols, France) as reported in [15].
SC
TR-FRET assays
TR-FRET assays were performed essentially as described previously [15]. The specificity of the FLAG-
M AN U
MW1, FLAG-4C9 and FLAG-MYC TR-FRET immunoassays was tested by a) omitting one of the antibodies of the TR-FRET-pair in each assay and b) performing the measurements on lysates overexpressing unrelated proteins (data not shown).
Results and discussion
TE D
Previous studies by us [15], and others [28] reported TR-FRET immunoassay detection of a conformational change imparted by mutant polyQ expansion on soluble HTT. These studies identified an absolute requirement on polyQ interrogation (with one of the TR-FRET antibodies in the antibody pair) for conformational change detection by TR-FRET immunoassay methods. Therefore, we decided to further
EP
investigate the exquisite polyQ dependence of the structural change detected by the conformational immunoassay and to determine its relevance to other polyQ disease proteins associated with
AC C
neurodegeneration, by studying proteins expressed in a biological context (transfected human cells). In order to do this, we designed different expression constructs (Fig. 1Ai) encoding either HTT with Q16 or Q72, an artificial protein encoding polyQ domains identical to those present in the HTT constructs employed (Q16 or Q72), or a comparable artificial construct where the polyQ domain is replaced by a multimerized MYC epitope (2xMYC or 8xMYC) to mimic theoretical epitope availability present for antipolyQ antibody MW1 on HTT Q16 and Q72, respectively. Correct expression of these constructs following transient transfection of HEK293T cells was verified by SDS-PAGE followed by immunoblotting (Fig. 1Aii). In these blots, detection with antibody MW1 predominantly detected polyQ expansion (Q72) in mutant HTT and mutant Art polyQ (Q72) over the wildtype (Q16) proteins, while detection with anti-MYC detected the presence of the MYC epitope in Art polyQ and Art polyMYC encoding constructs (Fig. 1Aii). Anti-FLAG immunoblotting detected expression of all constructs at the expected molecular weight (Fig. 1Aii). IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT Additional constructs (Fig. 1Bi) were designed, encoding androgen receptor (AR) and TATA binding protein (TBP) fragments comprising their wild type (Q19 for AR and Q36 for TBP) or mutant counterparts (Q65 and Q80, respectively). The AR and TBP protein regions encoded by the cDNAs were selected based on previous studies using cDNAs encoding protein fragments of the respective proteins ([36,37] for AR and [38] for TBP). Again, correct expression of these constructs following transient transfection of HEK293T cells was
RI PT
verified by SDS-PAGE followed by immunoblotting (Fig. 1Bii). MYC and FLAG epitopes at the N-terminus and C-terminus of these constructs were introduced in order to enable TR-FRET measurements independent of anti-HTT specific antibodies (including a FLAG epitope at the C-terminus of HTT exon 1-encoding constructs). These constructs were first used to investigate the role of the protein context on the structural rigidity imparted by polyQ expansion, as detected by the conformational immunoassay. Second, as polyQ
SC
expansions likely result in an increase in the number of epitopes for anti-polyQ antibodies we asked if increasing epitope number per se is sufficient to alter protein conformation as detected by TR-FRET based
M AN U
immunoassays, or if this is based on an intrinsic property of polyQ repeats.
