bims-unfpre 2022-01-09 papers

bims-unfpre

Biomed News

on Unfolded protein response

Issue of 2022–01–09
24 papers selected by
Susan Logue, University of Manitoba

Drosophila Unfolded Protein Response (UPR) Assays In Vitro and In Vivo.
HTS Identification of Activators and Inhibitors of Endoplasmic Reticulum (ER) Stress and the Unfolded Protein Response (UPR).
Assays to Study IRE1 Activation and Signaling.
Analysis of XBP1 Contribution to Hyperosmolarity-Induced Lipid Synthesis.
The interaction of SIRT4 and Calreticulin during ER stress in glia cells.
Protein Preparation for Proteomic Analysis of the Unfolded Protein Response in Arabidopsis thaliana.
Detecting the Non-conventional mRNA Splicing and Translational Activation of HAC1 in Budding Yeast.
The Use of Fluorescent Protein Fusions to Monitor the Unfolded Protein Response and Protein Foldase-Substrate Interactions in Plant Protoplasts.
A Platform Technology for Monitoring the Unfolded Protein Response.
Mfn2 Regulates High Glucose-Induced MAMs Dysfunction and Apoptosis in Podocytes via PERK Pathway.
TREX1 Deficiency Induces ER Stress-Mediated Neuronal Cell Death by Disrupting Ca2+ Homeostasis.
CNPY2 Contributes to Liver Oncogenesis by Promoting UPR-dependent destabilization of p53.
Integrating an ER Stress Reporter for Monitoring Genome-Wide UPR-ER in Budding Yeast.
Structure-Based Drug Discovery of IRE1 Modulators.
In Vitro Stimulation of IRE1α/XBP1-Deficient B Cells with LPS.
Methods to Study the Mitochondrial Unfolded Protein Response (UPRmt) in Caenorhabditis elegans.
Fluorescence-Based Biosensors for the Detection of the Unfolded Protein Response.
A new murine model of Barth Syndrome neutropenia links TAFAZZIN deficiency to increased ER stress induced apoptosis.
Effect Of XBP1 Deficiency In Cartilage On The Regulatory Network Of LncRNA/circRNA-miRNA-mRNA.
Detection of PERK Signaling in the Central Nervous System.
Determination of the Stability and Intracellular (Intra-Nuclear) Targeting and Recruitment of Pre-HAC1 mRNA in the Saccharomyces cerevisiae During the Activation of UPR.
Analyze Mouse Knockout Models of UPR Pathway Elements.
AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.
Detection of HAC1 mRNA Splicing by RT-PCR in Saccharomyces cerevisiae.

Methods Mol Biol. 2022 ;2378 261-277

Drosophila Unfolded Protein Response (UPR) Assays In Vitro and In Vivo.

Hidetaka Katow, Deepika Vasudevan, Hyung Don Ryoo.

  Wildtype or mutant proteins expressed beyond the capacity of a cell's protein folding system could be detrimental to general cellular function and survival. In response to misfolded protein overload in the endoplasmic reticulum (ER), eukaryotic cells activate the Unfolded Protein Response (UPR) that helps cells restore protein homeostasis in the endoplasmic reticulum (ER). As part of the UPR, cells attenuate general mRNA translation and activate transcription factors that induce stress-responsive gene expression.UPR signaling draws research interest in part because conditions that cause chronic protein misfolding in the ER or those that impair UPR signaling underlie several diseases including neurodegeneration, diabetes, and cancers. Model organisms are frequently employed in the field as the UPR pathways are generally well-conserved throughout phyla. Here, we introduce experimental procedures to detect UPR in Drosophila melanogaster.

Keywords:  ATF4 (crc); Drosophila melanogaster; ER stress; Eye discs; Fat body; IRE1; PERK; Unfolded Protein Response; Xbp1

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_17
Methods Mol Biol. 2022 ;2378 317-327

HTS Identification of Activators and Inhibitors of Endoplasmic Reticulum (ER) Stress and the Unfolded Protein Response (UPR).

Mehrnoosh Ghafouri, Chester B Gauss, Andrew M Fribley.

  The identification of small molecules and natural product extracts that enhance or interfere with the productivity of protein folding in the endoplasmic reticulum (ER) has the potential to improve a wide variety of human pathologies. Every protein that is destined for a lysosome, integral to the cell membrane, or secreted, is folded, post-translationally modified, and exported to the cytoplasm from the ER-Golgi complex. The following protocols have successfully employed several high-fidelity cell-based luciferase high-throughput screens (HTS) to identify activators and inhibitors of ER stress and the unfolded protein response (UPR).

