Detection of non‐native species formed during fibrillization of the myocilin olfactomedin domain

Scelsi, Hailee F.; Close, Emily G. S.; Huard, Dustin J. E.; Dunn, Elijah; Bogdanović, Nebojša; Mudiyanselage, Sonali H. W.; Grant, Arshay; Stagg, Scott M.; Schmidt‐Krey, Ingeborg; Van Horn, Wade D.; Lieberman, Raquel L.

doi:10.1002/pro.70063

Detection of non‐native species formed during fibrillization of the myocilin olfactomedin domain

Journal Article · Mon Mar 17 00:00:00 EDT 2025 · Protein Science

DOI:https://doi.org/10.1002/pro.70063· OSTI ID:2543177

Scelsi, Hailee F. ^[1]; Close, Emily G. S. ^[2]; Huard, Dustin J. E. ^[1]; Dunn, Elijah ^[1]; Bogdanović, Nebojša ^[3]; Mudiyanselage, Sonali H. W. ^[4]; Grant, Arshay ^[5]; Stagg, Scott M. ^[6]; Schmidt‐Krey, Ingeborg ^[5]; ^[4]; ^[1]

School of Chemistry and Biochemistry Georgia Institute of Technology Atlanta Georgia USA
School of Chemistry and Biochemistry Georgia Institute of Technology Atlanta Georgia USA, Pacific Northwest National Laboratory Richland Washington USA
Institute of Molecular Biophysics Florida State University Tallahassee Florida USA
Biodesign Center for Personalized Diagnostics Arizona State University Tempe Arizona USA, School of Molecular Sciences Arizona State University Tempe Arizona USA
School of Biological Sciences Georgia Institute of Technology Atlanta Georgia USA
Institute of Molecular Biophysics Florida State University Tallahassee Florida USA, Department of Biological Sciences Florida State University Tallahassee Florida USA

Abstract

Glaucoma is a group of neurodegenerative diseases that together are the leading cause of irreversible blindness worldwide. Myocilin‐associated glaucoma is an inherited form of this disease, caused by intracellular aggregation of misfolded mutant myocilin. In vitro, the myocilin C‐terminal olfactomedin domain (OLF), the relevant domain for glaucoma pathogenesis, can be driven to form amyloid‐like fibrils under mild conditions. Here we characterize a species present during in vitro fibrillization. Purified OLF was subjected to fibrillization at concentrations required for downstream electron microscopy imaging and NMR spectroscopy. Additional biophysical techniques, including analytical ultracentrifugation and X‐ray crystallography, were employed to further characterize the multicomponent mixture. Negative stain transmission electron microscopy (TEM) shows a non‐native species reminiscent of known prefibrillar oligomers from other amyloid systems, NMR indicates a minor population of partially misfolded species is present in solution, and cryo‐EM imaging shows two‐dimensional protein arrays. The predominant soluble species remaining in solution after the fibril reaction is natively folded, as evidenced by X‐ray crystallography. In summary, after incubating OLF under fibrillization‐promoting conditions, there is a heterogeneous mixture consisting of soluble folded protein, mature amyloid‐like fibrils, and partially misfolded intermediate species that at present belie additional molecular detail. The characterization of OLF fibrillar species illustrates the challenges associated with developing a comprehensive understanding of the fibrillization process for large, non‐model amyloidogenic proteins.

View Journal Article

Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0012704

OSTI ID:: 2543177

Alternate ID(s):: OSTI ID: 2545952

Journal Information:: Protein Science, Journal Name: Protein Science Journal Issue: 4 Vol. 34; ISSN 0961-8368

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United Kingdom

Language:: English

References (34)

