Abstract
Ubiquitin and its polymeric forms are conjugated to intracellular proteins to regulate diverse intracellular processes. Intriguingly, polyubiquitin has also been identified as a component of pathological protein aggregates associated with Alzheimer’s disease and other neurodegenerative disorders. We recently found that polyubiquitin can form amyloid-like fibrils, and that these fibrillar aggregates can be degraded by macroautophagy. Although the structural properties appear to function in recognition of the fibrils, no structural information on polyubiquitin fibrils has been reported so far. Here, we identify the core of M1-linked diubiquitin fibrils from hydrogen-deuterium exchange experiments using solution nuclear magnetic resonance (NMR) spectroscopy. Intriguingly, intrinsically flexible regions became highly solvent-protected in the fibril structure. These results indicate that polyubiquitin fibrils are formed by inter-molecular interactions between relatively flexible structural components, including the loops and edges of secondary structure elements.
Highlights
Ubiquitin is a post-translational modifier, and covalent modification of proteins with ubiquitin is related to myriad biological processes, including cell cycle progression and immune response [1]
A small amount of trifluoroacetic acid (TFA) was necessary to completely dissolve monoubiquitin in dimethyl sulfoxide (DMSO) and we observed no changes in cross-peak intensities caused by TFA at this low concentration
By using the quenched HD exchange method, we investigated to what extent the amide protons of M1-linked diubiquitin fibrils are solvent-protected
Summary
Ubiquitin is a post-translational modifier, and covalent modification of proteins with ubiquitin (ubiquitylation) is related to myriad biological processes, including cell cycle progression and immune response [1]. Ubiquitin is known to be an extremely stable and rigid protein; it is often found in intracellular aggregates associated with neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases [2,3]. We previously found that the thermodynamic stability of ubiquitin decreases as a function of the degree of polymerization, and that polyubiquitin forms amyloid-like fibrils upon application of heat or shear stress [4]. In living cells, polyubiquitin forms fibrillar aggregates that can be selectively degraded by macroautophagy [4,5]. In cells, ubiquitylation is related to the formation of fibrillar aggregates that can be substrates for macroautophagy; in the case of macroautophagy dysfunction, such aggregates accumulate, which can contribute to the development of neurodegenerative diseases [6]. In the proteolytic clearance of the polyubiquitin fibrillar aggregates, the structure of the fibrils may play a key role, possibly facilitating recognition by receptor proteins of the macroautophagy system; structural details of polyubiquitin fibrils remain scarce
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