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Cargo receptor-assisted endoplasmic reticulum export of pathogenic α1-antitrypsin polymers

ABSTRACT: Background: Circulating polymers of alpha1-antitrypsin (α1AT) are chemo-attractant for neutrophils and contribute to inflammation in pulmonary, vascular and adipose tissues. Cellular factors affecting the intracellular itinerary of mutant polymerogenic α1AT remain obscure. Methods: Here, we report on an unbiased genome-wide CRISPR/Cas9 screen for regulators of trafficking of the polymerogenic α1AT-H334D variant. Single guide RNAs targeting genes whose inactivation enhanced accumulation of polymeric α1AT were enriched by iterative construction of CRISPR libraries based on genomic DNA from fixed cells selected for high polymer content by fluorescence-activated cell sorting. This approach bypassed the limitation to conventional enrichment schemes imposed by cell fixation. Results: Our screen identified 121 genes involved in polymer retention at false discovery rate < 0.1. From that set of genes, the pathway ‘cargo loading into COPII-coated vesicles’ was overrepresented with 16 significant genes, including two transmembrane cargo receptors, LMAN1 (ERGIG-53) and SURF4. LMAN1 and SURF4-disrupted cells displayed a secretion defect extended beyond α1AT monomers to polymers, whose low-level secretion was especially dependent on SURF4 and correlated with SURF4-α1AT-H334D physical interaction and with enhanced co-localisation of polymeric α1AT-H334D with the endoplasmic reticulum (ER). Conclusions: These findings suggest that ER cargo receptors co-ordinate intracellular progression of α1AT out of the ER and modulate the accumulation of polymeric α1AT not only by controlling the concentration of precursor monomers but also through a previously-unrecognised role in secretion of the polymers themselves.

ORGANISM(S): Cricetulus griseus

PROVIDER: GSE158574 | GEO | 2021/06/02

REPOSITORIES: GEO

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Project description:This experiment was conducted to identify mRNA transcripts alteration in liver of Ern1 liver specific knockout mice. The following abstract from the submitted manuscript describes the major findings of this work. The IRE1a-XBP1s axis regulates the COPII-mediated secretory program in response to nutrient availability. Lin Liu, Jie Cai, Xijun Liang, Qian Zhou, Huimin Wang, Chenyun Ding, Yuangang Zhu, Liwei Xiao, Tingting Fu, Zhisheng Xu, Jing Liu, Yujing Yin, Lei Fang, Bin Xue, Yan Wang, Aibin He, Yong Liu, Xiao-Wei Chen, and Zhenji Gan The cytoplasmic coat protein complex-II (COPII) is an evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII-mediated secretion parallel the activation of XBP1s, the critical transcription factor in handling cellular ER stress, in both live cells and mouse liver upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent secretion, mediating the nutrient stimulatory effects. ChIP-seq and RNA-seq analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII secretion-related genes, thereby driving COPII-directed secretory process. Liver-specific disruption of the IRE1a-XBP1s signaling branch results in diminished COPII-mediated secretion. Reactivation of XBP1s in mice lacking hepatic IRE1a restores COPII-mediated lipoprotein secretion, and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid secretion: the IRE1a-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII secretory program.

Project description:The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. We have generated transgenic neural progenitor cell (NPC) cultures from mouse embryonic cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or deltaNS. We have characterised these cells in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. DeltaNS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used our model system to investigate which cellular pathways were activated by intracellular polymers of G392E NS by performing RNA sequencing of differentiated cells expressing G392E NS or the negative control protein GFP, and identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells. We focused our attention on genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS. Inhibition of these defences by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.

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Cargo receptor-assisted endoplasmic reticulum export of pathogenic α1-antitrypsin polymers

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