Structure and Assembly Properties of the N-Terminal Domain of the Prion Ure2p in Isolation and in Its Natural Context

Background: The aggregation of the baker’s yeast prion Ure2p is at the origin of the [URE3] trait. The Q- and N-rich N-terminal part of the protein is believed to drive Ure2p assembly into fibrils of amyloid nature and the fibrillar forms of fulllength Ure2p and its N-terminal part generated in vitro have been shown to induce [URE3] occurrence when introduced into yeast cells. This has led to the view that the fibrillar form of the N-terminal part of the protein is sufficient for the recruitment of constitutive Ure2p and that it imprints its amyloid structure to full-length Ure2p. Results: Here we generate a set of Ure2p N-terminal fragments, document their assembly and structural properties and compare them to that of full-length Ure2p. We identify the minimal region critical for the assembly of Ure2p N-terminal part into amyloids and show that such fibrils are unable to seed the assembly of full length Ure2p unlike fibrils made of intact Ure2p. Conclusion: Our results clearly indicate that fibrillar Ure2p shares no structural similarities with the amyloid fibrils made of Ure2p N-terminal part. Our results further suggest that the induction of [URE3] by fibrils made of full-length Ure2p is likely the consequence of fibrils growth by depletion of cytosolic Ure2p while it is the consequence of de novo formation of prion particles following, for example, titration within the cells of a specific set of molecular chaperones when fibrils made o

Extensive de novo solid-state NMR assignments of the 33kDa C-terminal domain of the Ure2 prion

We present the de novo resonance assignments for the crystalline 33kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly 13C, 15N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibril

DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios

Expanded polyglutamine (polyQ) stretches lead to protein aggregation and severe neurodegenerative diseases. A highly efficient suppressor of polyQ aggregation was identified, the DNAJB6, when molecular chaperones from the HSPH, HSPA, and DNAJ families were screened for huntingtin exon 1 aggregation in cells (Hageman et al. in Mol Cell 37(3):355-369, 2010). Furthermore, also aggregation of polyQ peptides expressed in cells was recently found to be efficiently suppressed by co-expression of DNAJB6 (Gillis et al. in J Biol Chem 288:17225-17237, 2013). These suppression effects can be due to an indirect effect of DNAJB6 on other cellular components or to a direct interaction between DNAJB6 and polyQ peptides that may depend on other cellular components. Here, we have purified the DNAJB6 protein to investigate the suppression mechanism. The purified DNAJB6 protein formed large heterogeneous oligomers, in contrast to the more canonical family member DNAJB1 which is dimeric. Purified DNAJB6 protein, at substoichiometric molar ratios, efficiently suppressed fibrillation of polyQ peptides with 45°Q in a thioflavin T fibrillation. No suppression was obtained with DNAJB1, but with the closest homologue to DNAJB6, DNAJB8. The suppression effect was independent of HSPA1 and ATP. These data, based on purified proteins and controlled fibrillation in vitro, strongly suggest that the fibrillation suppression is due to a direct protein-protein interaction between the polyQ peptides and DNAJB6 and that the DNAJB6 has unique fibrillation suppression properties lacking in DNAJB1. Together, the data obtained in cells and in vitro support the view that DNAJB6 is a peptide-binding chaperone that can interact with polyQ peptides that are incompletely degraded by and released from the proteasome.</p

Solid-state NMR sequential assignments of the amyloid core of Sup35pNM

Sup35pNM represents the N-terminal and middle (M) domains of the yeast Saccharomyces cerevisiae prion Sup35p. This fragment is commonly used for structural and functional studies of Sup35p. We here present a solid-state NMR study of fibrils formed by this fragment and show that sequential assignments can be obtained for the rigid and well-ordered parts of the protein using 3D spectroscopy. We describe in detail the sequential assignment of the 22 residues yielding strong, narrow signals with chemical shifts that correspond mostly to β-sheet secondary-structured amino acids that form the fibril core

Solid-state NMR sequential assignments of the amyloid core of full-length Sup35p

Sup35p is a yeast prion and is responsible for the [PSI +] trait in Saccharomyces cerevisiae. With 685 amino acids, full-length soluble and fibrillar Sup35p are challenging targets for structural biology as they cannot be investigated by X-ray crystallography or NMR in solution. We present solid-state NMR studies of fibrils formed by the full-length Sup35 protein. We detect an ordered and rigid core of the protein that gives rise to narrow and strong peaks, while large parts of the protein show either static disorder or dynamics on time scales which interfere with dipolar polarization transfer or shorten the coherence lifetime. Thus, only a small subset of resonances is observed in 3D spectra. Here we describe in detail the sequential assignments of the 22 residues for which resonances are observed in 3D spectra: their chemical shifts mostly corresponding to β-sheet secondary structure. We suspect that these residues form the amyloid core of the fibril

Solid-state NMR sequential assignments of the amyloid core of Sup35pNM

Sup35pNM represents the N-terminal and middle (M) domains of the yeast Saccharomyces cerevisiae prion Sup35p. This fragment is commonly used for structural and functional studies of Sup35p. We here present a solid-state NMR study of fibrils formed by this fragment and show that sequential assignments can be obtained for the rigid and well-ordered parts of the protein using 3D spectroscopy. We describe in detail the sequential assignment of the 22 residues yielding strong, narrow signals with chemical shifts that correspond mostly to β-sheet secondary-structured amino acids that form the fibril core.ISSN:1874-270XISSN:1874-271

Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

<div><p>An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, <em>in vitro</em>. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.</p> </div

Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

ISSN:0925-2738ISSN:1573-500

PolyQ amyloid fibrils.

<p>TEM micrograph of the products formed after TEV cleavage of the MBP from the polyQ-fusion protein complex. (A) Q45 proto-fibrilar precursors. (B) Mature Q45 fibrils.</p