Osumi, K. M. | Search bioRxiv

bioRxiv2026

Solid state NMR characterization of wild-type and mutant GFAP intermediate filament assemblies

GFAP is a type III intermediate filament primarily found within astrocytes and is known to maintain proper cell structure and mechanical strength. Mutations in GFAP are implicated in the pathology of Alexander disease, a neurodegenerative disease characterized by cytoplasmic inclusions of protein, known as Rosenthal fibers. GFAP has a typical type III intermediate filament domain structure, consisting of a highly conserved alpha-helical rod domain bracketed by an intrinsically disordered N-terminal head and C-terminal tail domains. While the general domain organization of monomeric GFAP and the assembly process for higher order quaternary structures are known, we lack an atomic resolution mechanistic understanding of GFAP assembly into mature filaments. Understanding the structure of GFAP filaments and how mutations disrupt this structure will provide vital information into how mutations produce Alexander disease pathology. As a first step towards a mechanistic description, we characterized GFAP wild type tetrameric and filamentous assemblies using solid state NMR and compared the results to those obtained from an assembly-deficient GFAP mutant. For wild-type GFAP, we observe surprisingly uniform rigid alpha helical structure and can spectroscopically resolve highly mobile intrinsically disordered regions in the filament assemblies. Wild type tetramers show increased mobility, likely arising from the head and tail domains. Mutation of the highly conserved cysteine at position 294 to serine results in an inability to form full-length filament assemblies. We show that the rigid regions of the C294S mutant assemblies largely remain structurally consistent with wild type tetrameric assemblies but differ from wild-type filament assemblies. There is an increase in highly mobile regions for the C294S mutant relative to the wild-type. Our results provide a foundation for developing solid state NMR approaches to characterize intermediate filament assembly mechanisms and the interfering effect of disease mutations.

biophysics↗

bioRxiv2025

Small heat shock protein HSPB8 interacts with a pre-fibrillar TDP43 low complexity domain species to delay fibril formation

The loss of cellular proteostasis through aberrant stress granule formation is implicated in neurodegenerative diseases. Stress granules are formed by biomolecular condensation involving protein-protein and protein-RNA interactions. These assemblies are protective, but can rigidify, leading to amyloid-like fibril formation, a hallmark of the disease pathology. Key proteins dictating stress granule formation and disassembly, such as TDP43, contain low-complexity (LC) domains that drive fibril formation. HSPB8, a small heat shock protein, localizes to stress granules, has known aggregation delaying activity, and helps direct aggregated proteins to protein degradation pathways. It is not known how HSPB8 interacts with aggregation prone LC domains in stress granules. Here, we examined the interaction between isolated HSPB8 and the TDP43 LC using thioflavin T (ThT) and fluorescence polarization (FP) aggregation assays, fluorescence microscopy and photobleaching experiments, and crosslinking mass spectrometry (XL-MS). Our results indicate that HSPB8 delays TDP43 LC aggregation through domain-specific interactions with fibril nucleating species, without affecting fibril elongation rates. These findings provide mechanistic insight into how HSPB8 mediates LC domain aggregation and provides bases for investigating how the TDP43 LC subverts chaperone activity in neurodegenerative disease and differing mechanisms between members of the HSPB protein family.

biophysics↗