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Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS.

Kim HJ, Kim NC, Wang YD, Scarborough EA, Moore J, Diaz Z, MacLea KS, Freibaum B, Li S, Molliex A, Kanagaraj AP, Carter R, Boylan KB, Wojtas AM, Rademakers R, Pinkus JL, Greenberg SA, Trojanowski JQ, Traynor BJ, Smith BN, Topp S, Gkazi AS, Miller J, Shaw CE, Kottlors M, Kirschner J, Pestronk A, Li YR, Ford AF, Gitler AD, Benatar M, King OD, Kimonis VE, Ross ED, Weihl CC, Shorter J, Taylor JP - Nature (2013)

Bottom Line: Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP.Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology.Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee 38120, USA.

ABSTRACT
Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

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Disease mutations accelerate hnRNPA2 and hnRNPA1 fibrillizationa. Synthetic hexapeptides A2 wild-type (NYNDFG) or mutant (NYNVFG) were incubated at 25°C for 2h. Fibrillization was monitored by ThT fluorescence. b. EM of A2 wild-type or mutant hexapeptides after 10min at 25°C. Bar, 0.1µm. c–d. Fibrillization analysis of A1 wild-type (SYNDFG) or mutant (SYNVFG) as in (a–b). e. Full-length hnRNPA2 WT, hnRNPA2 D290V, or hnRNP2Δ287–292 was incubated at 25 °C with agitation for 0–12h. At various times, the amount of aggregated hnRNPA2 was determined. Values represent means ± SEM (n=3). f. EM of hnRNPA2 fibrillization reactions after 0, 4, and 12h at 25°C. Note the absence of fibers after 4h for hnRNPA2 wild-type. Bar, 0.5µm. g–h. Fibrillization of full-length hnRNPA1 wild-type, hnRNPA1-D262V, hnRNPA1-D262N, or hnRNPA1Δ259–264 monitored as in (e, f).
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Figure 4: Disease mutations accelerate hnRNPA2 and hnRNPA1 fibrillizationa. Synthetic hexapeptides A2 wild-type (NYNDFG) or mutant (NYNVFG) were incubated at 25°C for 2h. Fibrillization was monitored by ThT fluorescence. b. EM of A2 wild-type or mutant hexapeptides after 10min at 25°C. Bar, 0.1µm. c–d. Fibrillization analysis of A1 wild-type (SYNDFG) or mutant (SYNVFG) as in (a–b). e. Full-length hnRNPA2 WT, hnRNPA2 D290V, or hnRNP2Δ287–292 was incubated at 25 °C with agitation for 0–12h. At various times, the amount of aggregated hnRNPA2 was determined. Values represent means ± SEM (n=3). f. EM of hnRNPA2 fibrillization reactions after 0, 4, and 12h at 25°C. Note the absence of fibers after 4h for hnRNPA2 wild-type. Bar, 0.5µm. g–h. Fibrillization of full-length hnRNPA1 wild-type, hnRNPA1-D262V, hnRNPA1-D262N, or hnRNPA1Δ259–264 monitored as in (e, f).

Mentions: We directly tested the predictions that (i) hnRNPA2 and hnRNPA1 are prone to fibrillization and (ii) this property is enhanced by disease-causing mutations. First, we experimentally assessed the ZipperDB prediction for the impact of disease mutations on steric zipper motifs found in hnRNPA2/B1 and hnRNPA1. Remarkably, the synthetic mutant hexapeptides of hnRNPA2/B1-D290V (NYNVFG) and hnRNPA1-D262V (SYNVFG) rapidly assembled into amyloid fibrils, as shown by thioflavin-T fluorescence and electron microscopy (EM), whereas the corresponding wild-type peptides did not even after several weeks (Fig. 4a–d). Thus, the disease mutations in hnRNPA2/B1 and hnRNPA1 generate highly amyloidogenic hexapeptides precisely as predicted. The more potent steric zippers that result from disease mutations are likely to be significant for two reasons. First, introduction of similarly potent steric zippers is sufficient to force fibril formation even in model proteins that do not ordinarily fibrillize17. Second, in hnRNPA1 and hnRNPA2 these potent steric zippers are centered in the intrinsically disordered PrLD and are thus available to make intermolecular contacts and drive fibril formation.


Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS.

