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1.
STAR Protoc ; 2(1): 100278, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33521680

ABSTRACT

The 26S proteasome is specialized for regulated protein degradation. It is formed by a regulatory particle (RP) that recognizes ubiquitinated substrates and caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Structural heterogeneity caused by dynamics makes it challenging to observe ubiquitin chains at the RP by cryogenic electron microscopy (cryo-EM). Here, we present a cryo-EM-based protocol we applied to study the human 26S proteasome with ubiquitin chains by using non-cleavable M1-linked hexaubiquitin (M1-Ub6) unanchored to a substrate. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).


Subject(s)
Cryoelectron Microscopy , Models, Molecular , Polyubiquitin , Proteasome Endopeptidase Complex , Humans , Polyubiquitin/chemistry , Polyubiquitin/ultrastructure , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/ultrastructure
2.
Nat Commun ; 6: 6116, 2015 Jan 20.
Article in English | MEDLINE | ID: mdl-25600778

ABSTRACT

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.


Subject(s)
Polyubiquitin/chemistry , Polyubiquitin/metabolism , Animals , Autophagy , Calorimetry, Differential Scanning , Circular Dichroism , Escherichia coli/metabolism , Immunohistochemistry , Mice , Microscopy, Electron, Transmission , Polyubiquitin/ultrastructure , Sf9 Cells , Spectrometry, Fluorescence , Ubiquitin/chemistry , Ubiquitin/metabolism
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