Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations

Abstract

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.

Transcriptomic analysis of Rhodococcus opacus R7 grown on polyethylene by RNA-seq

“…In addition, also the Amide I band components, due to the C=O stretch of the peptide bond and giving information on protein secondary structures, are observed at ~ 1657 cm −1 (assigned to protein alpha helix/random coil structures), and at ~ 1632 cm −1 with higher intensity in PE cells (due to protein beta -sheets) (Fig. 6 B) 56 .…”

“…6 A,D). In the spectral range 1760–1600 cm −1 , the second derivative spectra show two peaks mainly ascribable to carbonyl groups involved in ester bonds 56 , at ~ 1749 cm −1 and ~ 1738 cm −1 , with a different intensity in the two conditions (Fig. 6 B,D).…”

“…opacus R7 cell biomolecules induced by PE as the only carbon and energy source, compared to malate condition, FTIR microspectroscopy was employed. This vibrational approach enables to characterize not only isolated biomolecules but also complex biological systems, such as intact cells, providing a snapshot of the structure and content of the main molecules 56 . For sake of clarity, only the spectral bands displaying the most important spectral differences in the two growth conditions will be described.…”

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates

“…that in Huntington’s patients, a reduction of unsaturated lipids in the brain matter accompanies inclusion body formation and by Ami et al. who identified modifications to lipid membranes, peptidoglycans, and lipopolysaccharide barriers caused by misfolded human Ataxin-3 ( 83 , 85 ). While not directly providing high resolution structure, this information reports on the state of the cell and other aggregate components that may modulate aggregate structure.…”

“…Examples include those from patients with AD, PD, ALS, and HD ( 80 , 81 , 82 , 83 , 84 ). Analyses of these tissue samples often involve assessing the biochemical composition of aggregate/deposit structures by monitoring the lipid or DNA content; similar to proteins, these macromolecules have characteristic FTIR absorbances that can be spatially monitored ( 83 , 85 , 86 ). Lipid content is monitored using the C–H stretching vibration, which appears in the region 3000 to 2800 cm −1 and DNA or phosphorylation is monitored using the stretching vibrations of the P-O double bond appearing between 1237 and 1080 cm −1 ( 83 ).…”