Kim HJ, Kim NC, Wang YD, Scarborough EA, Moore J, Diaz Z, MacLea KS, Freibaum B, Li S, Molliex A, Kanagaraj AP, Carter R, Boylan KB, Wojtas AM, Rademakers R, Pinkus JL, Greenberg SA, Trojanowski JQ, Traynor BJ, Smith BN, Topp S, Gkazi AS, Miller J, Shaw CE, Kottlors M, Kirschner J, Pestronk A, Li YR, Ford AF, Gitler AD, Benatar M, King OD, Kimonis VE, Ross ED, Weihl CC, Shorter J, Taylor JP - Nature (2013)

Disease mutations accelerate hnRNPA2 and hnRNPA1 fibrillizationa. Synthetic hexapeptides A2 wild-type (NYNDFG) or mutant (NYNVFG) were incubated at 25°C for 2h. Fibrillization was monitored by ThT fluorescence. b. EM of A2 wild-type or mutant hexapeptides after 10min at 25°C. Bar, 0.1µm. c–d. Fibrillization analysis of A1 wild-type (SYNDFG) or mutant (SYNVFG) as in (a–b). e. Full-length hnRNPA2 WT, hnRNPA2 D290V, or hnRNP2Δ287–292 was incubated at 25 °C with agitation for 0–12h. At various times, the amount of aggregated hnRNPA2 was determined. Values represent means ± SEM (n=3). f. EM of hnRNPA2 fibrillization reactions after 0, 4, and 12h at 25°C. Note the absence of fibers after 4h for hnRNPA2 wild-type. Bar, 0.5µm. g–h. Fibrillization of full-length hnRNPA1 wild-type, hnRNPA1-D262V, hnRNPA1-D262N, or hnRNPA1Δ259–264 monitored as in (e, f).
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Figure 4: Disease mutations accelerate hnRNPA2 and hnRNPA1 fibrillizationa. Synthetic hexapeptides A2 wild-type (NYNDFG) or mutant (NYNVFG) were incubated at 25°C for 2h. Fibrillization was monitored by ThT fluorescence. b. EM of A2 wild-type or mutant hexapeptides after 10min at 25°C. Bar, 0.1µm. c–d. Fibrillization analysis of A1 wild-type (SYNDFG) or mutant (SYNVFG) as in (a–b). e. Full-length hnRNPA2 WT, hnRNPA2 D290V, or hnRNP2Δ287–292 was incubated at 25 °C with agitation for 0–12h. At various times, the amount of aggregated hnRNPA2 was determined. Values represent means ± SEM (n=3). f. EM of hnRNPA2 fibrillization reactions after 0, 4, and 12h at 25°C. Note the absence of fibers after 4h for hnRNPA2 wild-type. Bar, 0.5µm. g–h. Fibrillization of full-length hnRNPA1 wild-type, hnRNPA1-D262V, hnRNPA1-D262N, or hnRNPA1Δ259–264 monitored as in (e, f).
Mentions: We directly tested the predictions that (i) hnRNPA2 and hnRNPA1 are prone to fibrillization and (ii) this property is enhanced by disease-causing mutations. First, we experimentally assessed the ZipperDB prediction for the impact of disease mutations on steric zipper motifs found in hnRNPA2/B1 and hnRNPA1. Remarkably, the synthetic mutant hexapeptides of hnRNPA2/B1-D290V (NYNVFG) and hnRNPA1-D262V (SYNVFG) rapidly assembled into amyloid fibrils, as shown by thioflavin-T fluorescence and electron microscopy (EM), whereas the corresponding wild-type peptides did not even after several weeks (Fig. 4a–d). Thus, the disease mutations in hnRNPA2/B1 and hnRNPA1 generate highly amyloidogenic hexapeptides precisely as predicted. The more potent steric zippers that result from disease mutations are likely to be significant for two reasons. First, introduction of similarly potent steric zippers is sufficient to force fibril formation even in model proteins that do not ordinarily fibrillize17. Second, in hnRNPA1 and hnRNPA2 these potent steric zippers are centered in the intrinsically disordered PrLD and are thus available to make intermolecular contacts and drive fibril formation.

Bottom Line: Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP.Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology.Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee 38120, USA.

ABSTRACT
Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

Show MeSH
Related in: MedlinePlus