Biology subjects

Sakata, E.

Publications and source records attributed to Sakata, E..

6 recordsLinked to original sources

bioRxiv2026

The vacuolar tauopathy-associated mutation D395G confers redox sensitivity to p97/VCP

The multifunctional AAA+ ATPase p97/VCP is a pivotal regulator of cellular proteostasis, extracting polyubiquitinated substrates from protein complexes or organelle membranes for proteasomal degradation. Mutations in p97 are linked to a broad spectrum of neurodegenerative disorders, including multisystem proteinopathy and amyotrophic lateral sclerosis. Here, we provide insights into the basis of dysfunction in p97D395G, implicated in vacuolar tauopathy, using an integrated structural approach. The mutation destabilizes the interaction network in the transient ADP.Pi state previously identified in p97WT and alters the dynamics of the linker between the tandem ATPase domains, resulting in decreased ATPase activity. We further demonstrate that the D395G mutation sensitizes p97 to oxidative stress by enhancing C522 oxidation, thereby perturbing nucleotide binding in D2, and define the functional basis of this oxidative inactivation of p97. These findings reveal redox control as a key regulatory layer of the p97 ATPase cycle and provide a mechanistic framework for how oxidative stress contributes to p97-associated neurodegeneration.

molecular biology↗

bioRxiv2026

Blm10/PA200-activated 20S proteasomes promote α-synuclein degradation and bypass proteasome inhibition in Parkinson disease models

Protein homeostasis is essential for maintaining normal cellular function. However, protein homeostasis efficiency declines with age, leading to the accumulation of aberrant protein structures associated with neurodegenerative diseases such as Parkinsons disease (PD). PD is characterized by the aggregation of alpha-synuclein (Syn) into cytoplasmic inclusions. This process is accompanied by elevated phosphorylation at serine 129 (S129). The accumulation of Syn into aggregates and their propagation disrupts key proteostasis pathways, including the ubiquitin-proteasome system (UPS) or autophagy, contributing to cellular dysfunction and neuronal death. This study identified the proteasome activator Blm10 and its human ortholog PA200 as modulators of Syn degradation and toxicity. The conserved Blm10/PA200 protein plays a key role in regulating proteasome activity and assembly. The Syn expression increases Blm10 protein stability through autophagy inhibition, in a manner dependent on Syn phosphorylation at S129 in yeast. Overexpression of BLM10 or PA200 reduces Syn aggregation and enhances Syn turnover via activation of the 20S proteasome in yeast and mammalian cells. Blm10 and PA200-capped 20S proteasomes efficiently degrade both monomeric as well as oligomeric Syn in vitro. Notably, capped proteasomes retain proteolytic activities in presence of Syn, indicating resistance to Syn-induced inhibition, in contrast to 20S or 26S proteasomes. These results reveal a distinct proteasome subtype that bypasses UPS impairment and restores proteolytic capacity under proteotoxic stress. Our findings establish Blm10/PA200 as critical regulator of Syn proteostasis and highlight its protective role in maintaining protein homeostasis and cell viability under conditions of Syn toxicity.

cell biology↗

bioRxiv2025

Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

The 26S proteasome primarily degrades proteins marked by polyubiquitin chains. Although ubiquitin-independent pathways for proteasomal degradation exist, the mechanisms involved remain poorly understood. Bag1 links the Hsp70 chaperone to the 26S proteasome, recruiting Hsp70-bound aberrant proteins for degradation. Here, we present high-resolution cryo-EM structures of the Bag1-bound 26S proteasome, which reveal unprecedented conformational changes within the 19S regulatory particle. Bag1 binding to the Rpn1 subunit induces a dramatic reconfiguration of AAA+ ATPase subunits, disrupting the canonical spiral staircase conformation and remodeling the central channel architecture. This creates a large cavity above the substrate entry gate of the 20S core particle, enabling the direct entry of Hsp70 clients into the proteolytic chamber. Thus, in this ubiquitin-independent degradation pathway, unfolded proteins can bypass the need for both client ubiquitination and ATP hydrolysis for degradation.

molecular biology↗

bioRxiv2023

Unravelling ATP processing by the AAA+ protein p97 at the atomic level

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of homeostasis and impactful pharmacological target. The hexameric complex undergoes substantial conformational changes in the course of its catalytic cycle. Here, we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and MD simulations. p97 populates a metastable reaction intermediate, the ADP.Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

biophysics↗

bioRxiv2022

A mono- and intralink filter (mi-filter) to improve false-discovery rates in cross-linking mass spectrometry data

Cross-Linking Mass Spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter protein cross-links. This can be challenging, in particularly in samples where relatively few PPIs are detected, as is often the case in complex samples containing low abundant proteins or in in-vivo settings. In this manuscript we are describing a novel mono- and intralink filter (mi-filter) that is applicable to any kind of crosslinking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein crosslink has been identified within a given dataset are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of crosslinking-data ranging from single protein complexes, over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly lowers the number of false-positive identifications resulting in improved false-discovery rates for inter-protein links in all these types of XL-MS data.

biochemistry↗

bioRxiv2021

Allosteric control of Ubp6 and the proteasome via a bidirectional switch

The proteasome is the principal cellular protease, and recognizes target proteins that have been covalently marked by ubiquitin chains. The ubiquitin signal is subject to rapid editing at the proteasome, allowing it to reject substrates based on topological features of their attached ubiquitin chains. Editing is mediated by a key regulator of the proteasome, deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome-both by deubiquitinating proteasome-bound ubiquitin conjugates, and through a noncatalytic effect that does not involve deubiquitination. We report mutants in both Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations fall within its ILR element, defined here, which is conserved from yeast to mammals. The ILR is a component of the BL1 blocking loop, other parts of which obstruct ubiquitin access to the catalytic groove in free Ubp6. Rpt1 docking at the ILR opens the catalytic groove by rearranging not only BL1 but also a novel network of three directly interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition by Ubp6 are linked in that they were eliminated by the same Ubp6 and Rpt1 mutations. Ubp6 and ubiquitin together drive the proteasome into a unique conformational state associated with proteasome inhibition. Our results identify a multicomponent allosteric switch that exerts simultaneous control over the activity of both Ubp6 and the proteasome, and suggest that their active states are in general mutually exclusive. The findings lead to a new paradigm for allosteric control of deubiquitinating enzymes.

biochemistry↗