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Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

Fodale V, Kegulian NC, Verani M, Cariulo C, Azzollini L, Petricca L, Daldin M, Boggio R, Padova A, Kuhn R, Pacifici R, Macdonald D, Schoenfeld RC, Park H, Isas JM, Langen R, Weiss A, Caricasole A - PLoS ONE (2014)

Bottom Line: Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties.By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins.Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.

View Article: PubMed Central - PubMed

Affiliation: IRBM Promidis, Pomezia, Rome, Italy.

ABSTRACT

Background: In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays.

Methodology/principal findings: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.

Conclusions/significance: The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.

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Related in: MedlinePlus

Reversibility of the temperature-dependent effect on TR-FRET signal.Following assembly of the TR-FRET cocktail (2B7-Tb and MW1-d2, 1∶10 ratio), the same samples were subjected to 2 successive cycles of incubation (for 1 hr) and measurement, at the two different temperatures (RT and 4°C). Note that the difference between fluorescence ratio values obtained at RT or 4°C is inversely correlated with polyQ length. A-F. Reversibility for HTT N548 HTT of increasing polyQ length (Q16, Q19, Q25, Q33, Q39, Q55). G. Correlation between fluorescence ratio values obtained in the two RT readings performed during the cycles. H. Correlation between fluorescence ratio values obtained in the two 4°C readings performed during the cycles.
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pone-0112262-g004: Reversibility of the temperature-dependent effect on TR-FRET signal.Following assembly of the TR-FRET cocktail (2B7-Tb and MW1-d2, 1∶10 ratio), the same samples were subjected to 2 successive cycles of incubation (for 1 hr) and measurement, at the two different temperatures (RT and 4°C). Note that the difference between fluorescence ratio values obtained at RT or 4°C is inversely correlated with polyQ length. A-F. Reversibility for HTT N548 HTT of increasing polyQ length (Q16, Q19, Q25, Q33, Q39, Q55). G. Correlation between fluorescence ratio values obtained in the two RT readings performed during the cycles. H. Correlation between fluorescence ratio values obtained in the two 4°C readings performed during the cycles.