A conformational constraint is imparted on proteins by repeat-length expansion of the polyQ domain We therefore asked if a polyQ domain can determine conformational changes by itself, irrespective of protein context, by interrogating wholly artificial constructs bearing a short (Q16) or mutant-like (Q72) polyQ expansion, as well as AR and TBP protein fragments, comprising their respective polyQ domains (wild
TE D
type or mutant), as their non-polyQ sequences are unrelated to each other and HTT. To do this, we individually transfected each plasmid of the four pairs of constructs into HEK293T cells (Fig. 2A). The first pair of constructs (HTT Ex1 Q16 or Q72) encoded HTT exon 1 with a Q16 or a Q72 repeat (Fig. 2Ai). This pair served as a reference, control pair, and yielded comparable signals for lysates expressing the Q16 and
EP
Q72 HTT exon 1 proteins with the FLAG-4C9 TR-FRET antibody pair, indicative of comparable expression levels of the two constructs in HEK293T cells (Fig. 2Aii). As expected, the FLAG-MW1 antibody pair
AC C
produced a TR-FRET signal which was polyQ-dependent (Fig. 2Aiii), as previously reported for a HTT-specific polyQ-dependent TR-FRET antibody pair (2B7-MW1; [15,28]). The second pair of constructs encoded a Q16 or Q72 polyQ expansion flanked at the N-terminus and C-terminus by artificial sequences, and bearing an N-terminus MYC epitope and a C-terminal FLAG epitope (Fig. 2Bi). This pair of constructs (Art Q16 and Art Q72) was designed to determine if HTT sequences N-terminal or C-terminal of the polyQ are absolutely required for HTT’s conformational plasticity detected by the TR-FRET 2B7/MW1 immunoassay. Again comparable signals for lysates expressing the Q16 and Q72 Art proteins were obtained with the FLAG-4C9 TR-FRET antibody pair (Fig. 2Bii), while a polyQ-dependent TR-FRET signal was obtained with the FLAGMW1 antibody pair (Fig. 2Biii). The third and fourth pairs of constructs encoded wild type or mutant AR and TBP proteins, respectively (Fig. 2Ci and 2Di). As observed for HTT proteins and for the Art polyQ proteins, the FLAG-4C9 TR-FRET antibody pair did not result in increased signal in the expanded (mutant) proteins IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT (Fig. 2Cii and 2Dii). Significantly, however, the FLAG-MW1 TR-FRET pair showed a strong increase in lysates expressing the mutant AR protein (Fig. 2Ciii), and the polyQ-dependent increase was evident even in lysates expressing mutant TBP, although a significant polyQ expansion is present in wild type TBP already (Fig. 2Diii). Collectively, these data are consistent with the capacity of MW1-based TR-FRET immunoassays to detect repeat length-dependent changes in polyQ epitopes in different proteins, including HTT. We next
RI PT
turned to determining if the FLAG-MW1 TR-FRET immunoassay can detect the temperature- and polyQdependent conformational change previously reported in HTT for the 2B7-MW1 TR-FRET immunoassay [15,28] and if this is detectable in artificial proteins (Art polyQ) and in polyQ disease-associated proteins other than HTT (AR and TBP). As shown in Fig. 3Aii, a control (FLAG-4C9) TR-FRET immunoassay (which does not interrogate the polyQ domain) yields an essentially identical and polyQ-independent signal in Q16 and
SC
Q72 HTT proteins (Fig. 3A), as previously reported for the 4C9-MW1 TR-FRET immunoassay [15,28], while the FLAG-MW1 TR-FRET immunoassay easily detects the conformational change imparted by polyQ
M AN U
expansion on HTT exon 1 protein, as previously reported for the 2B7-MW1 TR-FRET immunoassay [15,28]. Similar results were obtained for lysates expressing Art polyQ (Fig. 3B), AR (Fig. 3C) and TBP (Fig. 3D), where the control (FLAG-4C9) TR-FRET immunoassay yields a polyQ-independent signal in wild type and mutant proteins (Fig. 3Bii, 3Cii and 3Dii), while the FLAG-MW1 TR-FRET immunoassay easily detects the conformational change imparted by polyQ expansion on all proteins. Together with the previously reported requirement of the conformational TR-FRET immunoassays for polyQ interrogation and its independence
TE D
on HTT protein fragment length [15,28], these data are strongly supportive of the notion that the polyQ domain is necessary and sufficient to produce a repeat-length conformational constraint on proteins. Importantly, the data demonstrate that polyQ expansion can alter the conformational properties of other
EP
proteins associated with CAG repeat-disorders in a manner similar to that observed for HTT. Intrinsic nature of the conformational constraint imparted on proteins by repeat-length expansion of the
AC C
polyQ domain
