d16e402f186c9bb349e3c38f02b8403c pmc_33477953.pdf f65e6ac10c93306c3429f5fbe114a42970d81229 pmc_33477953.pdf 3591edd5bd2268d0ee31f07535ccc7047ad7ccb69eb7b6f1c9c67dcc9eef36d9 pmc_33477953.pdf Title: Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations Subject: 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. Keywords: ataxin-3 expression; amyloids; Escherichia coli; FTIR microspectroscopy; multivariate analysis; oligomer toxicity; protein aggregation Author: Diletta Ami, Barbara Sciandrone, Paolo Mereghetti, Jacopo Falvo, Tiziano Catelani, Cristina Visentin, Paolo Tortora, Salvador Ventura, Antonino Natalello and Maria Elena Regonesi Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jan 19 10:56:29 2021 CET ModDate: Tue Jan 19 12:17:27 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 21 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5572655 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CMFVHJ+VnURWPalladioL Type 1 Custom yes yes no 10 0 ZFDDQN+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 GUHZGS+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 PXFODL+URWPalladioL-BoldItal Type 1 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application/pdf PDFMetadata: Objects: 638 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Toxic Effect of the Pathogenic Ataxin-3 Variant on E. coli Growth Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: ATX3-Q55 Toxicity Is Not Correlated to a Higher Protein Expression or Increase of the Soluble Fraction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: FTIR Analysis of E. coli Cell Modifications Induced by the Expression of ATX3-Q24 and ATX3-Q55 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: FTIR Study of Protein Secondary Structure Modifications: Analysis of Amide I Band Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: FTIR Study of Lipid Modifications: Analysis of the 3050 2800 cm-1 Spectral Range Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: FTIR Study of Lipid and Peptidoglycan Modifications: Analysis of the 1500 1200 cm-1 Spectral Range Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: FTIR Study of Lipopolysaccharide and Peptidoglycans: Analysis of the 1200 900 cm-1 Spectral Range Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Electron Microscopy Analysis of ATX3 Expressing Strains Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Bacterial Strain and Plasmid Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Growth Curves Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Colony-Formation Unit (CFU) Count Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Western Blot Analysis of Total Extract and Soluble Protein Fractions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Dot-Blot Analysis of Total Extract Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: FTIR Microspectroscopy Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Multivariate Analysis of the FTIR Data Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Transmission Electron Microscopy Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Conclusions and Future Perspectives Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Info: Title: Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations Author: Diletta Ami, Barbara Sciandrone, Paolo Mereghetti, Jacopo Falvo, Tiziano Catelani, Cristina Visentin, Paolo Tortora, Salvador Ventura, Antonino Natalello and Maria Elena Regonesi Subject: 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. 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