Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP

ARTICLE https://doi.org/10.1038/s41467-022-30238-2 OPEN Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP 1234567890():,; Eduardo Pinho Melo 1,2,6 ✉, Tasuku Konno 1, Ilaria Farace1, Mosab Ali Awadelkareem 1, Lise R. Skov Fernando Teodoro 2, Teresa P. Sancho2, Adrienne W. Paton3, James C. Paton 3, Matthew Fares4, Pedro M. R. Paulo5, Xin Zhang 4 & Edward Avezov 1,6 ✉ 1, Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates’ formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone – BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response. 1 Department of Clinical Neurosciences, UK Dementia Research Institute, University of Cambridge, Cambridge, UK. 2 CCMAR-Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, Faro, Portugal. 3 Research Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia. 4 Department of Chemistry, The Pennsylvania State University, University Park, State College, PA, USA. 5 Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, Lisboa, Portugal. 6These authors jointly supervised this work: Eduardo Pinho Melo, Edward Avezov. ✉email: ; NATURE COMMUNICATIONS | (2022)13:2501 | https://doi.org/10.1038/s41467-022-30238-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30238-2 N ewly synthesised polypeptides must attain the functional three-dimensional conformation, thermodynamically encoded by the amino acid sequence1. Protein folding describes the process where polypeptides seek the minimum point in energy-funnel but can be trapped in local minima separated by low barriers (misfolding traps2). Furtherer, the chemical diversity and crowdedness of cellular compartments create conditions for reactions competing with native folding, inducing local unfolding and stabilising non-functional, misfolding-trapped conformations. Unfolded and misfolded protein species tend to form insoluble aggregates3. To avoid the accumulation of cytotoxic aggregates, the biosynthetic organelles (i.e. cytoplasm, the Endoplasmic Reticulum, ER, and mitochondria) evolved a multi-layer proteostasis network (PN) that chaperones nascent proteins and rescues or recycles misfolded intermediates4. The PN is endowed with a capability to feedback to the gene-transcription and translation systems to interactively adjust the protein production rate to its foldingassistance and quality control capability. The ER branch of the PN, the Unfolded Protein Response (UPR), alleviates un/misfolded protein load during stress by transient attenuation of protein synthesis and posttranslational chaperone activation, followed by transcriptional upregulation of protein maturation and quality control factors5,6. Shortfalls of the PN machinery may lead to pathology: protein aggregation accompanies neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Amyotrophic Lateral Sclerosis and Frontotemporal Dementia7. Neuronally expressed metastable proteins (e.g. Aβ, Tau, or α-synuclein)—with a tendency to oligomerise and consequently aggregate—underlie these conditions. The late onset of the aggregation-related sub-populational neurodegeneration is consistent with age- and environmental factor-dependent decline in the efficiency of the PN machinery. Understanding how the cell handles aggregation-prone proteins on an organellar level is crucial to rationalising the disease-related proteostasis impairment. The recent discovery of the cytosolic Hsp70 chaperone and its assisting system’s capacity to resolve cytoplasmic aggregates8 (including amyloids9,10) adds another functional layer to the PN for rectifying imperfections of the quality control system or prionlike transition from native folding to aggregation. However, it is unknown whether such a capability exists in the ER—a site responsible for the manufacturing of about one-third of the cell’s protein repertoire (including disease determinants, e.g. amyloid precursor protein, APP). Observations suggest that the chaperonerich environment of the ER is more immune to protein aggregation than the cytoplasm11. How the organelle maintains high fidelity protein folding, avoiding aggregation throughout the lifetime of the cell, particularly the non-dividing neurons, remains an open question. Explaining this in terms of protein folding quality control alone would rest on the assumption that the system can achieve zero aggregation and absolute quality control through astronomically large numbers of protein folding cycles. In this work, we use a purpose-developed protein aggregation probing system with sub-organellar resolution to show accumulation of protein aggregates in the ER. Strikingly, ER stress, induced by N-glycosilation or Ca2+ pump inhibitors, triggers an aggregates’ clearance activity. We find that protein dissagregation is catalysed by the stress-responsive ER molecular chaperone— BiP. Modulating its amount in the ER reveal an inverse correlation between BiP’s presence and the aggregation load. Further, we reconstruct the disaggregation reaction in vitro by a minimal system of ATP-fuelled BiP with a J-domain cofactor. Results and discussion Monitoring ER protein aggregation in live cells. We sought an optical single-cell protein aggregate probing approach with 2 subcellular space resolution to surmount limitations associated with biochemical, post-lysis cell population extracts. The lysateprobing techniques can detect aggregation events in a population involving drastic changes in a high proportion of an aggregating protein. However, the heterogenous population averaging effect in ensemble analyses, combined with the lack of information on the probe’s distribution in space and the reliance on protein status preservation in lysis limit the ability to resolve folded/misfolded/ aggregated species of metastable substrate. The folding status (...truncated)