We next asked if a multimerized artificial epitope (MYC), designed to mimic epitope recognition by MW1 of a Q16 or a Q72 repeat can impart a conformational constraint detectable by a relevant TR-FRET immunoassay. The multimerized MYC epitope presents either 2 or 8 epitopes for its specific (anti-MYC) antibody, in order to mimic MW1 recognition of a Q16 and Q72 polyQ region (which appears to involve a linear epitope of 8-10 glutamines [42,43]), and is flanked at the N-terminus and C-terminus by artificial sequences (the same as in Art polyQ). This pair of constructs (Art 2xMYC and 8xMYC; Fig. 4Ai) was then used to determine the dependence of the polyQ- and temperature-dependent conformational changes on epitope multimerization rather than intrinsic polyQ properties. If interrogation of a multimerized, nonpolyQ epitope with a specific antibody could substitute for the polyQ-anti polyQ antibody interaction, then epitope multimerization rather than polyQ conformation would be relevant for the observed effect. IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT Analysis of the HEK293T cells lysates with the FLAG-MYC pair detected a relatively higher signal in the sample expressing the Art polyMYC 8xMYC construct, as might be expected given the presence of a multimerized MYC epitope. Interestingly, when the lysates where interrogated using this TR-FRET immunoassay at the two temperatures (20°C and 4°C, as done for the 2B7-MW1 and FLAG-MW1 TR-FRET immunoassays to detect the repeat-length conformational constraint imposed by polyQ on HTT and other
RI PT
polyQ proteins), no MYC epitope-length dependent effect was observed, unlike results obtained when polyQ repeat proteins are interrogated with relevant TR-FRET immunoassays (where one antibody in the TR-FRET pair interrogates the polyQ domain). This discordance in behaviour between multimerized MYC and polyQ epitopes indicates that conformational differences detected by TR-FRET immunoassays at the two temperatures are due to some inherent properties of the polyQ region rather than simple
SC
multimerization of an epitope.
In conclusion, we have provided evidence that polyQ expansion can also influence the
M AN U
conformation of other polyQ repeat-associated proteins. Consistent with previous reports, our data also show that an intrinsic property of polyQ repeats, rather than simple epitope multimerization, is associated with this effect. The conformational, TR-FRET based immunoassays may prove useful, practical and scalable tools to identify and further develop modifiers of conformation for a range of CAG repeat-associated proteins, which could constitute the basis for novel therapeutic development in different CAG repeat
Figure legends
TE D
expansion diseases.
EP
Figure 1. Schemes of the different constructs, and confirmation of their correct expression in transiently transfected HEK293T cells. A. Details and characterization of constructs encoding HTT exon 1 (HTT Ex1; Q16
AC C
and Q72) and similarly sized artificial proteins encoding multimeric MW1 (Art polyQ; Q16 and Q72) or antiMYC epitopes (Art polyMYC; 2x and 8x). i) Schemes of constructs ii). Representative immunoblots of whole cell lysates from HEK293T cells transiently transfected with the constructs in Ai and probed for different antibodies to confirm presence of polyQ expansions (MW1), MYC epitopes (anti-MYC), expression of all constructs (anti-FLAG) and GAPDH (anti-GAPDH) to control for total protein content. B. Details and characterization of constructs encoding androgen receptor (AR) and TATA binding protein (TBP) protein fragments. i) Schemes of constructs encoding wild type and mutant AR (Q19 and Q65) and TATA binding protein (TBP; Q36 and Q80). ii) Representative immunoblots of whole cell lysates from HEK293T cells transiently transfected with the constructs in Ai and probed for different antibodies to confirm presence of polyQ expansions (MW1), MYC epitopes (anti-MYC), expression of all constructs (anti-FLAG) and GAPDH (anti-GAPDH) to control for total protein content. Numbers indicate MW (in kDa). IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT Figure 2. TR-FRET analysis of HEK293T cell lysates expressing the different constructs (HTT Ex1, Art polyQ, AR and TBP; Ai, Bi, Ci, Di, respectively) with wild type or mutant polyQ repeats, using the FLAG-4C9 (HTT ex1; Ai) or FLAG-MYC antibody pairs (Bii, Cii, Dii, respectively), which do not interrogate the polyQ repeat. TR-FRET analysis of HEK293T cell lysates expressing the different constructs with the FLAG-MW1 antibody pair, which interrogates the polyQ repeat, is shown in Aiii, Biii, Ciii, Diii. Data obtained from a
RI PT
representative experiment are shown. At least three independent biological replicates were performed. Figure 3. Conformational immunoassay analysis of cell lysates expressing the different constructs (HTT Ex1, Art polyQ, AR and TBP. Data represent ratiometric values of TR-FRET signals obtained for each construct at the two temperatures (4°C and 20°C). Ai, Bi, Ci, Di, respectively) with wild type or mutant polyQ repeats.
