34 research outputs found
Polyglutamine expansion affects huntingtin conformation in multiple Huntington's disease models
Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine-and temperaturedependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperatureand polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients
ULTRASENSITIVE MEASUREMENT OF MUTANT HUNTINGTIN IN HUMAN CSF AND RODENT SAMPLES
Huntington disease (HD) is a neurodegenerative disorder caused by a genetic defect in Huntingtin gene (<i>HTT</i>) that leads to the expression of an expanded poly-glutamine protein. HD has a broad impact on a person's functional abilities due to progressive motor dysfunction, cognitive decline, and psychiatric disturbance, probably caused by both neuronal dysfunction and neuronal cell death. New strategies for drug discovery and new therapeutic approaches are now leading to slow the progression of HD; one of these promising strategies consists in reducing the mutant protein level through its direct reduction (gene silencing) or increasing its clearance (compound treatment). In this framework mutant HTT protein can be a biomarker for treatment efficacy and disease progression, raising the necessity to develop highly sensitive immunoassays to quantify its level in biological fluids. The Single Molecule Counting (SMC?) technology, powered by Singulex?, enabled the ultrasensitive measurement of mutant HTT at levels previously undetectable in human Cerebrospinal fluid (CSF). The immunoassay was developed using the 2B7 and the MW1 antibodies that recognize the N-terminal region and the poly-Q trait of the protein respectively and was successfully applied to human and murine derived fluids and murine tissues. Here is presented a summary of results obtained from the assay validation process, including the evaluation of calibration curve performance, specificity, matrix effect (spike recovery, parallelism and dilution linearity), selectivity, accuracy, precision and stabilit
Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.
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
Quantifying autophagy using novel LC3B and p62 TR-FRET assays
<div><p>Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.</p></div
LC3B-II and p62 protein responses after tool compound.
<p>HEK293T cells (A), rat cortico-striatal neurons (B) and rat astrocytes (C) were treated with an autophagy inhibitor (5 nM bafilomycin A1) or upregulator (KU0063794) and examined at 2, 6 and 24 hours post-treatment, compared to DMSO. Western blot analysis is presented for LC3B-I/II and p62 levels, with a GAPDH loading control.</p
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Polyglutamine expansion affects huntingtin conformation in multiple Huntington's disease models.
Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients
Evaluation of LC3B-II and p62 TR-FRET specificity.
<p>Genetic validation of the LC3B-II and p62 readouts was achieved by gene silencing of LC3B and p62 in HEK293T cells using shRNA. <i>LC3B</i> and <i>p62</i> mRNA were reduced after shRNA of each gene, compared to shRNA scramble, as verified by qRT-PCR (N = 3; avg±SD; Studentâs t-test (unpaired; two-tailed); *p<0.001), expression levels were calculated using the 2^-ÎÎCT method and expressed relative to scramble control (A). A corresponding reduction of protein levels was observed by western blot (B) and TR-FRET (N = 4; avg±SD; Studentâs t-test (unpaired; two-tailed); *p<0.005; expression relative to scramble control; C). ATG4B overexpression in HEK293T cells was confirmed with western blot (D). ATG4B overexpression did not alter LC3B mRNA levels as seen by qRT-PCR (N = 3, avg±SD; Studentâs t-test (unpaired, two-tailed) p>0.05, expression levels were calculated using the 2^-ÎÎCT method and expressed relative to scramble control (E) but clearly reduced LC3B-II detection, as measured by TR-FRET (expressed relative to empty vector; N = 3, unpaired t-test, *p<0.001 (F) and western blot (D). LC3B-II quantification by TR-FRET (fluorescence ratio of 665/615 nm) in HEK293 cells showed detection with as few as 2000 cells/well (N = 2; avg±SD; G). Different concentrations of purified p62 were measured and the p62 TR-FRET assay is sensitive enough to detect 1ng/ml purified recombinant p62 protein (signal expressed as fluorescence ratio of 665/615 nm; N = 2; avg±SEM; H).</p
<i>In vivo</i> mTOR inhibition resulted in a measurable stimulation of autophagy.
<p>6 month mice were treated one time with a mTOR inhibitor and sacrificed 0.5 or 2 hours afterwards. mTOR inhibition increased LC3B-II (A) and reduced p62 (B) levels in the mouse liver, as measured by TR-FRET and expressed as the fluorescence ration (665/615 nm) ÎF; N = 5; one-way ANOVA, p<0.01; Tukeyâs multiple comparison test, *p<0.05, **pâ€0.01.</p
Co-treatment with bafilomycin A1 to distinguish autophagy enhancers versus blockers.
<p>Rat primary astrocytes were treated with 10 ÎŒM KU0063794, 1 ÎŒM SU11652 or 5 ÎŒM NVP-TAE684 (concentration selected from information in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194423#pone.0194423.g002" target="_blank">Fig 2</a>) followed by either 50 nM bafilomycin A1 (+Baf) or vehicle (DMSO, -Baf) for an additional 4 hours. Control samples were treated with vehicle (DMSO) for 2 hours followed by either bafilomycin A1 (50 nM) or vehicle (DMSO) for an additional 4 hours. LC3B-II TR-FRET signals are reported as fold increase with respect to the vehicle. Co-treatment of KU0063794 and bafilomycin A1 increased LC3B-II, compared to KU0063794 or bafilomycin A1 alone (N = 2, one-way ANOVA, p<0.01; Tukeyâs multiple comparison test, *p<0.05); co-treatment of SU11652 and bafilomycin A1 increased LC3B-II, compared to SU11652 or bafilomycin A1 alone (N = 2, one-way ANOVA, p<0.01; Tukeyâs multiple comparison test, *p<0.05); co-treatment of NVP-TAE684 and bafilomycin A1 did not alter the LC3B-II signal (N = 2, one-way ANOVA, p>0.05; (A). Western blots (B) confirm the TR-FRET data.</p