title: An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation.
abstract: The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltose-binding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an intein-based strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1-7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties.
authors.affiliation: From the Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland and.
authors.forename: Sophie
authors.initials: S
authors.lastname: Vieweg
authors.affiliation: From the Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland and.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Ansaloni
authors.affiliation: From the Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland and.
authors.forename: Zhe-Ming
authors.initials: ZM
authors.lastname: Wang
authors.affiliation: From the Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland and.
authors.forename: John B
authors.initials: JB
authors.lastname: Warner
authors.affiliation: From the Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland and Qatar Biomedical Research Institute (QBRI), Hamad bin Khalifa University (HBKU), 5825 Doha, Qatar hilal.lashuel@epfl.ch.
authors.forename: Hilal A
authors.initials: HA
authors.lastname: Lashuel
mesh_terms: D020816:Amino Acid Motifs; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D005091:Exons; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism*; D047668:Inteins; D007700:Kinetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D066329:Protein Aggregates
chemical_list: D000682:Amyloid; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D010455:Peptides; D066329:Protein Aggregates; C097188:polyglutamine
keywords: Huntington disease; circular dichroism (CD); electron microscopy (EM); fibril; protein aggregation; protein purification; protein structure