| 1. |
- Baldo, Barbara, et al.
(författare)
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Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay
- 2018
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Ingår i: eNeuro. - 2373-2822. ; 5:4
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Tidskriftsartikel (refereegranskat)abstract
- The neurodegenerative Huntington's disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pm and optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cells in vitro and in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.
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| 2. |
- Jung, Taeyang, et al.
(författare)
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The Polyglutamine Expansion at the N-Terminal of Huntingtin Protein Modulates the Dynamic Configuration and Phosphorylation of the C-Terminal HEAT Domain
- 2020
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Ingår i: Structure. - : Cell Press. - 0969-2126 .- 1878-4186. ; 28:9, s. 1035-1050.e8
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Tidskriftsartikel (refereegranskat)abstract
- The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural information on its HEAT-repeat domains. Here, we present analyses of the impact of polyQ length on the structure and function of HTT via an integrative structural and biochemical approach. The cryo-EM analysis of normal (Q23) and disease (Q78) type HTTs shows that the structures of apo HTTs significantly differ from the structure of HTT in a HAP40 complex and that the polyQ expansion induces global structural changes in the relative movements among the HTT domains. In addition, we show that the polyQ expansion alters the phosphorylation pattern across HTT and that Ser2116 phosphorylation in turn affects the global structure and function of HTT These results provide a molecular basis for the effect of the polyQ segment on HTT structure and activity, which may be important for HTT pathology.
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| 3. |
- Vijayvargia, Ravi, et al.
(författare)
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Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function
- 2016
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS. - 2050-084X. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- The polyglutamine expansion in huntingtin protein causes Huntington's disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical alpha-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases alpha-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.
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