SC
Data obtained using the FLAG-4C9 (HTT ex1; Ai) or FLAG-MYC antibody pairs (Bii, Cii, Dii, respectively), which do not interrogate the polyQ repeat. In Aiii, Biii, Ciii, Diii the data obtained by TR-FRET analysis of HEK293T cell lysates expressing the different constructs with the FLAG-MW1 antibody pair, which
M AN U
interrogates the polyQ repeat, are shown. Data represent the means and standard deviations of the means of at least three independent experiments (one-way ANOVA Tukey's Multiple Comparison Test, degrees of significance are indicated).
Figure 4. A. Standard and conformational TR-FRET analysis of the Art-polyMYC construct.. A i) Details of the Art polyMYC constructs, designed to mimick the presence of a small (2xMYC) or larger (8xMYC) number of
TE D
epitopes for the interrogating antibody (as in the case of Q16 and Q72 for MW1). ii) Data obtained by TRFRET analysis of HEK293T cell lysates expressing the two constructs with the FLAG-MYC antibody pair, which interrogates the MYC repeat from one representative experiment are shown. iii) ratio of TR-FRET signals obtained at the two temperatures (4°C/20°C) using the FLAG-MYC antibody pair on these constructs
EP
(similar to FLAG-MW1 conformational TR-FRET analysis of Art polyQ). Data represent the means and standard deviations of the means of at least three independent experiments ((one-way ANOVA Tukey's degrees of significance are indicated). B. Schematic interpretation of the lack of
AC C
Multiple Comparison Test,
conformational effects of temperature and epitope multimerization on TR-FRET signals obtained with the FLAG-MYC antibody pair on Art polyMYC constructs, compared to the effects observed on TR-FRET signals with the FLAG-MW1 antibody pair on Art polyQ constructs with i) short (Q16) and ii) long (Q72) polyQ repeats.
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[5]
[6]
[7]
[8]
[9]
[10]
SC
M AN U
[4]
TE D
[3]
EP
[2]
S.L. Butland, R.S. Devon, Y. Huang, C.L. Mead, A.M. Meynert, S.J. Neal, et al., CAG-encoded polyglutamine length polymorphism in the human genome, BMC Genomics. 8 (2007) 126. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17519034. C.M. Everett, N.W. Wood, Trinucleotide repeats and neurodegenerative disease, Brain. 127 (2004) 2385–2405. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=15329351. J.R. Gatchel, H.Y. Zoghbi, Diseases of unstable repeat expansion: mechanisms and common principles, Nat Rev Genet. 6 (2005) 743–755. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16205714. S.W. Davies, K. Beardsall, M. Turmaine, M. DiFiglia, N. Aronin, G.P. Bates, Are neuronal intranuclear inclusions the common neuropathology of triplet-repeat disorders with polyglutamine-repeat expansions?, Lancet. 351 (1998) 131–133. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=9439509. M.J. Friedman, A.G. Shah, Z.H. Fang, E.G. Ward, S.T. Warren, S. Li, et al., Polyglutamine domain modulates the TBP-TFIIB interaction: implications for its normal function and neurodegeneration, Nat Neurosci. 10 (2007) 1519–1528. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17994014. N.A. Di Prospero, K.H. Fischbeck, Therapeutics development for triplet repeat expansion diseases, Nat Rev Genet. 6 (2005) 756–765. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16205715. E. Cattaneo, D. Rigamonti, D. Goffredo, C. Zuccato, F. Squitieri, S. Sipione, Loss of normal huntingtin function: new developments in Huntington’s disease research, Trends Neurosci. 24 (2001) 182–188. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=11182459. P.S. Thomas Jr., G.S. Fraley, V. Damian, L.B. Woodke, F. Zapata, B.L. Sopher, et al., Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy, Hum Mol Genet. 15 (2006) 2225–2238. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16772330. C. Zuccato, M. Valenza, E. Cattaneo, Molecular mechanisms and potential therapeutical targets in Huntington’s disease, Physiol Rev. 90 (2010) 905–981. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=20664076. H.T. Orr, Polyglutamine neurodegeneration: expanded glutamines enhance native functions, Curr Opin Genet Dev. 22 (2012) 251–255. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=22284692.
