Detection of ubiquitinated huntingtin species in intracellular aggregates

Abstract

Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

Associations between Prenatal and Early Childhood Fish and Processed Food Intake, Conduct Problems, and Co-Occurring Difficulties

“…While the previous studies did not find associations between junk food and emotional difficulties, our findings differ in three important ways: first, we focused on EOP CP children who already have a high risk of co-occurring difficulties (Connor 2002), rather than focusing on typically developing children. Second, we limited our analyses to savoury processed foods, as excluding sugary processed foods may help to reduce the possible confounding and contradictory effects of sugar on brain development (Beilharz, Maniam & Morris 2014; Maniam, Antoniadis, Youngson, Sinha, & Morris 2015). Third, we investigated associations between prenatal and 3 year diet – time periods that are more susceptible to nutritional insults – and early-adolescent outcomes, rather than focusing on a less critical age range for junk food (4.5 years and 7 years).…”

Cellular models for Parkinson's disease

“…Thus, this method specifically measures aggregates large enough to stick in the filter, but does not allow for discrimination between specific and nonspecific immunoreactivity based on molecular weight. While the filtration assay works well for many aggregating proteins (Myeku et al 2011;Juenemann et al 2015), it has been difficult for a-synuclein from cultured cells in our hands.…”

“…large ones and aggregates attached to membranes) may remain in the pellet during preparation and thus escape the analysis. At the same time, one may use these differences to define subpopulations of a-synuclein aggregates, such as a-synuclein aggregates soluble in urea but not in triton (Trimmer et al 2004;Juenemann et al 2015). Cross-linking a-synuclein before cell lysis can stabilize oligomers and has been used to demonstrate the tetrameric nature of a-synuclein (Dettmer et al 2013).…”

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

“…Filter trap assays have been used extensively in the past to assess inclusion formation in cells (Chang and Kuret 2008; cells following each treatment is displayed as mean ± SEM (n = 3) and was analysed via a one-way ANOVA with a Dunnett's post-test compared to the untreated EGFP control, where ***p < 0.001 Juenemann et al 2015;Nasir et al 2015). Typically, soluble protein moves through the membrane pores, whilst aggregated protein is retained on the membrane such that proteins in the aggregates can be detected by immunoblotting.…”