Keywords:  ATF4; CHOP; Cell-based assay; ER stress; GRP78/BiP; UPR; XBP1

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_20
Methods Mol Biol. 2022 ;2378 141-168

Assays to Study IRE1 Activation and Signaling.

Paloma Moraga, Raul Aravena, Hery Urra, Claudio Hetz.

  The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.  In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known as regulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress. Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

Keywords:  ER stress; IRE1 activation; IRE1 oligomerization; IRE1 phosphorylation; Regulated IRE1-dependent decay (RIDD); Unfolded protein response; XBP1 mRNA splicing

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_10
Methods Mol Biol. 2022 ;2378 169-187

Analysis of XBP1 Contribution to Hyperosmolarity-Induced Lipid Synthesis.

Cecilia I Casali, Leandro Parra, Luciana C Erjavec, María Del Carmen Fernández Tome.

  The unfolded protein response (UPR) is a complex network of intracellular pathways that transmits signals from ER lumen and/or ER bilayer to the nuclear compartment in order to activate gene transcription. UPR is activated by the loss of ER capacities, known as ER stress, and occurs to restore ER properties. In this regard, glycerolipid (GL) synthesis activation contributes to ER membrane homeostasis and IRE1α-XBP1, one UPR pathway, has a main role in lipogenic genes transcription. Herein, we describe the strategy and methodology used to evaluate whether IRE1α-XBP1 pathway regulates lipid metabolism in renal epithelial cells subjected to hyperosmolar environment. XBP1s activity was hindered by blocking IRE1α RNAse activity and by impeding its expression; under these conditions, we determined GL synthesis and lipogenic enzymes expression.

Keywords:  Endoplasmic reticulum (ER) stress; Glycerolipid synthesis; Hyperosmolarity; Inositol-requiring enzyme kinase 1α (IRE1α); Unfolded-protein response (UPR); X-box-binding protein 1 (XBP1)

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_11
Gene. 2021 Dec 29. pii: S0378-1119(21)00730-7. [Epub ahead of print]814 146135

The interaction of SIRT4 and Calreticulin during ER stress in glia cells.

Aysenur Akkulak, Gizem Donmez Yalcin.

  Endoplasmic Reticulum (ER) stress is the response that occurs after the dysfunction of ER and its structure. Activated UPR triggers a stress response using ER membrane proteins such as PERK, IRE-1, GRP78, ATF5 ve ATF6. Sirtuins are enzymes that carry out post-translational modifications such as deacetylation and ADP-ribosylation. In our previous study, we identified Calreticulin as a SIRT4-interacting protein via mass spectrometry. Calreticulin binds to misfolded proteins, prevents them from leaving ER, which results in the reduction of ER stress. In this study, we aimed to investigate the interaction between SIRT4 and Calreticulin during ER stress in glia cells (IHA-immortalized human astrocytes). To trigger ER stress in glia cells, we first optimized the dose and the duration of the Tunicamycin which is 2.5 μg/ml concentration for 16 h. SIRT4 gene was silenced with lentiviral particles using 4 MOI (Multiplicity of Infection). In SIRT4-silenced cells, when treated with 2.5 μg/ml Tunicamycin for 16 h, the increase in the expressions of ATF6, GRP78 and the ratio of spliced/unspliced XBP1 mRNA were reduced. This shows that silencing SIRT4 may decrease ER stress. SIRT4-Calreticulin interaction was shown both in control and ER-stress induced glia cells. Additionally, this interaction did not change with the ER stress. SIRT4 only ADP-ribosylates Calreticulin during ER stress. Normally, SIRT4 ADP-ribosylates and deactivates Calreticulin during ER stress condition. When SIRT4 is silenced, the ADP-ribosylation level of Calreticulin decreases resulting in the activation of Calreticulin and the reduction of ER stress. In summary, SIRT4 inhibitors may be investigated as protective agents or drug candidates in neurodegenerative diseases where ER stress mostly underlies as one of the molecular mechanisms.

Keywords:  ADP-ribosylation; ER stress; Post-translational modification; SIRT4

DOI:  https://doi.org/10.1016/j.gene.2021.146135
Methods Mol Biol. 2022 ;2378 279-289

Protein Preparation for Proteomic Analysis of the Unfolded Protein Response in Arabidopsis thaliana.

