Examination of Ataxin-3 (atx-3) Aggregation by Structural Mass Spectrometry Techniques: A Rationale for Expedited Aggregation upon Polyglutamine (polyQ) Expansion

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73–96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided.

[1]  Vidya N. Nukala,et al.  The first 17 amino acids of Huntingtin modulate its sub-cellular localization, aggregation and effects on calcium homeostasis. , 2007, Human molecular genetics.

[2]  Jeremy L. England,et al.  Polyglutamine shows a urea‐like affinity for unfolded cytosolic protein , 2011, FEBS letters.

[3]  I. A. Abreu,et al.  Trinucleotide Repeats: A Structural Perspective , 2013, Front. Neurol..

[4]  R. Grandori,et al.  Charge-surface correlation in electrospray ionization of folded and unfolded proteins. , 2011, Analytical chemistry.

[5]  Lisa D. Cabrita,et al.  Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability , 2004, Journal of Biological Chemistry.

[6]  Giuseppe Nicastro,et al.  The solution structure of the Josephin domain of ataxin-3: structural determinants for molecular recognition. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  H. Zoghbi,et al.  Glutamine repeats and neurodegeneration. , 2000, Annual review of neuroscience.

[8]  C. MacPhee,et al.  Mass spectrometry methods for intrinsically disordered proteins. , 2013, The Analyst.

[9]  C. Robinson,et al.  Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. , 2010, Analytical chemistry.

[10]  Masahiko Watanabe,et al.  Machado–Joseph disease gene products carrying different carboxyl termini , 1997, Neuroscience Research.

[11]  H. Braak,et al.  Intranuclear Aggregation of Nonexpanded Ataxin-3 in Marinesco Bodies of the Nonhuman Primate Substantia Nigra , 2002, Experimental Neurology.

[12]  Dalaver H. Anjum,et al.  Polyglutamine disruption of the huntingtin exon1 N-terminus triggers a complex aggregation mechanism , 2009, Nature Structural &Molecular Biology.

[13]  Sandra Macedo-Ribeiro,et al.  Towards a structural understanding of the fibrillization pathway in Machado-Joseph's disease: trapping early oligomers of non-expanded ataxin-3. , 2005, Journal of molecular biology.

[14]  S. Doglia,et al.  The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli , 2012, PLoS ONE.

[15]  Giuseppe Nicastro,et al.  The Josephin domain determines the morphological and mechanical properties of ataxin-3 fibrils. , 2011, Biophysical journal.

[16]  H. Zoghbi,et al.  Fourteen and counting: unraveling trinucleotide repeat diseases. , 2000, Human molecular genetics.

[17]  Alessandra Gliozzi,et al.  A Major Role for Side-Chain Polyglutamine Hydrogen Bonding in Irreversible Ataxin-3 Aggregation , 2011, PloS one.

[18]  Franca Fraternali,et al.  Josephin domain of ataxin‐3 contains two distinct ubiquitin‐binding sites , 2009, Biopolymers.

[19]  Ryan D. Morin,et al.  The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). , 2004, Genome research.

[20]  James R. Burke,et al.  Inhibition of Polyglutamine Protein Aggregation and Cell Death by Novel Peptides Identified by Phage Display Screening* , 2000, The Journal of Biological Chemistry.

[21]  A. Ashcroft.  Mass spectrometry and the amyloid problem—How far can we go in the gas phase? , 2010, Journal of the American Society for Mass Spectrometry.

[22]  Stephen P Bottomley,et al.  Multi-domain misfolding: understanding the aggregation pathway of polyglutamine proteins. , 2009, Protein engineering, design & selection : PEDS.

[23]  M. Tyers,et al.  BoxPlotR: a web tool for generation of box plots , 2014, Nature Methods.

[24]  A. Pastore,et al.  Expansion of amino acid homo-sequences in proteins: Insights into the role of amino acid homo-polymers and of the protein context in aggregation , 2006, Cellular and Molecular Life Sciences CMLS.

