Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome

Abstract

a b s t r a c tAtaxin-3 (AT3) is the protein that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It cleaves isopeptide bonds between ubiquitin monomers, an event involved in protein quality control mechanisms. AT3 has been implicated in the pathway that sorts aggregated protein to aggresomes via microtubules, in which dynein and histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of small angle X-ray scattering (SAXS) and surface plasmon resonance (SPR), we have investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS results, the AT3 oligomer, consisting of 6-7 subunits, tightly binds to the tubulin hexameric oligomer in a "parallel" fashion. By SPR analysis we have demonstrated that AT3 binds to tubulin dimer with a 50 nM affinity. Binding fits with a Langmuir 1:1 model and involves a single binding interface. Nevertheless, the interaction surface consists of three distinct, discontinuous tubulin-binding regions (TBR), one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. In the absence of any of the three TBRs, the affinity is drastically reduced. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, with affinity in the range 0.1-1 M. These results shed light on the interactions among the components of the transport machinery that sorts aggregate protein to the aggresome, and pave the way to in vivo studies aimed at further clarifying their roles.

The polyglutamine protein ataxin-3 enables normal growth under heat shock conditions in the methylotrophic yeast Pichia pastoris

“…Available data suggest that ATX3 fulfills this task as a part of a multiprotein complex wherein, along with K63-ubiquinated aggregates, HDAC6 is also included. The latter recruits them to dynein motor complexes that subsequently transport the aggregated cargo to the aggresomes via microtubules 17 – 21 . On the whole, this picture highlights ATX3 as a sorting device of proteins committed to degradation, including those residing in the ER, by destining them for either the proteasome or the aggresome.…”

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

“…To date, most of the published structural studies on ataxin-3 have focused on the Josephin domain (Nicastro et al, 2005(Nicastro et al, , 2006(Nicastro et al, , 2010Robertson et al, 2010;Sanfelice et al, 2014;Satoh et al, 2014;Weeks et al, 2011), which contains the catalytic site of the enzyme (Nicastro et al, 2009(Nicastro et al, , 2010. The importance of the ataxin-3 C terminus cannot nevertheless be underestimated: this region is involved in key interactions that modulate the physiological function of ataxin-3 (Bonanomi et al, 2014;Rao et al, 2017;Song et al, 2010). The C terminus also contains the polyQ tract that modulates the aggregation propensity of ataxin-3 (Ellisdon et al, 2006) and is directly linked to disease (Paulson et al, 1997).…”

“…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 Molecular Physiopathogenesis of Islet Amyloidosis

“…Aggresomes, a protein complex consisting of proteasome, ubiquitin, heat shock proteins (HSP) and γ-tubulin, serve as alternative degrading/sequestering center to lysosomes for many misfolded and potentially toxic cytosolic proteins (Blair et al 2014; Bonanomi et al 2014; Junn et al 2002). To determine if the cytosolic pool of internalized hA is targeted to aggresomes for degradation, immuno-confocal approach was again employed.…”