Yunting Pu, Federica Brandizzi.

  Excessive accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) leads to a potentially cytotoxic condition known as the ER stress. Upon ER stress, cells initiate a homeostatic response called unfolded protein response (UPR) to assist proper folding the unfolded or misfolded proteins. Proteomics have been broadly used in plants with Liquid Chromatography coupled to tandem MS (LC-MS/MS) technologies. LC-MS/MS techniques have also been a great tool for studies of posttranslational modifications (PTMs). Here we describe our protocol of a fast method for large amount of seedling treatment and collection for UPR study in Arabidopsis thaliana and the preparation of total proteins for proteomic analysis.

Keywords:  Arabidopsis; Endoplasmic reticulum stress; Protein extraction; Proteomics; Unfolded protein response

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_18
Methods Mol Biol. 2022 ;2378 113-120

Detecting the Non-conventional mRNA Splicing and Translational Activation of HAC1 in Budding Yeast.

Weihan Li, Robert H Singer.

  Protein-folding homeostasis in the endoplasmic reticulum (ER) is maintained by the unfolded protein response (UPR). UPR in Saccharomyces cerevisiae is regulated by a bZIP transcription factor, Hac1p. Under non-stress condition, HAC1 mRNA is translationally repressed. When un- or mis-folded proteins accumulate in the ER, HAC1 mRNA undergoes non-conventional mRNA splicing. The spliced HAC1 mRNA is translationally active and produces functional Hac1p, which initiates a transcriptional response that restores ER protein-folding homeostasis. Thus, the activation of yeast UPR is tightly regulated by HAC1 mRNA splicing. Here, we describe two methods that are used to monitor the splicing and translational status of HAC1 mRNA in budding yeast.

Keywords:  Budding yeast; ER stress; HAC1; Non-conventional mRNA splicing; Unfolded protein response

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_8
Methods Mol Biol. 2022 ;2378 69-81

The Use of Fluorescent Protein Fusions to Monitor the Unfolded Protein Response and Protein Foldase-Substrate Interactions in Plant Protoplasts.

Rina Carrillo, Elizabeth Feldeverd, David A Christopher.

  Endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR) are critical stress response pathways in eukaryotes. To study these types of interactions in plants, a wide range of methods have been used, including generation of transgenic plants, subcellular immunolocalization of protein foldases, and co-immunoprecipitation (co-IP) assays. Although these more time-consuming methods have been successfully implemented, there is a need for a versatile and rapid in vivo system to investigate ER stress and UPR. Here, we describe a transient expression system that uses plant protoplasts to define in vivo subcellular localizations and protein-protein interactions of protein foldases and their substrates fused to fluorescent protein reporters. This accurate and robust assay utilizes a variety of analyses, such as subcellular localization, FLIM-FRET, co-IP, mutagenesis, and RT-PCR in the genetically amenable Arabidopsis model system. We demonstrate the methodology by using the representative protein foldase, protein disulfide isomerase-9 (PDI9), as well as subcellular markers, secretory proteins, and dithiothreitol (DTT)-mediated induction of the UPR as monitored by RT-PCR. Together, these methods yield reliable high output results for investigating subcellular localization and protein-protein interactions in plants to decipher the UPR pathways.

Keywords:  FLIM-FRET; Fluorescent protein fusion; Protein disulfide isomerase; Protein foldase; Protoplasts; Transient expression analysis; Unfolded protein response

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_5
Methods Mol Biol. 2022 ;2378 45-67

A Platform Technology for Monitoring the Unfolded Protein Response.

Bhagyashree Bachhav, Carlos A Origel Marmolejo, Yafet Arefeayne, Laura Segatori.

  The unfolded protein response (UPR) is a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER) and is linked to the development of a range of diseases, including Alzheimer's disease, diabetes, and several types of cancer. UPR induction is typically monitored by measuring the expression level of UPR marker genes. Most tools for quantifying gene expression, including DNA microarrays and quantitative PCR with reverse transcription (RT-PCR), produce snapshots of the cell transcriptome, but are not ideal for measurements requiring temporal resolution of gene expression dynamics. Reporter assays for indirect detection of the UPR typically rely on extrachromosomal expression of reporters under the control of minimal or synthetic regulatory sequences that do not recapitulate the native chromosomal context of the UPR target genes. To address the need for tools to monitor chromosomal gene expression that recapitulate gene expression dynamics from the native chromosomal context and generate a readily detectable signal output, we developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal gene marker of the UPR. The platform is based on a genetic circuit that amplifies the output signal with high sensitivity and dynamic resolution and is implemented through chromosomal integration of the gene encoding the main control element of the genetic circuit to link its expression to that of the target gene, thereby generating a platform that can be easily adapted to monitor any UPR target through integration of the main control element at the appropriate chromosomal locus. By recapitulating the transcriptional and translational control mechanisms underlying the expression of UPR targets with high sensitivity, this platform provides a novel technology for monitoring the UPR with superior sensitivity and dynamic resolution.