[25]  E. Wilson,et al.  An Interdomain Interaction of the Androgen Receptor Is Required for Its Aggregation and Toxicity in Spinal and Bulbar Muscular Atrophy* , 2010, The Journal of Biological Chemistry.

[26]  Giuseppe Nicastro,et al.  Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3. , 2004, Journal of molecular biology.

[27]  Masino Laura,et al.  Functional interactions as a survival strategy against abnormal aggregation , 2011, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[28]  S. Hirai,et al.  Ataxin-3 Is Translocated into the Nucleus for the Formation of Intranuclear Inclusions in Normal and Machado–Joseph Disease Brains , 2000, Experimental Neurology.

[29]  Brandon T Ruotolo,et al.  Ion mobility–mass spectrometry analysis of large protein complexes , 2008, Nature Protocols.

[30]  C. MacPhee,et al.  A mass-spectrometry-based framework to define the extent of disorder in proteins. , 2014, Analytical chemistry.

[31]  Susan Lindquist,et al.  Flanking sequences profoundly alter polyglutamine toxicity in yeast. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[32]  K. Fischbeck,et al.  Intranuclear Inclusions of Expanded Polyglutamine Protein in Spinocerebellar Ataxia Type 3 , 1997, Neuron.

[33]  H. Paulson,et al.  Toward understanding Machado–Joseph disease , 2012, Progress in Neurobiology.

[34]  T. Wyttenbach,et al.  Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to beta-sheet in amyloid fibril formation , 2010, Nature chemistry.

[35]  Marianne Rooman,et al.  Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein , 2011, Protein science : a publication of the Protein Society.

[36]  Stephen P Bottomley,et al.  The Two-stage Pathway of Ataxin-3 Fibrillogenesis Involves a Polyglutamine-independent Step* , 2006, Journal of Biological Chemistry.

[37]  A. Pastore,et al.  Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein. , 2005, Journal of molecular biology.

[38]  Michele Vendruscolo,et al.  Characterization of the Conformational Fluctuations in the Josephin Domain of Ataxin-3 , 2014, Biophysical journal.

[39]  E. L. Guenther,et al.  Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS , 2013, Proceedings of the National Academy of Sciences.

[40]  C. Robinson,et al.  Evidence for Macromolecular Protein Rings in the Absence of Bulk Water , 2005, Science.

[41]  S. Radford,et al.  Elongated oligomers in β2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry , 2010, Proceedings of the National Academy of Sciences.

[42]  Stephen P Bottomley,et al.  Mechanisms of ataxin-3 misfolding and fibril formation: kinetic analysis of a disease-associated polyglutamine protein. , 2007, Journal of molecular biology.

[43]  P. Loll,et al.  An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel β-fibrils , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Marco Leite,et al.  Transfer Function between EEG and BOLD Signals of Epileptic Activity , 2013, Front. Neur..

[45]  E. Snapp,et al.  Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells , 2010, PloS one.

[46]  Michelle K. M. Chow,et al.  Structural and functional analysis of the Josephin domain of the polyglutamine protein ataxin-3. , 2004, Biochemical and biophysical research communications.

[47]  Zoya Ignatova,et al.  Extended Polyglutamine Tracts Cause Aggregation and Structural Perturbation of an Adjacent β Barrel Protein* , 2006, Journal of Biological Chemistry.

[48]  Justin L. P. Benesch,et al.  Two decades of studying non-covalent biomolecular assemblies by means of electrospray ionization mass spectrometry , 2012, Journal of The Royal Society Interface.

[49]  C. Robinson,et al.  Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease. , 2009, Nature chemistry.

[50]  Carlos A. Matos,et al.  Polyglutamine diseases: The special case of ataxin-3 and Machado–Joseph disease , 2011, Progress in Neurobiology.

[51]  Sheena E. Radford,et al.  A tale of a tail: Structural insights into the conformational properties of the polyglutamine protein ataxin-3 , 2013, International journal of mass spectrometry.