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RNA Binds to Tau Fibrils and Sustains Template-Assisted Growth.

Dinkel PD, Holden MR, Matin N, Margittai M - Biochemistry (2015)

Bottom Line: These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents.Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin.These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States.

ABSTRACT
Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

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EPR analysis of Tau fibrils. Single cysteinesin K18 and K19 monomerswere spin-labeled with the nitroxide label MTSL and allowed to fibrillizein the presence of polyanionic cofactors. All spectra were taken at150 G and normalized to the same number of spins. K18 and K19 fibrilswere formed in the presence of (A) polyA, (B) polyU, polyAU, tRNA,and yeast RNA extract, and (C) polydA and polyGlu. The single-lineEPR spectra observed for all fibrils indicate that, regardless ofcofactor, β-strands in the proteins are aligned parallel andin-register.
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fig1: EPR analysis of Tau fibrils. Single cysteinesin K18 and K19 monomerswere spin-labeled with the nitroxide label MTSL and allowed to fibrillizein the presence of polyanionic cofactors. All spectra were taken at150 G and normalized to the same number of spins. K18 and K19 fibrilswere formed in the presence of (A) polyA, (B) polyU, polyAU, tRNA,and yeast RNA extract, and (C) polydA and polyGlu. The single-lineEPR spectra observed for all fibrils indicate that, regardless ofcofactor, β-strands in the proteins are aligned parallel andin-register.

Mentions: A first step in the fibrillizationof Tau is the formation of a multimeric nucleus.20,38,39 In this step, negatively charged cofactorsare thought to induce conformational changes in the intrinsicallydisordered Tau monomers40 and to increasethe local Tau concentration by simultaneously binding to multiplepositively charged monomers.25 We previouslyshowed that fibrils formed in the presence of heparin assume a parallel,in-register β-sheet structure.35,41 Since cofactorsplay a critical role in fibril nucleation, an important, yet unanswered,question is whether these auxiliary factors modulate overall fibrilstructure. In order to address this question, we first examined whethersubstitution of heparin by RNA affects β-strand arrangementand registry. For this purpose, six single-cysteine mutants of K18and K19 (cysteines in positions 309, 310, 311, 317, 322, and 326)were labeled with the paramagnetic nitroxide label MTSL.42 The sites were chosen to represent the stablecore of the third microtubule binding repeat, which is critical forfibril formation.43 Spin-labeled Tau wasallowed to aggregate in the presence of RNA (polyA) for 3 days underagitation (see Materials and Methods). Continuous-waveEPR measurements produced single-line spectra for all fibrils (Figure 1A). The collapseof the three-line spectra that are normally observed for MTSL-labeledproteins can be explained by the exchange interaction between multiplespin labels. This is achieved by parallel, in-register arrangementof β-strands in which labels attached to identical positionsin different Tau molecules are stacked along the fibril axis.44 Fibrils formed in the presence of other RNAspecies such as polyU, double-stranded RNA (UA), tRNA, and whole yeastRNA extract produced similar results (Figure 1B). Also, different negatively charged polymerssuch as single-stranded DNA and polyglutamate produced fibrils thatresulted in similar single-line characteristics (Figure 1C). Combined, these data suggestthat the parallel, in-register arrangement of β-strands is acommon feature of Tau fibrils, independent of the cofactor that isused to induce aggregation.


RNA Binds to Tau Fibrils and Sustains Template-Assisted Growth.

Dinkel PD, Holden MR, Matin N, Margittai M - Biochemistry (2015)

EPR analysis of Tau fibrils. Single cysteinesin K18 and K19 monomerswere spin-labeled with the nitroxide label MTSL and allowed to fibrillizein the presence of polyanionic cofactors. All spectra were taken at150 G and normalized to the same number of spins. K18 and K19 fibrilswere formed in the presence of (A) polyA, (B) polyU, polyAU, tRNA,and yeast RNA extract, and (C) polydA and polyGlu. The single-lineEPR spectra observed for all fibrils indicate that, regardless ofcofactor, β-strands in the proteins are aligned parallel andin-register.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526887&req=5

fig1: EPR analysis of Tau fibrils. Single cysteinesin K18 and K19 monomerswere spin-labeled with the nitroxide label MTSL and allowed to fibrillizein the presence of polyanionic cofactors. All spectra were taken at150 G and normalized to the same number of spins. K18 and K19 fibrilswere formed in the presence of (A) polyA, (B) polyU, polyAU, tRNA,and yeast RNA extract, and (C) polydA and polyGlu. The single-lineEPR spectra observed for all fibrils indicate that, regardless ofcofactor, β-strands in the proteins are aligned parallel andin-register.
Mentions: A first step in the fibrillizationof Tau is the formation of a multimeric nucleus.20,38,39 In this step, negatively charged cofactorsare thought to induce conformational changes in the intrinsicallydisordered Tau monomers40 and to increasethe local Tau concentration by simultaneously binding to multiplepositively charged monomers.25 We previouslyshowed that fibrils formed in the presence of heparin assume a parallel,in-register β-sheet structure.35,41 Since cofactorsplay a critical role in fibril nucleation, an important, yet unanswered,question is whether these auxiliary factors modulate overall fibrilstructure. In order to address this question, we first examined whethersubstitution of heparin by RNA affects β-strand arrangementand registry. For this purpose, six single-cysteine mutants of K18and K19 (cysteines in positions 309, 310, 311, 317, 322, and 326)were labeled with the paramagnetic nitroxide label MTSL.42 The sites were chosen to represent the stablecore of the third microtubule binding repeat, which is critical forfibril formation.43 Spin-labeled Tau wasallowed to aggregate in the presence of RNA (polyA) for 3 days underagitation (see Materials and Methods). Continuous-waveEPR measurements produced single-line spectra for all fibrils (Figure 1A). The collapseof the three-line spectra that are normally observed for MTSL-labeledproteins can be explained by the exchange interaction between multiplespin labels. This is achieved by parallel, in-register arrangementof β-strands in which labels attached to identical positionsin different Tau molecules are stacked along the fibril axis.44 Fibrils formed in the presence of other RNAspecies such as polyU, double-stranded RNA (UA), tRNA, and whole yeastRNA extract produced similar results (Figure 1B). Also, different negatively charged polymerssuch as single-stranded DNA and polyglutamate produced fibrils thatresulted in similar single-line characteristics (Figure 1C). Combined, these data suggestthat the parallel, in-register arrangement of β-strands is acommon feature of Tau fibrils, independent of the cofactor that isused to induce aggregation.

Bottom Line: These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents.Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin.These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States.

ABSTRACT
Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

Show MeSH
Related in: MedlinePlus