Keywords:  CRISPR-Cas9; ER stress; Gene signal amplifier; NanoDeg; Unfolded protein response (UPR)

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_4
Front Cell Dev Biol. 2021 ;9 769213

Mfn2 Regulates High Glucose-Induced MAMs Dysfunction and Apoptosis in Podocytes via PERK Pathway.

Yun Cao, Zhaowei Chen, Jijia Hu, Jun Feng, Zijing Zhu, Yanqin Fan, Qiaoxuan Lin, Guohua Ding.

  The endoplasmic reticulum (ER) stress and mitochondrial dysfunction in high glucose (HG)-induced podocyte injury have been demonstrated to the progression of diabetic kidney disease (DKD). However, the pathological mechanisms remain equivocal. Mitofusin2 (Mfn2) was initially identified as a dynamin-like protein involved in fusing the outer mitochondrial membrane (OMM). More recently, Mfn2 has been reported to be located at the ER membranes that contact OMM. Mitochondria-associated ER membranes (MAMs) is the intercellular membrane subdomain, which connects the mitochondria and ER through a proteinaceous tether. Here, we observed the suppression of Mfn2 expression in the glomeruli and glomerular podocytes of patients with DKD. Streptozotocin (STZ)-induced diabetic rats exhibited abnormal mitochondrial morphology and MAMs reduction in podocytes, accompanied by decreased expression of Mfn2 and activation of all three unfolded protein response (UPR) pathways (IRE1, ATF6, and PERK). The HG-induced mitochondrial dysfunction, MAMs reduction, and increased apoptosis in vitro were accompanied by the downregulation of Mfn2 and activation of the PERK pathway. Mfn2 physically interacts with PERK, and HG promotes a decrease in Mfn2-PERK interaction. In addition, Mfn2-silenced podocytes showed mitochondrial dysfunction, MAMs reduction, activation of PERK pathway, and increased apoptosis. Conversely, all these effects of HG stimulation were alleviated significantly by Mfn2 overexpression. Furthermore, the inhibition of PERK phosphorylation protected mitochondrial functions but did not affect the expression of Mfn2 in HG-treated podocytes. Therefore, this study confirmed that Mfn2 regulates the morphology and functions of MAMs and mitochondria, and exerts anti-apoptotic effects on podocytes by inhibiting the PERK pathway. Hence, the Mfn2-PERK signaling pathway may be a new therapeutic target for preventing podocyte injury in DKD.

Keywords:  DKD; ER stress; MAMs; Mfn2; apoptosis; podocyte

DOI:  https://doi.org/10.3389/fcell.2021.769213
Mol Neurobiol. 2022 Jan 07.

TREX1 Deficiency Induces ER Stress-Mediated Neuronal Cell Death by Disrupting Ca2+ Homeostasis.

Debasish Halder, Su-Jin Jeon, Ji-Yong Yoon, Jeong-Ju Lee, Soo Young Jun, Min-Hyuk Choi, Bohyeon Jeong, Duk Hyun Sung, Da Yong Lee, Byoung Joon Kim, Nam-Soon Kim.

  TREX1 is an exonuclease that degrades extranuclear DNA species in mammalian cells. Herein, we show a novel mechanism by which TREX1 interacts with the BiP/GRP78 and TREX1 deficiency triggers ER stress through the accumulation of single-stranded DNA and activates unfolded protein response (UPR) signaling via the disruption of the TREX1-BiP/GRP78 interaction. In TREX1 knockdown cells, the activation of ER stress signaling disrupted ER Ca2+ homeostasis via the ERO1α-IP3R1-CaMKII pathway, leading to neuronal cell death. Moreover, TREX1 knockdown dysregulated the Golgi-microtubule network through Golgi fragmentation and decreased Ac-α-tubulin levels, contributing to neuronal injury. These alterations were also observed in neuronal cells harboring a TREX1 mutation (V91M) that has been identified in hereditary spastic paraplegia (HSP) patients in Korea. Notably, this mutation leads to defects in the TREX1-BiP/GRP78 interaction and mislocalization of TREX1 from the ER and possible disruption of the Golgi-microtubule network. In summary, the current study reveals TREX1 as a novel regulator of the BiP/GRP78 interaction and shows that TREX1 deficiency promotes ER stress-mediated neuronal cell death, which indicates that TREX1 may hold promise as a therapeutic target for neurodegenerative diseases such as HSP.