Mentions: We reasoned that the conformational flexibility of the N-terminal portion of HTT may be detected by TR-FRET assays by interrogating relevant epitopes. In the appropriate context, TR-FRET signals can inform on epitope accessibility as well as relative distance between the antibodies themselves ([28], [29]), and therefore can provide conformational information on the protein analyte. In the simplest context, we elected to investigate conformational flexibility in recombinant, purified HTT proteins by TR-FRET using an antibody pair comprising previously characterized monoclonals (2B7 and MW1; Fig. 1A; [13], [27], [30]–[32]) interrogating two regions of known relevance for HTT conformation and biological properties, namely the N17 domain and the polyQ domain (e.g. see [17], [24], [24], [26]). As one of the major factors influencing the stability of protein conformation is temperature (e.g. see[33]), we pragmatically chose this variable and decided to investigate HTT behaviour at a low temperature (4°C) and at the temperature at which TR-FRET assays are normally performed (room temperature, 20°C, RT). In order to avoid possible confounding effects due to aggregation, we chose to employ recombinant, purified N548 HTT fragments of different polyQ length, which should not aggregate or aggregate at a lower rate respective to the exon 1 under the conditions employed (Fig. 1B and C, [13]). Initially, we selected HTT proteins bearing two polyQ lengths, representing a wild type (Q16) and an expanded, or mutant (Q55), context. We then performed TR-FRET using antibodies 2B7 (labeled with a donor fluorophore, namely terbium) and MW1 (labeled with the acceptor fluorophore, D2), performing the incubation prior to reading at RT and then shifting the sample to 4°C for 2 hrs, first on a fixed (3.3 ng) and then on a range of HTT protein analyte concentrations (Fig. 2 A–E). As expected, the specific (donor-acceptor energy transfer dependent) fluorescence signal was significantly higher for mutant (Q55) HTT protein relative to wild-type (Q16) HTT protein (Fig. 2A). On this substrate, at RT the fluorescence signal obtained with the 2B7-Tb MW1-D2 antibody pair was close to that obtained from acceptor labeled MW1 alone (Fig. 2A). This is consistent with the specificity of the assay for mutant HTT protein ([13]). Interestingly, the TR-FRET signal obtained with 2B7-MW1 on wild type (Q16) N548 HTT protein can be significantly affected by temperature, where sample incubation at 4°C results in significantly higher TR-FRET signal than samples incubated at RT (Fig. 2B and C). However, when the samples containing the mutant (Q55) N548 HTT protein are incubated at 4°C the increase in TR-FRET signal was significantly reduced relative to the increase observed for the Q16 counterpart (Fig. 2B and C), suggesting that polyQ expansion influences the effects of temperature on protein conformation and/or antibody epitope recognition. The observed effect is not influenced by the stoichiometry of the donor and acceptor antibody pair, as performing the experiments with a 1∶1 instead of a 1∶10 ratio of donor to acceptor antibody pair, in which the acceptor-labeled MW1 is more limiting, still results in a temperature- and polyQ dependent variation in the obtained TR-FRET signal (Fig. 2D and E). A clear polyQ dependence of the temperature effect in the 2B7-MW1 TR-FRET signal was confirmed using HTT substrates of increasing polyQ length (Fig. 3A–C). Additionally, to further avoid the possibility of different MW1 binding sites between wild type and expanded HTT influencing the findings when using acceptor-labeled MW1, experiments were performed where 2B7 was labeled with acceptor label and MW1 with donor label. The results (Fig. 3D) indicate that the influence of temperature on TR-FRET signal using 2B7 and MW1 antibodies is independent of the nature of the acceptor antibody. The effect is rapidly (1 hr-cycles) reversible (Fig. 4), indicating its dependence on fast operating events compatible with conformational changes. We therefore concluded that, under these experimental conditions, temperature can influence HTT epitope exposure/recognition and/or protein analyte conformation, and investigated this further employing a non immunoassay-based orthogonal readout.


Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

Fodale V, Kegulian NC, Verani M, Cariulo C, Azzollini L, Petricca L, Daldin M, Boggio R, Padova A, Kuhn R, Pacifici R, Macdonald D, Schoenfeld RC, Park H, Isas JM, Langen R, Weiss A, Caricasole A - PLoS ONE (2014)

Reversibility of the temperature-dependent effect on TR-FRET signal.Following assembly of the TR-FRET cocktail (2B7-Tb and MW1-d2, 1∶10 ratio), the same samples were subjected to 2 successive cycles of incubation (for 1 hr) and measurement, at the two different temperatures (RT and 4°C). Note that the difference between fluorescence ratio values obtained at RT or 4°C is inversely correlated with polyQ length. A-F. Reversibility for HTT N548 HTT of increasing polyQ length (Q16, Q19, Q25, Q33, Q39, Q55). G. Correlation between fluorescence ratio values obtained in the two RT readings performed during the cycles. H. Correlation between fluorescence ratio values obtained in the two 4°C readings performed during the cycles.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4251833&req=5

