Zubiri, I. | Search bioRxiv

bioRxiv2021

Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1a.

Endoplasmic reticulum (ER) to nucleus homeostatic signalling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1. While IRE1 can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1 complexes as well as the targeting mechanism that recruits XBP1 to IRE1 oligomers, remain unknown. Here, we used a single molecule imaging approach to directly monitor the recruitment of individual XBP1 transcripts to the ER surface. We confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2-dependent targeting and relies on active translation. In addition, we show that IRE1-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1 clusters, which only assemble upon overexpression of fluorescently-tagged IRE1 during ER stress. Our findings support a model where ribosome-engaged, ER-poised XBP1 mRNA is processed by functional IRE1 assemblies that are homogenously distributed throughout the ER membrane.

cell biology↗

bioRxiv2020

Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

Blood-based biomarkers can be informative of brain disorders where protein aggregation play a major role. The proteome of plasma and circulating protein aggregates (CPA) reflect the inflammatory and metabolic state of the organism and can be predictive of system-level and/or organ-specific pathologies. CPA are enriched with heavy chain neurofilaments (NfH), key axonal constituents involved in brain aggregates formation and biomarkers of the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Here we show that CPA and brain protein aggregates (BPA) from ALS differ in protein composition and appear as a combination of electron-dense large globular and small filamentous formations on transmission electron microscopy. CPA are highly enriched with proteins involved in the proteasome and energy metabolism. Compared to the human proteome, proteins within aggregates show distinct and tissue-dependent chemical features of aggregation propensity. The use of a TMTcalibrator proteomics workflow with ALS brain as calibrant reveals 4973 brain-derived low-abundance proteins in CPA, including the products of translation of 24 ALS risk genes. 285 of these (5.7%) are regulated in ALS CPA including FUS (p<0.05). CPA from both ALS and healthy controls affect cell viability when testing endothelial and PC12 neuronal cell lines, while CPA from ALS exert a more toxic effect at lower concentrations. The analysis of resistance to protease enzymes hydrolysis indicates an ALS-specific digestion pattern for NfH using enterokinase. This study reveals how peripheral protein aggregates are significantly enriched with brain proteins which are highly representative of ALS pathology and a potential alternative source of biomarkers and therapeutic targets for this incurable disorder. Significance StatementMolecular mechanism of neurodegeneration like protein aggregation are important brain-specific alterations which need to be addressed therapeutically. Recently described fluid biomarkers of neurodegenerative disorders provide means for stratification and monitoring of disease progression. Here we show that circulating protein aggregates are easily accessible in blood and reproduce important features of brain pathology for an incurable disorder like amyotrophic lateral sclerosis. They represent a source of biomarkers and of novel therapeutics for ALS.

neuroscience↗