Keywords:  BiP/GRP78; Ca2+ homeostasis; ER stress; Hereditary spastic paraplegia; Neuronal cells; Three prime repair exonuclease 1

DOI:  https://doi.org/10.1007/s12035-021-02631-3
Hepatology. 2022 Jan 05.

CNPY2 Contributes to Liver Oncogenesis by Promoting UPR-dependent destabilization of p53.

Feng Hong, Ching Ying Lin, Jinyue Yan, Yizhou Dong, Yuli Ouyang, Doyeon Kim, Xiaoli Zhang, Bei Liu, Shaoli Sun, Wei Gu, Zihai Li.

   BACKGROUD & AIMS: Abnormalities in the p53 gene and overexpression of Mouse double minute 2 homolog (MDM2), a negative regulator of p53, are commonly observed in cancers. p53 destabilization is regulated by ER stress and unfolded protein response (UPR) in cancer. However, the mechanisms remain enigmatic. Canopy 2 (CNPY2) is a key UPR initiator that primarily involved in ER stress and is highly expressed in liver, but its functional role in regulating liver carcinogenesis is poorly understood. Therefore, we aimed to investigate the role of CNPY2 in hepartocarcinogenesis through the URP-dependent p53 destabilization.
APPROACH & RESULTS: Here we showed that CNPY2 expression is upregulated in Hepatocellular carcinoma (HCC), and negatively correlated with liver cancer patients' survival rate. Deletion of Cnpy2 obliterates diethyl nitrosamine (DEN)-induced HCC in mice. Mechanistic studies demonstrated that CNPY2 binds and prevents ribosome proteins from inhibiting MDM2, and enhances UPR activity of PERK and IRE1α, leading to p53 destabilization and cell cycle progression. In addition, transcriptome analyses uncovered that CNPY2 is also required for DEN-induced expression of oncogenes, including c-Jun and FGF21. Intratumoral injection of the nanoparticle-based Crispr sgRNA/Cas9 mRNA against Cnpy2 has antitumor effects in HCC.
CONCLUSION: These findings demonstrate that CNPY2 is crucial for liver oncogenesis through the UPR-dependent repression of p53 and activation of oncogenes, providing insights into the design of new therapeutic target for HCC.

Keywords:  CNPY2; FGF21; Hepatocellular carcinoma; c-Jun; p53; ribosome biogenesis; unfolded protein response

DOI:  https://doi.org/10.1002/hep.32318
Methods Mol Biol. 2022 ;2378 189-201

Integrating an ER Stress Reporter for Monitoring Genome-Wide UPR-ER in Budding Yeast.

Shuvadeep Maity, Asmita Ghosh, Kausik Chakraborty.

  Genetic interaction studies have been instrumental in understanding and organizing cellular pathways. This has been helpful in identifying and arranging genes according to pathways, identifying novel pathways, ascribing gene function, and providing information regarding redundant and antagonistic pathways. Synthetic Genetic Array (SGA) uses growth to identify genome scale gene interaction networks. While this has provided most of the genetic interaction data available, SGA coupled to other reporters have the potential to identify components of pathways that specifically affect the probed reporter. The method described here utilizes SGA principles to understand conserved elements of endoplasmic reticulum (ER) homeostasis in the presence and absence of ER stress. The use of a fluorescent reporter of ER stress allows quantitative measurements and provides a handle to measure the proteostasis capacity of the ER in a high-throughput manner. The integration of such a fluorescent reporter in the background of different mutant/deletion strains is sufficient to identify genetic modules in a high-throughput manner.

Keywords:  Endoplasmic reticulum stress; Fluorescent reporter; Synthetic genetic array; Unfolded protein response

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_12
Methods Mol Biol. 2022 ;2378 293-315

Structure-Based Drug Discovery of IRE1 Modulators.

Diana Pelizzari Raymundo, Leif A Eriksson, Eric Chevet, Xavier Guillory.

