The Machado-Joseph disease-associated expanded form of ataxin-3: Overexpression, purification, and preliminary biophysical and structural characterization

Capturing the Conformational Ensemble of the Mixed Folded Polyglutamine Protein Ataxin-3

“…NMR, on the other hand, despite being the most appropriate technique for flexible proteins, is affected by challenging technical limitations, mainly dictated by the heterogeneity of the backbone dynamics, the exchange regime, and the low spectral dispersion of the C terminus (Sicorello et al, 2018). Simulations of isolated fragments of the tail (Invernizzi et al, 2013), low-resolution structural characterizations based solely on SAXS and ab initio methods (Bonanomi et al, 2014;Contessotto et al, 2018), or native mass spectrometry (Scarff et al, 2013) have provided a valuable yet fragmentary and incomplete picture of ataxin-3 structure.…”

The Machado–Joseph disease‐associated form of ataxin‐3 impacts dynamics of clathrin‐coated pits

“…The human ATXN3 gene was amplified as described elsewhere (Contessotto et al, 2018). Wild‐type ataxin‐3 (amino acids 1–336) and expanded ataxin‐3 (amino acids 1–421) genes, containing 19 and 74 CAG repeats, respectively, were subcloned into pEFGP‐N1 (Clontech) and pLVX (Clontech) modified in house to encode enhanced green fluorescent protein (EGFP) at the C‐terminus of the target protein.…”

“…After an initial biophysical and structural characterization of expanded ataxin‐3 (Contessotto et al, 2018), we further examined whether and how the expanded ataxin‐3 could affect CME and CCP dynamics and found that the expanded form of ataxin‐3 had significantly higher proportion of short‐lived abortive CCPs and lower nucleation rate, consistent with inhibition of CME. To the best of our knowledge, our work shows for the first time, in an overexpression model, that ataxin‐3 affects CME dynamics.…”