AC C
[1]
RI PT
Reference list
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[16]
[17]
[18]
[19]
[20]
[21]
[22] [23]
RI PT
SC
[15]
M AN U
[14]
TE D
[13]
EP
[12]
J.C. Jacobsen, G.C. Gregory, J.M. Woda, M.N. Thompson, K.R. Coser, V. Murthy, et al., HD CAGcorrelated gene expression changes support a simple dominant gain of function, Hum Mol Genet. 20 (2011) 2846–2860. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=21536587. J. Shao, M.I. Diamond, Polyglutamine diseases: emerging concepts in pathogenesis and therapy, Hum Mol Genet. 16 Spec No (2007) R115–23. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17911155. Y. Nagai, H.A. Popiel, Conformational changes and aggregation of expanded polyglutamine proteins as therapeutic targets of the polyglutamine diseases: exposed beta-sheet hypothesis, Curr Pharm Des. 14 (2008) 3267–3279. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19075705. B. Almeida, S. Fernandes, I.A. Abreu, S. Macedo-Ribeiro, Trinucleotide repeats: a structural perspective, Front Neurol. 4 (2013) 76. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=23801983. V. Fodale, N.C. Kegulian, M. Verani, C. Cariulo, L. Azzollini, L. Petricca, et al., Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy, PLoS One. 9 (2014) e112262. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=25464275. N. Hazeki, K. Nakamura, J. Goto, I. Kanazawa, Rapid aggregate formation of the huntingtin Nterminal fragment carrying an expanded polyglutamine tract, Biochem Biophys Res Commun. 256 (1999) 361–366. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=10079189. A. Lunkes, K.S. Lindenberg, L. Ben-Haiem, C. Weber, D. Devys, G.B. Landwehrmeyer, et al., Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions, Mol Cell. 10 (2002) 259–269. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=12191472. E. Roze, F. Saudou, J. Caboche, Pathophysiology of Huntington’s disease: from huntingtin functions to potential treatments, Curr Opin Neurol. 21 (2008) 497–503. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=18607213. C. Landles, K. Sathasivam, A. Weiss, B. Woodman, H. Moffitt, S. Finkbeiner, et al., Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease, J Biol Chem. 285 (2010) 8808–8823. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=20086007. D.E. Ehrnhoefer, L. Sutton, M.R. Hayden, Small changes, big impact: posttranslational modifications and function of huntingtin in Huntington disease, Neuroscientist. 17 (2011) 475–492. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=21311053. C.A. Ross, S.J. Tabrizi, Huntington’s disease: from molecular pathogenesis to clinical treatment, Lancet Neurol. 10 (2011) 83–98. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=21163446. G.P. Bates, R. Dorsey, J.F. Gusella, M.R. Hayden, C. Kay, B.R. Leavitt, et al., Huntington disease, Nat. Rev. Dis. Prim. (2015) 15005. http://dx.doi.org/10.1038/nrdp.2015.5. K. Sathasivam, A. Neueder, T.A. Gipson, C. Landles, A.C. Benjamin, M.K. Bondulich, et al., Aberrant
AC C
[11]
IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
RI PT
SC
[28]
M AN U
[27]
TE D
[26]
EP
[25]
AC C
[24]
splicing of HTT generates the pathogenic exon 1 protein in Huntington disease., Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 2366–70. doi:10.1073/pnas.1221891110. T.A. Gipson, A. Neueder, N.S. Wexler, G.P. Bates, D. Housman, Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis, RNA Biol. 10 (2013) 1647–1652. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=24256709. B.A. Barbaro, T. Lukacsovich, N. Agrawal, J. Burke, D.J. Bornemann, J.M. Purcell, et al., Comparative study of naturally occurring huntingtin fragments in Drosophila points to exon 1 as the most pathogenic species in Huntington’s disease, Hum Mol Genet. 24 (2015) 913–925. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=25305076. B. Baldo, P. Paganetti, S. Grueninger, D. Marcellin, L.S. Kaltenbach, D.C. Lo, et al., TR-FRET-based duplex immunoassay reveals an inverse correlation of soluble and aggregated mutant huntingtin in Huntington’s disease, Chem. Biol. 19 (2012) 264–275. doi:10.1016/j.chembiol.2011.12.020. A. Weiss, D. Abramowski, M. Bibel, R. Bodner, V. Chopra, M. DiFiglia, et al., Single-step detection of mutant huntingtin in animal and human tissues: a bioassay for Huntington’s disease, Anal Biochem. 