  IRE1α (inositol-requiring enzyme 1 alpha, referred to IRE1 hereafter) is an Endoplasmic Reticulum (ER) resident transmembrane enzyme with cytosolic kinase/RNAse activities. Upon ER stress IRE1 is activated through trans-autophosphorylation and oligomerization, resulting in a conformational change of the RNase domain, thereby promoting two signaling pathways: i) the non-conventional splicing of XBP1 mRNA and ii) the regulated IRE1-dependent decay of RNA (RIDD). IRE1 RNase activity has been linked to diverse pathologies such as cancer or inflammatory, metabolic, and degenerative diseases and the modulation of IRE1 activity is emerging as an appealing therapeutic strategy against these diseases. Several modulators of IRE1 activity have been reported in the past, but none have successfully translated into the clinics as yet. Based on our expertise in the field, we describe in this chapter the approaches and protocols we used to discover novel IRE1 modulators and characterize their effect on IRE1 activity.

Keywords:  Endoplasmic reticulum; IRE1; Structure-based drug discovery (SBDD); Unfolded protein response computer assisted drug design (CADD)

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_19
Methods Mol Biol. 2022 ;2378 221-231

In Vitro Stimulation of IRE1α/XBP1-Deficient B Cells with LPS.

Huabin Zhu, Chen Jiang, Randal J Kaufman, Honglin Li, Nagendra Singh.

  During immune responses, pathogen-specific B cells differentiate into plasma cells. Plasma cells synthesize and secrete large amounts of immunoglobulin (Ig) molecules which play a central role in immunity against pathogens. The synthesis, proper folding, and secretion of these Ig molecules require expansion of the extensive endoplasmic reticulum (ER) network. Accumulation of unfolded or misfolded proteins in the ER is sensed by three sensors: IRE1/XBP1, PERK, and ATF6, which coordinate with each other and initiate the unfolded protein response (UPR) pathway to expand the ER network and its protein folding and secretion capability. The expansion and maintenance of the ER network in plasma cells is triggered by activation of the IRE1/XBP1 branch of the UPR pathway. Here, we discuss the methods to stimulate the differentiation of B cells into plasma cells, measure the activation of the XBP1 pathway, and quantify the ER network.

Keywords:  B cells; Unfolded protein response pathway (UPR); XBP1

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_14
Methods Mol Biol. 2022 ;2378 249-259

Methods to Study the Mitochondrial Unfolded Protein Response (UPRmt) in Caenorhabditis elegans.

Simon Haeussler, Barbara Conradt.

  The nematode Caenorhabditis elegans is a powerful model to study cellular stress responses. Due to its transparency and ease of genetic manipulation, C. elegans is especially suitable for fluorescence microscopy. As a result, studies of C. elegans using different fluorescent reporters have led to the discovery of key players of cellular stress response pathways, including the mitochondrial unfolded protein response (UPRmt). UPRmt is a protective retrograde signaling pathway that ensures mitochondrial homeostasis. The nuclear genes hsp-6 and hsp-60 encode mitochondrial chaperones and are highly expressed upon UPRmt induction. The transcriptional reporters of these genes, hsp-6::gfp and hsp-60::gfp, have been instrumental for monitoring this pathway in live animals. Additional tools for studying UPRmt include fusion proteins of ATFS-1 and DVE-1, ATFS-1::GFP and DVE-1::GFP, key players of the UPRmt pathway. In this protocol, we discuss advantages and limitations of currently available methods and reporters, and we provide detailed instructions on how to image and quantify reporter expression.

Keywords:  UPRmt reporters; atfs-1; dve-1; gfp; hsp-6; hsp-60

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_16
Methods Mol Biol. 2022 ;2378 19-30

Fluorescence-Based Biosensors for the Detection of the Unfolded Protein Response.

Heinrich Kroukamp, Kai Peng, Ian T Paulsen, Riaan den Haan.

  The unfolded protein response (UPR) is a highly conserved protein quality control mechanism of eukaryotic cells. Aberrations in this response have been linked to several human diseases, including retinitis pigmentosa and several cancers, and have been shown to have a drastic impact on recombinant protein yields in fungal, insect, and mammalian cell lines. Here, we describe the use of in vivo biosensors to measure and characterize this dynamic cellular response, specifically for detecting the UPR induced by protein overproduction stress in the model cell factory Saccharomyces cerevisiae.