pone-0112262-g004: Reversibility of the temperature-dependent effect on TR-FRET signal.Following assembly of the TR-FRET cocktail (2B7-Tb and MW1-d2, 1∶10 ratio), the same samples were subjected to 2 successive cycles of incubation (for 1 hr) and measurement, at the two different temperatures (RT and 4°C). Note that the difference between fluorescence ratio values obtained at RT or 4°C is inversely correlated with polyQ length. A-F. Reversibility for HTT N548 HTT of increasing polyQ length (Q16, Q19, Q25, Q33, Q39, Q55). G. Correlation between fluorescence ratio values obtained in the two RT readings performed during the cycles. H. Correlation between fluorescence ratio values obtained in the two 4°C readings performed during the cycles.
Mentions: We reasoned that the conformational flexibility of the N-terminal portion of HTT may be detected by TR-FRET assays by interrogating relevant epitopes. In the appropriate context, TR-FRET signals can inform on epitope accessibility as well as relative distance between the antibodies themselves ([28], [29]), and therefore can provide conformational information on the protein analyte. In the simplest context, we elected to investigate conformational flexibility in recombinant, purified HTT proteins by TR-FRET using an antibody pair comprising previously characterized monoclonals (2B7 and MW1; Fig. 1A; [13], [27], [30]–[32]) interrogating two regions of known relevance for HTT conformation and biological properties, namely the N17 domain and the polyQ domain (e.g. see [17], [24], [24], [26]). As one of the major factors influencing the stability of protein conformation is temperature (e.g. see[33]), we pragmatically chose this variable and decided to investigate HTT behaviour at a low temperature (4°C) and at the temperature at which TR-FRET assays are normally performed (room temperature, 20°C, RT). In order to avoid possible confounding effects due to aggregation, we chose to employ recombinant, purified N548 HTT fragments of different polyQ length, which should not aggregate or aggregate at a lower rate respective to the exon 1 under the conditions employed (Fig. 1B and C, [13]). Initially, we selected HTT proteins bearing two polyQ lengths, representing a wild type (Q16) and an expanded, or mutant (Q55), context. We then performed TR-FRET using antibodies 2B7 (labeled with a donor fluorophore, namely terbium) and MW1 (labeled with the acceptor fluorophore, D2), performing the incubation prior to reading at RT and then shifting the sample to 4°C for 2 hrs, first on a fixed (3.3 ng) and then on a range of HTT protein analyte concentrations (Fig. 2 A–E). As expected, the specific (donor-acceptor energy transfer dependent) fluorescence signal was significantly higher for mutant (Q55) HTT protein relative to wild-type (Q16) HTT protein (Fig. 2A). On this substrate, at RT the fluorescence signal obtained with the 2B7-Tb MW1-D2 antibody pair was close to that obtained from acceptor labeled MW1 alone (Fig. 2A). This is consistent with the specificity of the assay for mutant HTT protein ([13]). Interestingly, the TR-FRET signal obtained with 2B7-MW1 on wild type (Q16) N548 HTT protein can be significantly affected by temperature, where sample incubation at 4°C results in significantly higher TR-FRET signal than samples incubated at RT (Fig. 2B and C). However, when the samples containing the mutant (Q55) N548 HTT protein are incubated at 4°C the increase in TR-FRET signal was significantly reduced relative to the increase observed for the Q16 counterpart (Fig. 2B and C), suggesting that polyQ expansion influences the effects of temperature on protein conformation and/or antibody epitope recognition. The observed effect is not influenced by the stoichiometry of the donor and acceptor antibody pair, as performing the experiments with a 1∶1 instead of a 1∶10 ratio of donor to acceptor antibody pair, in which the acceptor-labeled MW1 is more limiting, still results in a temperature- and polyQ dependent variation in the obtained TR-FRET signal (Fig. 2D and E). A clear polyQ dependence of the temperature effect in the 2B7-MW1 TR-FRET signal was confirmed using HTT substrates of increasing polyQ length (Fig. 3A–C). Additionally, to further avoid the possibility of different MW1 binding sites between wild type and expanded HTT influencing the findings when using acceptor-labeled MW1, experiments were performed where 2B7 was labeled with acceptor label and MW1 with donor label. The results (Fig. 3D) indicate that the influence of temperature on TR-FRET signal using 2B7 and MW1 antibodies is independent of the nature of the acceptor antibody. The effect is rapidly (1 hr-cycles) reversible (Fig. 4), indicating its dependence on fast operating events compatible with conformational changes. We therefore concluded that, under these experimental conditions, temperature can influence HTT epitope exposure/recognition and/or protein analyte conformation, and investigated this further employing a non immunoassay-based orthogonal readout.

Bottom Line: Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties.By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins.Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.

View Article: PubMed Central - PubMed

Affiliation: IRBM Promidis, Pomezia, Rome, Italy.

ABSTRACT

Background: In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays.

Methodology/principal findings: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.

Conclusions/significance: The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.

Show MeSH
Related in: MedlinePlus