NMRPipe: A multidimensional spectral processing system based on UNIX pipes Delaglio, Frank; Grzesiek, Stephan; Vuister, GeertenW. Journal of Biomolecular NMR, Vol. 6, Issue 3 https://doi.org/10.1007/BF00197809	journal	November 1995
SEDNTERP: a calculation and database utility to aid interpretation of analytical ultracentrifugation and light scattering data Philo, John S. European Biophysics Journal, Vol. 52, Issue 4-5 https://doi.org/10.1007/s00249-023-01629-0	journal	February 2023
CcpNmr AnalysisAssign: a flexible platform for integrated NMR analysis Skinner, Simon P.; Fogh, Rasmus H.; Boucher, Wayne Journal of Biomolecular NMR, Vol. 66, Issue 2 https://doi.org/10.1007/s10858-016-0060-y	journal	September 2016
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Bradford, Marion M. Analytical Biochemistry, Vol. 72, Issue 1-2 https://doi.org/10.1016/0003-2697(76)90527-3	journal	May 1976
Negative staining of proteins Kiselev, N. A.; Sherman, M. B.; Tsuprun, V. L. Electron Microscopy Reviews, Vol. 3, Issue 1 https://doi.org/10.1016/0892-0354(90)90013-I	journal	January 1990
[20] Processing of X-ray diffraction data collected in oscillation mode Otwinowski, Zbyszek; Minor, Wladek Macromolecular Crystallography Part A, 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X	book	January 1997
Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells Joe, Myung Kuk; Sohn, Seongsoo; Hur, Wonhee Biochemical and Biophysical Research Communications, Vol. 312, Issue 3 https://doi.org/10.1016/j.bbrc.2003.10.162	journal	December 2003
A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co-solutes Schuck, Peter Biophysical Chemistry, Vol. 108, Issue 1-3 https://doi.org/10.1016/j.bpc.2003.10.016	journal	March 2004
HullRad: Fast Calculations of Folded and Disordered Protein and Nucleic Acid Hydrodynamic Properties Fleming, Patrick J.; Fleming, Karen G. Biophysical Journal, Vol. 114, Issue 4 https://doi.org/10.1016/j.bpj.2018.01.002	journal	February 2018
The many faces of the trabecular meshwork cell Stamer, W. Daniel; Clark, Abbot F. Experimental Eye Research, Vol. 158 https://doi.org/10.1016/j.exer.2016.07.009	journal	May 2017
Amyloid Fibril Formation by the Glaucoma-Associated Olfactomedin Domain of Myocilin Orwig, Susan D.; Perry, Christopher W.; Kim, Laura Y. Journal of Molecular Biology, Vol. 421, Issue 2-3 https://doi.org/10.1016/j.jmb.2011.12.016	journal	August 2012
Oligomeric Intermediates in Amyloid Formation: Structure Determination and Mechanisms of Toxicity Fändrich, Marcus Journal of Molecular Biology, Vol. 421, Issue 4-5 https://doi.org/10.1016/j.jmb.2012.01.006	journal	August 2012
The Glaucoma-Associated Olfactomedin Domain of Myocilin Forms Polymorphic Fibrils That Are Constrained by Partial Unfolding and Peptide Sequence Hill, Shannon E.; Donegan, Rebecca K.; Lieberman, Raquel L. Journal of Molecular Biology, Vol. 426, Issue 4 https://doi.org/10.1016/j.jmb.2013.12.002	journal	February 2014
Stable, Metastable, and Kinetically Trapped Amyloid Aggregate Phases Miti, Tatiana; Mulaj, Mentor; Schmit, Jeremy D. Biomacromolecules, Vol. 16, Issue 1 https://doi.org/10.1021/bm501521r	journal	December 2014
Amyloid pores from pathogenic mutations Lashuel, Hilal A.; Hartley, Dean; Petre, Benjamin M. Nature, Vol. 418, Issue 6895 https://doi.org/10.1038/418291a	journal	July 2002
cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination Punjani, Ali; Rubinstein, John L.; Fleet, David J. Nature Methods, Vol. 14, Issue 3 https://doi.org/10.1038/nmeth.4169	journal	February 2017
Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming β-propeller Saccuzzo, Emily G.; Mebrat, Mubark D.; Scelsi, Hailee F. Nature Communications, Vol. 15, Issue 1 https://doi.org/10.1038/s41467-023-44479-2	journal	January 2024
Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs Bepler, Tristan; Morin, Andrew; Rapp, Micah Nature Methods, Vol. 16, Issue 11 https://doi.org/10.1038/s41592-019-0575-8	journal	October 2019
Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation Huard, Dustin J. E.; Jonke, Alex P.; Torres, Matthew P. Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-019-48751-8	journal	September 2019
Structural and functional properties of prefibrillar α-synuclein oligomers Pieri, Laura; Madiona, Karine; Melki, Ronald Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep24526	journal	April 2016
Amyloid ion channels: A common structural link for protein-misfolding disease Quist, A.; Doudevski, I.; Lin, H. Proceedings of the National Academy of Sciences, Vol. 102, Issue 30 https://doi.org/10.1073/pnas.0502066102	journal	July 2005
The Glaucoma-associated Olfactomedin Domain of Myocilin Is a Novel Calcium Binding Protein Donegan, Rebecca K.; Hill, Shannon E.; Turnage, Katherine C. Journal of Biological Chemistry, Vol. 287, Issue 52 https://doi.org/10.1074/jbc.M112.408906	journal	November 2012
Recurrent Mutations in a Single Exon Encoding the Evolutionarily Conserved Olfactomedin-Homology Domain of TIGR in Familial Open-Angle Glaucoma Adam, M. F.; Belmouden, A.; Binisti, P. Human Molecular Genetics, Vol. 6, Issue 12 https://doi.org/10.1093/hmg/6.12.2091	journal	November 1997
Structural basis for misfolding in myocilin-associated glaucoma Donegan, Rebecca K.; Hill, Shannon E.; Freeman, Dana M. Human Molecular Genetics, Vol. 24, Issue 8 https://doi.org/10.1093/hmg/ddu730	journal	December 2014
Genetics of glaucoma Wiggs, Janey L.; Pasquale, Louis R. Human Molecular Genetics, Vol. 26, Issue R1 https://doi.org/10.1093/hmg/ddx184	journal	May 2017
Evidence for S₃₃₁-G-S-L within the amyloid core of myocilin olfactomedin domain fibrils based on low-resolution 3D solid-state NMR spectra Saccuzzo, Emily G.; Robang, Alicia S.; Gao, Yuan https://doi.org/10.1101/2024.08.09.606901	preprint	August 2024
Topaz-Denoise: general deep denoising models for cryoEM and cryoET Bepler, Tristan; Kelley, Kotaro; Noble, Alex J. https://doi.org/10.1101/838920	preprint	May 2020
Solving structures of protein complexes by molecular replacement with Phaser McCoy, Airlie J. Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 1 https://doi.org/10.1107/S0907444906045975	journal	December 2006
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Towards automated crystallographic structure refinement with phenix.refine Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4 https://doi.org/10.1107/S0907444912001308	journal	March 2012
Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis Kayed, R. Science, Vol. 300, Issue 5618 https://doi.org/10.1126/science.1079469	journal	April 2003
Protein Misfolding, Amyloid Formation, and Human Disease: A Summary of Progress Over the Last Decade Chiti, Fabrizio; Dobson, Christopher M. Annual Review of Biochemistry, Vol. 86, Issue 1 https://doi.org/10.1146/annurev-biochem-061516-045115	journal	June 2017
Aggregated Myocilin Induces Russell Bodies and Causes Apoptosis Yam, Gary Hin-Fai; Gaplovska-Kysela, Katarina; Zuber, Christian The American Journal of Pathology, Vol. 170, Issue 1 https://doi.org/10.2353/ajpath.2007.060806	journal	January 2007
Amyloid oligomers as on-pathway precursors or off-pathway competitors of fibrils Muschol, Martin; Hoyer, Wolfgang Frontiers in Molecular Biosciences, Vol. 10 https://doi.org/10.3389/fmolb.2023.1120416	journal	February 2023

Similar Records

Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming β-propeller

Journal Article · Mon Jan 01 19:00:00 EST 2024 · Nature Communications · OSTI ID:2470105

Differential Misfolding Properties of Glaucoma-Associated Olfactomedin Domains from Humans and Mice

Journal Article · Sun Feb 24 19:00:00 EST 2019 · Biochemistry · OSTI ID:1506495

Detection of non‐native species formed during fibrillization of the myocilin olfactomedin domain

Citation Formats

References (34)

Similar Records

Related Subjects