Keywords:  Biosensors; Protein folding capacity; Saccharomyces cerevisiae; Unfolded protein response

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_2
Blood Adv. 2022 Jan 03. pii: bloodadvances.2021005720. [Epub ahead of print]

A new murine model of Barth Syndrome neutropenia links TAFAZZIN deficiency to increased ER stress induced apoptosis.

Jihee Sohn, Jelena Milosevic, Thomas Brouse, Najihah Aziz, Jenna Elkhoury, Suya Wang, Alexander Hauschild, Nick van Gastel, Murat Cetinbas, Sara F Tufa, Douglas R Keene, Ruslan Sadreyev, William T Pu, David Brian Sykes.

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knock-out animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN KO hematopoietic stem cells. We also used the ER Hoxb8 system of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis, and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress mediated and non ER stress mediated triggers of apoptosis. While the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some Barth syndrome patients remains to be clarified, our studies and new model system set a foundation for further investigation.

DOI: https://doi.org/10.1182/bloodadvances.2021005720
Int J Biol Sci. 2022 ;18(1): 315-330

Effect Of XBP1 Deficiency In Cartilage On The Regulatory Network Of LncRNA/circRNA-miRNA-mRNA.

Xiaoli Li, Yuyou Yang, Li Liang, Mengtian Fan, Xingyue Li, Naibo Feng, Yiming Pan, Qiaoyan Tan, Qingbo Xu, Yangli Xie, Fengjin Guo.

  X-box binding protein 1(XBP1) is a critical component for unfolded protein response (UPR) in ER stress. According to previous studies performed with different XBP1-deficient mice, the XBP1 gene affects mouse cartilage development and causes other related diseases. However, how the complete transcriptome, including mRNA and ncRNAs, affects the function of cartilage and other tissues when XBP1 is deficient in chondrocytes is unclear. In this study, we aimed to screen the differentially expressed (DE) mRNAs, circRNAs, lncRNAs and miRNAs in XBP1 cartilage-specific knockout (CKO) mice using high throughput sequencing and construct the circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA regulatory networks. DE LncRNAs (DE-LncRNAs), circRNAs (DE-circRNAs), miRNAs (DE-miRNAs), and mRNAs [differentially expressed genes (DEGs)] between the cartilage tissue of XBP1 CKO mice and controls were identified, including 441 DE-LncRNAs, 15 DE-circRNAs, 6 DE-miRNAs, and 477 DEGs. Further, 253,235 lncRNA-miRNA-mRNA networks and 1,822 circRNA-miRNA-mRNA networks were constructed based on the correlation between lncRNAs/circRNAs, miRNAs, mRNAs. The whole transcriptome analysis revealed that XBP1 deficiency in cartilage affects the function of cartilage and other different tissues, as well as associated diseases. Overall, our findings may provide potential biomarkers and mechanisms for the diagnosis and treatment of cartilage and other related diseases.

Keywords:  XBP1; cartilage; circRNA-miRNA-mRNA; lncRNA-miRNA-mRNA; whole transcriptome analysis

DOI:  https://doi.org/10.7150/ijbs.64054
Methods Mol Biol. 2022 ;2378 233-245

Detection of PERK Signaling in the Central Nervous System.

Zhixin Lei, Sarrabeth Stone, Wensheng Lin.

  In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) signaling adapts cells to stressful conditions by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). Phosphorylation of eIF2α inhibits global protein translation but stimulates the expression of numerous stress-responsive genes by inducing the transcription factor ATF4. A large number of studies have shown that activation of PERK signaling has beneficial or detrimental effects in various diseases of the central nervous system (CNS), including neurodegenerative diseases, myelin disorders, CNS injuries, among others. This chapter is devoted to describing the practical methods for the detection of PERK signaling in CNS diseases.

Keywords:  ATF4; CHOP; GADD34; Immunofluorescence; Neuron; Oligodendrocyte; PERK; Phosphorylated eIF2α; Real-time PCR; Western blot

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_15
Methods Mol Biol. 2022 ;2378 121-140

Determination of the Stability and Intracellular (Intra-Nuclear) Targeting and Recruitment of Pre-HAC1 mRNA in the Saccharomyces cerevisiae During the Activation of UPR.

Sunirmal Paira, Biswadip Das.

