A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins

Abstract

Aim:The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemical modulators of degradation. For example, Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of mHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies.Methods:The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads.Results:We validated that the signals were within the linear detection range and were HTT-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates. The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers.Conclusion:We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies.

Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer

“…In addition to targeting the machinery regulating AR polyubiquitination, small molecules targeting AR could also modulate AR polyubiquitination. Androgen was previously reported to suppress AR polyubiquitination 24 and is shown here to suppress AR polyubiquitination in the nucleus. In contrast, the small molecule CPPI is a novel AR antagonist capable of enhancing AR polyubiquitination in the nucleus.…”

“…A second approach was to pulse-chase exogenous or endogenous AR with the methionine analog homopropargylglycine (HPG) coupled with Click chemistry (24)(25)(26), nucleocytoplasmic fractionation, and Western blot analysis (Fig. 2D).…”

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models

“…The Click-iT AHA (ℒ-azidohomoalaine) kit (Thermo Fisher Scientific, #C10102) was used and the assay was similar to that previously described 30 , 88 . Briefly, cells were starved with methionine-free media for 1 h prior to adding Click-iT AHA for 12 h (pulse, 50 μM).…”

“…The Click-iT AHA labeled proteins were then conjugated with biotin by treatment with biotin-DIBO Alkyne (Thermo Fisher Scientific, #C10412). The biotin conjugated proteins were then pulled-down using streptavidin beads (Pierce, #20349), and the HTT level is determined by HTRF 88 .…”

Modeling the Degradation Effects of Autophagosome Tethering Compounds (ATTEC)

“…In our study demonstrating the concept of ATTEC, we observed increased degradation of mutant HTT protein (mHTT) induced by ATTEC. Thus, we used mHTT as the POI for our modeling, and its half-life has been reported to be around 33~40 hours [13][14][15] . In order to facilitate the math calculation, we estimate the mHTT half-life as 36 hours, 6 times of the half-life of mHTT tethered to autophagosomes.…”