Insight into Molecular Basis of Curing of [PSI+] Prion by Overexpression of 104-kDa Heat Shock Protein (Hsp104)

Helsen, Christopher W.; Glover, John R.

doi:10.1074/jbc.m111.302869

Cited by 80 publications

(91 citation statements)

References 71 publications

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“…1). To do so, we prepared chimeric His 6 -NM[ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) ]-Npu[ (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) ]-His 6 , expressed in natural-abundance media (Fig. 1C).…”

Section: Resultsmentioning

confidence: 99%

“…The final product was greater than 90% pure by SDS/PAGE and contained a single cysteine mutation (or "scar") at the ligation site. We prepared more than 8 mg of segmentally labeled NM molecules per liter of isotopically labeled growth for a practical yield of about 50% of the His 6 -NM[ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) ]-DnaE[ ] precursor. To avoid sample heterogeneity derived from multiple fiber forms, the segmentally labeled NM was templated into amyloid fibers using amyloid seeds derived from lysates of yeast carrying the strong [PSI + ] phenotype (11).…”

Section: Resultsmentioning

confidence: 99%

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Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Frederick

Michaelis

Caporini

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C-and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. (2), is an amyloid form of the translation termination factor Sup35 (3). The amyloid fold is polymeric fiber of protein monomers that is rich in β-sheets that run perpendicular to the fiber axis. Amyloid formation sequesters Sup35 from the ribosome, changing the rate of stop-codon read-through and creating a variety of new yeast phenotypes (4,5). Different regions of the Sup35 protein are responsible for prion templating, prion inheritance, and translation termination. The N-terminal domain "N" is Q/N-rich and required for the formation and templating of amyloid. The highly charged K/E-rich middle domain "M" promotes solubility in the nonprion form and is required for chaperone-mediated prion inheritance (6, 7). The C-terminal domain is necessary and sufficient for translation termination. The N and M domains together, NM, contain all of the necessary features to act as an amyloid-based prion.A wide variety of approaches have been used to investigate Sup35 prion structures, but a consensus structural picture has yet to emerge. Amyloids are notoriously difficult to study, mainly because they are insoluble yet noncrystalline and are therefore not easily amenable to traditional structural biology methods. In all models, at least some part of the N domain is involved in the amyloid fold (8-11), and most of the M domain is thought to be solvent-accessible and intrinsically disordered (11-13). However, there are two conflicting models of how monomers of NM are arranged in amyloid fibers. In one model, called the β-helical model, monomers make intermolecular contacts in discrete regions, with the region in-between making intramolecular contacts. This model is supported by the patterns of interaction and cross-linking determined from a series of site-directed mutants (8). The work has the caveat that mutations may perturb the fibril structure. However, fibers made from the mutated versions of the protein ca...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Frederick

Michaelis

Caporini

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Sup35M is enriched in charged residues and is suspected of helping to maintain a balance between aggregated and non-aggregated states, possibly via interaction with Hsp's (Liu et al 2002). Indeed, Sup35M interacts with Hsp104 in vitro and is involved in [PSI + ] curing by excess Hsp104 in vivo (Helsen and Glover 2012). Yeast PrDs may confer a prion state to a different protein when fused to it artificially.…”

Section: Prion Domainsmentioning

confidence: 99%

Prions in Yeast

2012

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The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the "protein only" model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-b aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions.

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“…26,27 The ability of Ssa1 to transiently bind a segment of Sup35 involved in β-strand formation could also explain why Ssa1 inhibits Sup35 assembly into fibrils in vitro. 1,22,28 Incomplete extraction of Sup35 protomers could also result in Hsp104 becoming stalled with partially processed substrates stuck in the axial channel. However, Hsp104, like its bacterial ortholog ClpB, may release partially threaded substrates when it encounters an intractably stably-folded domain.…”

Section: Probing the Hsp104/sup35 Interactionmentioning

confidence: 99%

“…Glover J.R., Michalowska M., Lum R., unpublished). In experiments that probed the interaction between Hsp104 and Sup35, 1 we found that the full-length M-domain (we used Sup35 amino acids 105-253 as the full-length M-domain) also has these properties. We found that the binding affinity between M-domain and Hsp104 (about 540 nM) is substantially weaker than that of p370 or RCMLA.…”

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confidence: 99%

Insight into Molecular Basis of Curing of [PSI+] Prion by Overexpression of 104-kDa Heat Shock Protein (Hsp104)

Cited by 80 publications

References 71 publications

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Prions in Yeast

A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI⁺]

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Insight into Molecular Basis of Curing of [PSI+] Prion by Overexpression of 104-kDa Heat Shock Protein (Hsp104)

Cited by 80 publications

References 71 publications

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Prions in Yeast

A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI+]

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Product

Resources

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A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI⁺]