  Nuclear degradation of pre-HAC1 mRNA and its subsequent targeting plays a vital role in the activation as well as attenuation of Unfolded Protein Response (UPR) in Saccharomyces cerevisiae. Accurate measurement of the degradation of precursor HAC1 mRNA therefore appears vital to determine the phase of activation or attenuation of this important intracellular signaling pathway. Typically, pre-HAC1 mRNA degradation is measured by the transcription shut-off experiment in which RNA Polymerase II transcription is inhibited by a potent transcription inhibitor to prevent the de novo synthesis of all Polymerase II transcripts followed by the measurement of the steady-state levels of a specific (e.g., pre-HAC1) mRNA at different times after the inhibition of the transcription. The rate of the decay is subsequently determined from the slope of the decay curve and is expressed as half-life (T1/2). Estimation of the half-life values and comparison of this parameter determined under different physiological cues (such as in absence or presence of redox/ER/heat stress) gives a good estimate of the stability of the mRNA under these conditions and helps gaining an insight into the mechanism of the biological process such as activation or attenuation of UPR.Intra-nuclear targeting of the pre-HAC1 mRNA from the site of its transcription to the site of non-canonical splicing, where the kinase-endonuclease Ire1p clusters into the oligomeric structures constitutes an important aspect of the activation of Unfolded Protein Response pathway. These oligomeric structures are detectable as the Ire1p foci/spot in distinct locations across the nuclear-ER membrane under confocal micrograph using immunofluorescence procedure. Extent of the targeting of the pre-HAC1 mRNA is measurable in a quantified manner by co-expressing fluorescent-labeled pre-HAC1 mRNA and Ire1p protein followed by estimating their co-localization using FACS (Fluorescence-Activated Cell Sorter) analysis. Here, we describe detailed protocol of both determination of intra-nuclear decay rate and targeting-frequency of pre-HAC1 mRNA that were optimized in our laboratory.

Keywords:  HAC1; Unfolded Protein Response; mRNA Recruitment; mRNA degradation; mRNA targeting

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_9
Methods Mol Biol. 2022 ;2378 205-219

Analyze Mouse Knockout Models of UPR Pathway Elements.

Chao Zheng, Cheng Wang, Qiang Jie, Liu Yang.

  Evidence from genetic studies in human and mice indicates that defective skeletal development is one of the major phenotypic outcomes for aberrant UPR signaling. Visualization of morphological alterations in whole-mount skeleton and protein secretion and UPR activation on tissue sections is the very first step to investigate skeletal phenotypes of UPR-related mouse models. In this chapter, we introduce the major techniques that have been frequently used in our laboratory to study UPR-induced skeletal disorders with genetically modified mice and provide descriptive directions of mouse genotyping, bone tissue grossing, whole-mount skeletal staining, immunostaining assays of matrix secretion, and UPR activation.

Keywords:  Chondrocyte; Immunostaining assay; Matrix secretion; Mouse genotyping; Skeletal preparation; UPR

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_13
Cell Rep. 2022 Jan 04. pii: S2211-1247(21)01701-0. [Epub ahead of print]38(1): 110197

AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.

Adrien Grenier, Laury Poulain, Johanna Mondesir, Arnaud Jacquel, Claudie Bosc, Lucille Stuani, Sarah Mouche, Clement Larrue, Ambrine Sahal, Rudy Birsen, Victoria Ghesquier, Justine Decroocq, Fetta Mazed, Mireille Lambert, Mamy Andrianteranagna, Benoit Viollet, Patrick Auberger, Andrew A Lane, Pierre Sujobert, Didier Bouscary, Jean-Emmanuel Sarry, Jerome Tamburini.

  AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Keywords:  AML; AMPK; GSK621; PERK; mitochondrial apoptosis; unfolded protein response; venetoclax

DOI:  https://doi.org/10.1016/j.celrep.2021.110197
Methods Mol Biol. 2022 ;2378 101-110

Detection of HAC1 mRNA Splicing by RT-PCR in Saccharomyces cerevisiae.

Jagadeesh Kumar Uppala, Madhusudan Dey.

  HAC1 mRNA remains translationally repressed in the cytoplasm of the budding yeast Saccharomyces cerevisiae. Under conditions of cellular stress, a dual kinase RNase IRE1 (Inositol Requiring Enzyme-1) cleaves out an intervening sequence from the HAC1 mRNA. Cleaved mRNAs are then ligated by tRNA ligase, thus generating a spliced mRNA that translates an active transcription factor. This unconventional splicing of HAC1 mRNA in the cytoplasm is a molecular marker for various cellular stresses including oxidative stress and endoplasmic reticulum (ER) stress. This article describes a PCR-based protocol to detect the HAC1 mRNA splicing.

Keywords:  HAC1; IRE1; Protein folding; Stress

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_7