Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

The inherited form of open angle glaucoma arises due to a toxic gain-of-function intracellular misfolding event involving a mutated myocilin olfactomedin domain (OLF). Mutant myocilin is recognized by the endoplasmic reticulum (ER)-resident heat shock protein 90 paralog, glucose regulated protein 94...

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Bibliographic Details
Published in:Scientific reports 2019-09, Vol.9 (1), p.12769-12, Article 12769
Main Authors: Huard, Dustin J E, Jonke, Alex P, Torres, Matthew P, Lieberman, Raquel L
Format: Article
Language:English
Subjects:
Amyloid
Amyloid - chemistry
Amyloid - genetics
Amyloid - metabolism
Animals
Cytoskeletal Proteins - chemistry
Cytoskeletal Proteins - genetics
Cytoskeletal Proteins - metabolism
Dogs
Endoplasmic reticulum
Eye Proteins - chemistry
Eye Proteins - genetics
Eye Proteins - metabolism
Fluorescence polarization
Glaucoma
Glycoproteins - chemistry
Glycoproteins - genetics
Glycoproteins - metabolism
Heat shock proteins
Hsp90 protein
Humans
Mass spectroscopy
Membrane Glycoproteins - chemistry
Membrane Glycoproteins - genetics
Membrane Glycoproteins - metabolism
Mutation
Olfactomedin
Protein Aggregation, Pathological
Protein Domains
siRNA
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Summary:The inherited form of open angle glaucoma arises due to a toxic gain-of-function intracellular misfolding event involving a mutated myocilin olfactomedin domain (OLF). Mutant myocilin is recognized by the endoplasmic reticulum (ER)-resident heat shock protein 90 paralog, glucose regulated protein 94 (Grp94), but their co-aggregation precludes mutant myocilin clearance by ER-associated degradation. When the Grp94-mutant myocilin interaction is abrogated by inhibitors or siRNA, mutant myocilin is efficiently degraded. Here we dissected Grp94 into component domains (N, NM, MC) to better understand the molecular factors governing its interaction with OLF. We show that the Grp94 N-terminal nucleotide-binding N domain is responsible for accelerating OLF aggregation in vitro. Upon inhibiting the isolated N domain pharmacologically or removing the Pre-N terminal 57 residues from full-length Grp94, OLF aggregation rates revert to those seen for OLF alone, but only pharmacological inhibition rescues co-aggregation. The Grp94-OLF interaction is below the detection limit of fluorescence polarization measurements, but chemical crosslinking paired with mass spectrometry analyses traps a reproducible interaction between OLF and the Grp94 N domain, as well as between OLF and the Grp94 M domain. The emerging molecular-level picture of quinary interactions between Grp94 and myocilin points to a role for the far N-terminal sequence of the Grp94 N domain and a cleft in the M domain. Our work further supports drug discovery efforts to inhibit these interactions as a strategy to treat myocilin-associated glaucoma.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-019-48751-8