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Identifying amyloid-related diseases by mapping mutations in low-complexity protein domains to pathologies

Murray, Kevin A. and Hughes, Michael P. and Hu, Carolyn J. and Sawaya, Michael R. and Salwinski, Lukasz and Pan, Hope and French, Samuel W. and Seidler, Paul M. and Eisenberg, David S. (2022) Identifying amyloid-related diseases by mapping mutations in low-complexity protein domains to pathologies. Nature Structural & Molecular Biology, 29 (6). pp. 529-536. ISSN 1545-9993. PMCID PMC9205782. doi:10.1038/s41594-022-00774-y. https://resolver.caltech.edu/CaltechAUTHORS:20220531-843690000

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Image (JPEG) (Extended Data Fig. 2: Thioflavin T aggregation kinetics assay of WT KRT8_(1-90) aggregation) - Supplemental Material
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Image (JPEG) (Extended Data Fig. 3: Radial profiles of x-ray powder diffraction of KRT8 aggregates) - Supplemental Material
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Image (JPEG) (Extended Data Fig. 5: The effects of pathogenic LARKS mutations on FUS aggregation) - Supplemental Material
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Image (JPEG) (Extended Data Fig. 6: KRT8 Western blots of patient-derived liver tissue extracts for alcoholic steatohepatitis (ASH) and hepatocellular carcinoma) - Supplemental Material
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Image (JPEG) (Extended Data Fig. 7: Seeded aggregation of KRT8 by preformed recombinant KRT8 aggregates) - Supplemental Material
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Image (JPEG) (Extended Data Fig. 10: Atomic structures of FUS and TDP-43) - Supplemental Material
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Abstract

Proteins including FUS, hnRNPA2, and TDP-43 reversibly aggregate into amyloid-like fibrils through interactions of their low-complexity domains (LCDs). Mutations in LCDs can promote irreversible amyloid aggregation and disease. We introduce a computational approach to identify mutations in LCDs of disease-associated proteins predicted to increase propensity for amyloid aggregation. We identify several disease-related mutations in the intermediate filament protein keratin-8 (KRT8). Atomic structures of wild-type and mutant KRT8 segments confirm the transition to a pleated strand capable of amyloid formation. Biochemical analysis reveals KRT8 forms amyloid aggregates, and the identified mutations promote aggregation. Aggregated KRT8 is found in Mallory–Denk bodies, observed in hepatocytes of livers with alcoholic steatohepatitis (ASH). We demonstrate that ethanol promotes KRT8 aggregation, and KRT8 amyloids co-crystallize with alcohol. Lastly, KRT8 aggregation can be seeded by liver extract from people with ASH, consistent with the amyloid nature of KRT8 aggregates and the classification of ASH as an amyloid-related condition.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/s41594-022-00774-yDOIArticle
https://doi.org/10.2210/pdb7K3C/pdbDOIData
https://doi.org/10.2210/pdb7K3X/pdbDOIData
https://doi.org/10.2210/pdb7K3Y/pdbDOIData
https://services.mbi.ucla.edu/zipperdb/Related ItemCalculations of zipper
https://srv.mbi.ucla.edu/LARKSdb/Related ItemCalculations of LARKS
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc9205782/PubMed CentralArticle
ORCID:
AuthorORCID
Murray, Kevin A.0000-0003-1969-7701
Hu, Carolyn J.0000-0001-6218-4034
Sawaya, Michael R.0000-0003-0874-9043
Salwinski, Lukasz0000-0003-4522-1969
Pan, Hope0000-0001-6509-6224
Seidler, Paul M.0000-0001-8530-7244
Eisenberg, David S.0000-0003-2432-5419
Additional Information:© 2022 The Authors. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 24 May 2021. Accepted 08 April 2022. Published 30 May 2022. We thank S. French Sr. for advice regarding tissue extraction of KRT8, J. Treanor and E. Marcus for discussions, D. Cascio and M. Collazo in the UCLA Department of Energy Institute Macromolecular Crystallization Core Technology Center for assistance in structure determination, and the UCLA Translational Pathology Core Laboratory for their assistance in acquiring tissue samples. We acknowledge USPHS National Research Service Award 5T32GM008496 (K. A. M.), the UCLA-Caltech Medical Scientist Training Program (K. A. M.), and HHMI for support. L. S. is supported by NIH GM123126. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on the 24-ID-E beamline is funded by a NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We acknowledge support from NIH AG 054022, and AG 048120 and DOE DE-FC02-02ER63421 (D. S. E.). Contributions. The project was conceived and designed by K. A. M. and D. S. Identification of LARKS in the proteome was done by M. P. H., identification of disease mutations within LARKS was done by L. S., and assessment of steric-zipper propensity for each mutation was measured by K. A. M. Crystallization and determination of KRT8 segment structures were performed by K. A. M. with assistance from M. R. S. Protein purification was performed by K. A. M. and C. J. H. Aggregation experiments, X-ray diffraction and SDS denaturation was performed by K. A. M. Liver extraction was performed by K. A. M. H. P. performed western blot analysis. Liver disease tissue samples were acquired and characterized with assistance from S. W. F. Electron microscopy was performed by P. M. S. and K. A. M. The manuscript was written by K. A. M. and D. S. E., with contributions from all other authors. Data availability. The datasets generated and/or analyzed during the current study are provided as source data. Any additional data are available from the corresponding author. All structural data have been deposited into the Worldwide Protein Data Bank (wwPDB) with the following accession codes: PDB 7K3C for SGMGGIT, PDB 7K3X for SGMGCIT, and PDB 7K3Y for GGYAGAS. Calculations of zipper and LARKS propensity can be performed using our online web servers, which can be found at https://services.mbi.ucla.edu/zipperdb/ and https://srv.mbi.ucla.edu/LARKSdb/ Atomic coordinates for KRT8 segments SGMGGIT, SGMGCIT, and GGYAGAS have been deposited in the Protein Data Bank under accession codes 7K3C, 7K3X, and 7K3Y, respectively. Source data are provided with this paper. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Competing interests. D. S. E. is SAB chair and equity holder in ADRx, Inc. The remaining authors declare no competing interests. Peer review. Nature Structural and Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.
Funders:
Funding AgencyGrant Number
NIH Predoctoral Fellowship5T32GM008496
UCLA-Caltech Medical Scientist Training ProgramUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIHGM123126
NIHP30 GM124165
NIHS10OD021527
Department of Energy (DOE)DE-AC02-06CH11357
NIHAG 054022
NIHAG 048120
Department of Energy (DOE)DE-FC02-02ER63421
Issue or Number:6
PubMed Central ID:PMC9205782
DOI:10.1038/s41594-022-00774-y
Record Number:CaltechAUTHORS:20220531-843690000
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20220531-843690000
Official Citation:Murray, K.A., Hughes, M.P., Hu, C.J. et al. Identifying amyloid-related diseases by mapping mutations in low-complexity protein domains to pathologies. Nat Struct Mol Biol 29, 529–536 (2022). https://doi.org/10.1038/s41594-022-00774-y
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:114966
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:01 Jun 2022 16:18
Last Modified:30 Jun 2022 17:58

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