395 (2009) 8–15. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19664996. X. Cui, Q. Liang, Y. Liang, M. Lu, Y. Ding, B. Lu, TR-FRET assays of Huntingtin protein fragments reveal temperature and polyQ length-dependent conformational changes, Sci Rep. 4 (2014) 5601. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=24998512. N.S. Caron, C.R. Desmond, J. Xia, R. Truant, Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin., Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 14610–5. doi:10.1073/pnas.1301342110. A. Poletti, The polyglutamine tract of androgen receptor: from functions to dysfunctions in motor neurons, Front Neuroendocr. 25 (2004) 1–26. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=15183036. I. Palazzolo, A. Gliozzi, P. Rusmini, D. Sau, V. Crippa, F. Simonini, et al., The role of the polyglutamine tract in androgen receptor, J Steroid Biochem Mol Biol. 108 (2008) 245–253. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17945479. J.P. Chua, A.P. Lieberman, Pathogenic mechanisms and therapeutic strategies in spinobulbar muscular atrophy, CNS Neurol Disord Drug Targets. 12 (2013) 1146–1156. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=24040817. W.M. van Roon-Mom, S.J. Reid, R.L. Faull, R.G. Snell, TATA-binding protein in neurodegenerative disease, Neuroscience. 133 (2005) 863–872. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=15916858. R. Gao, T. Matsuura, M. Coolbaugh, C. Zuhlke, K. Nakamura, A. Rasmussen, et al., Instability of expanded CAG/CAA repeats in spinocerebellar ataxia type 17, Eur J Hum Genet. 16 (2008) 215–222. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=18043721. T.K. Kerppola, Bimolecular fluorescence complementation: visualization of molecular interactions in living cells, Methods Cell Biol. 85 (2008) 431–470. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=18155474. Y. Takeshita, R. Fujinaga, C. Zhao, A. Yanai, K. Shinoda, Huntingtin-associated protein 1 (HAP1) interacts with androgen receptor (AR) and suppresses SBMA-mutant-AR-induced apoptosis, Hum Mol Genet. 15 (2006) 2298–2312. IRBM-CONFIDENTIAL
ACCEPTED MANUSCRIPT
[41]
[42]
[43]
RI PT
SC
M AN U
[40]
TE D
[39]
EP
[38]
AC C
[37]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=16782802. J.E. Young, G.A. Garden, R.A. Martinez, F. Tanaka, C.M. Sandoval, A.C. Smith, et al., Polyglutamineexpanded androgen receptor truncation fragments activate a Bax-dependent apoptotic cascade mediated by DP5/Hrk, J Neurosci. 29 (2009) 1987–1997. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19228953. P.J. Kung, Y.C. Tao, H.C. Hsu, W.L. Chen, T.H. Lin, D. Janreddy, et al., Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models, Drug Des Devel Ther. 8 (2014) 1929–1939. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=25342886. M.B. Bustamante, A. Ansaloni, J.F. Pedersen, L. Azzollini, C. Cariulo, Z.M. Wang, et al., Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKK??, Biochem. Biophys. Res. Commun. 463 (2015) 1317–1322. doi:10.1016/j.bbrc.2015.06.116. J. Ko, S. Ou, P.H. Patterson, New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins, Brain Res Bull. 56 (2001) 319–329. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=11719267. P. Paganetti, A. Weiss, M. Trapp, I. Hammerl, D. Bleckmann, R.A. Bodner, et al., Development of a method for the high-throughput quantification of cellular proteins, Chembiochem. 10 (2009) 1678– 1688. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=19492395. P. Li, K.E. Huey-Tubman, T. Gao, X. Li, A.P. West Jr., M.J. Bennett, et al., The structure of a polyQanti-polyQ complex reveals binding according to a linear lattice model, Nat Struct Mol Biol. 14 (2007) 381–387. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui ds=17450152. G.E. Owens, D.M. New, A.P. West, P.J. Bjorkman, Anti-PolyQ Antibodies Recognize a Short PolyQ Stretch in Both Normal and Mutant Huntingtin Exon 1., J. Mol. Biol. 427 (2015) 2507–19. doi:10.1016/j.jmb.2015.05.023.
IRBM-CONFIDENTIAL
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT