bims-unfpre 2023-11-26 papers

bims-unfpre

Biomed News

on Unfolded protein response

Issue of 2023–11–26
twelve papers selected by
Susan Logue, University of Manitoba

A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells.
GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells.
Increased activity of IRE1 improves the clinical presentation of EAE.
HIV-1 replication requires optimal activation of the unfolded protein response.
Animal Models for the Study of Gaucher Disease.
Reticulons bind sphingolipids to activate the endoplasmic reticulum cell cycle checkpoint, the ER surveillance pathway.
Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.
The Unfolded Protein Response in a Murine Model of Alzheimer's Disease: Looking for Predictors.
Intricate subcellular journey of nanoparticles to the enigmatic domains of endoplasmic reticulum.
ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation.
Higher matrix stiffness promotes VSMC senescence by affecting mitochondria-ER contact sites and mitochondria/ER dysfunction.
Adaptive changes in tumor cells in response to reductive stress.

Clin Cancer Res. 2023 Nov 20. OF1-OF18

A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells.

Veronica Calvo, Wei Zheng, Anna Adam-Artigues, Kirk A Staschke, Xin Huang, Julie F Cheung, Ana Rita Nobre, Sho Fujisawa, David Liu, Maria Fumagalli, David Surguladze, Michael E Stokes, Ari Nowacek, Mark Mulvihill, Eduardo F Farias, Julio A Aguirre-Ghiso.

PURPOSE: The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis.
EXPERIMENTAL DESIGN: A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR.
RESULTS: HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF.
CONCLUSIONS: Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested "persister" cells that escape anti-proliferative therapies.

DOI: https://doi.org/10.1158/1078-0432.CCR-23-1427
Cell Rep Med. 2023 Nov 21. pii: S2666-3791(23)00491-3. [Epub ahead of print]4(11): 101297

GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells.

Jorge Ibanez, Nikhil Hebbar, Unmesha Thanekar, Zhongzhen Yi, Haley Houke, Meghan Ward, Chris Nevitt, Liqing Tian, Stephen C Mack, Heather Sheppard, Jason Chiang, M Paulina Velasquez, Giedre Krenciute.

  Lack of targetable antigens is a key limitation for developing successful T cell-based immunotherapies. Members of the unfolded protein response (UPR) represent ideal immunotherapy targets because the UPR regulates the ability of cancer cells to resist cell death, sustain proliferation, and metastasize. Glucose-regulated protein 78 (GRP78) is a key UPR regulator that is overexpressed and translocated to the cell surface of a wide variety of cancers in response to elevated endoplasmic reticulum (ER) stress. We show that GRP78 is highly expressed on the cell surface of multiple solid and brain tumors, making cell surface GRP78 a promising chimeric antigen receptor (CAR) T cell target. We demonstrate that GRP78-CAR T cells can recognize and kill GRP78+ brain and solid tumors in vitro and in vivo. Additionally, our findings demonstrate that GRP78 is upregulated on CAR T cells upon T cell activation; however, this expression is tumor-cell-line specific and results in heterogeneous GRP78-CAR T cell therapeutic response.

Keywords:  CAR T cells; ER stress; GRP78; brain tumors; immune synapse; immunotherapy; solid tumors

DOI:  https://doi.org/10.1016/j.xcrm.2023.101297
FASEB J. 2023 Dec;37(12): e23283

Increased activity of IRE1 improves the clinical presentation of EAE.

Valerie Bracchi-Ricard, Kayla Nguyen, Daniela Ricci, Brian Gaudette, Jorge Henao-Mejia, Roberta Brambilla, Tetyana Martynyuk, Tali Gidalevitz, David Allman, John R Bethea, Yair Argon.

  Activation of the endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme-1α (IRE1α) contributes to neuronal development and is known to induce neuronal remodeling in vitro and in vivo. On the contrary, excessive IRE1 activity is often detrimental and may contribute to neurodegeneration. To determine the consequences of increased activation of IRE1α, we used a mouse model expressing a C148S variant of IRE1α with increased and sustained activation. Surprisingly, the mutation did not affect the differentiation of highly secretory antibody-producing cells but exhibited a beneficial effect in a mouse model of experimental autoimmune encephalomyelitis (EAE). Although mechanical allodynia was unaffected, significant improvement in motor function was found in IRE1C148S mice with EAE relative to wild type (WT) mice. Coincident with this improvement, there was reduced microgliosis in the spinal cord of IRE1C148S mice, with reduced expression of proinflammatory cytokine genes. This was accompanied by reduced axonal degeneration and enhanced 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) levels, suggesting improved myelin integrity. Interestingly, while the IRE1C148S mutation is expressed in all cells, the reduction in proinflammatory cytokines and in the microglial activation marker ionized calcium-binding adapter molecule (IBA1), along with preservation of phagocytic gene expression, all point to microglia as the cell type contributing to the clinical improvement in IRE1C148S animals. Our data suggest that sustained increase in IRE1α activity can be beneficial in vivo, and that this protection is cell type and context dependent. Considering the overwhelming but conflicting evidence for the role of ER stress in neurological diseases, a better understanding of the function of ER stress sensors in physiological contexts is clearly needed.

Keywords:  alternatively activated microglia; augmented IRE1α activity; experimental autoimmune encephalomyelitis; neuroinflammation; unfolded protein response

DOI:  https://doi.org/10.1096/fj.202300769RR
FEBS Lett. 2023 Nov 20.

HIV-1 replication requires optimal activation of the unfolded protein response.

Anjali Tripathi, Kruthika Iyer, Debashis Mitra.

  Several human diseases including viral infections activate the unfolded protein response (UPR) due to abnormal accumulation of unfolded/misfolded proteins. However, UPR modulation and its functional relevance in HIV-1 infection lack comprehensive elucidation. This study reveals that HIV-1 activates IRE1, PERK, and ATF6 signaling pathways of UPR. The knockdown of PERK and ATF6 reduces HIV-1 long terminal repeat (LTR)-driven gene expression, whereas the endoplasmic reticulum (ER) chaperone HSPA5 prevents proteasomal degradation of HIV-1 p24 through its chaperone activity. Interestingly, overstimulation of UPR by a chemical inducer leads to anti-HIV activity through an enhanced type-1 interferon response. Also, treatment with a chemical ER stress inhibitor reduces HIV-1 replication. These findings suggest that an optimal UPR activation is crucial for effective viral replication, as either overstimulating UPR or inhibiting ER stress leads to viral suppression.

Keywords:  ATF6; ER stress; HIV-1; HSPA5; PERK; unfolded protein response

DOI:  https://doi.org/10.1002/1873-3468.14772
Int J Mol Sci. 2023 Nov 07. pii: 16035. [Epub ahead of print]24(22):

Animal Models for the Study of Gaucher Disease.

Or Cabasso, Aparna Kuppuramalingam, Lindsey Lelieveld, Martijn Van der Lienden, Rolf Boot, Johannes M Aerts, Mia Horowitz.

  In Gaucher disease (GD), a relatively common sphingolipidosis, the mutant lysosomal enzyme acid β-glucocerebrosidase (GCase), encoded by the GBA1 gene, fails to properly hydrolyze the sphingolipid glucosylceramide (GlcCer) in lysosomes, particularly of tissue macrophages. As a result, GlcCer accumulates, which, to a certain extent, is converted to its deacylated form, glucosylsphingosine (GlcSph), by lysosomal acid ceramidase. The inability of mutant GCase to degrade GlcSph further promotes its accumulation. The amount of mutant GCase in lysosomes depends on the amount of mutant ER enzyme that shuttles to them. In the case of many mutant GCase forms, the enzyme is largely misfolded in the ER. Only a fraction correctly folds and is subsequently trafficked to the lysosomes, while the rest of the misfolded mutant GCase protein undergoes ER-associated degradation (ERAD). The retention of misfolded mutant GCase in the ER induces ER stress, which evokes a stress response known as the unfolded protein response (UPR). GD is remarkably heterogeneous in clinical manifestation, including the variant without CNS involvement (type 1), and acute and subacute neuronopathic variants (types 2 and 3). The present review discusses animal models developed to study the molecular and cellular mechanisms underlying GD.

Keywords:  ER stress; GBA1; glucocerebrosidase (GCase); glucosylceramide (GlcCer); inflammation; knockin animals; knockout animals; misfolding; unfolded protein response (UPR)

DOI:  https://doi.org/10.3390/ijms242216035
Cell Rep. 2023 Nov 17. pii: S2211-1247(23)01415-8. [Epub ahead of print]42(12): 113403

Reticulons bind sphingolipids to activate the endoplasmic reticulum cell cycle checkpoint, the ER surveillance pathway.

Francisco Piña, Bing Yan, Junjie Hu, Maho Niwa.

  The inheritance of a functional endoplasmic reticulum (ER) is ensured by the ER stress surveillance (ERSU) pathway. Here, we made the unexpected discovery that reticulon 1 (Rtn1) and Yop1, well-known ER-curvature-generating proteins, each possess two sphingolipid-binding motifs within their transmembrane domains and that these motifs recognize the ER-stress-induced sphingolipid phytosphingosine (PHS), resulting in an ER inheritance block. Upon binding PHS, Rtn1/Yop1 accumulate on the ER tubule, poised to enter the emerging daughter cell, and cause its misdirection to the bud scars (i.e., previous cell division sites). Amino acid changes in the conserved PHS-binding motifs preclude Rtn1 or Yop1 from binding PHS and diminish their enrichment on the tubular ER, ultimately preventing the ER-stress-induced inheritance block. Conservation of these sphingolipid-binding motifs in human reticulons suggests that sphingolipid binding to Rtn1 and Yop1 represents an evolutionarily conserved mechanism that enables cells to respond to ER stress.

Keywords:  CP: Cell biology; ER; ER inheritance; ER stress; ER surveillance; cell cycle checkpoint; cytokinesis block; endoplasmic reticulum; phytosphingosine; reticulons; sphingolipids

DOI:  https://doi.org/10.1016/j.celrep.2023.113403
Int J Mol Sci. 2023 Nov 17. pii: 16467. [Epub ahead of print]24(22):

Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

Ching-Sheng Hung, Kun-Lin Lee, Wei-Jan Huang, Fang-He Su, Yu-Chih Liang.

  The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

Keywords:  apoptosis; autophagy; ferroptosis; pancreatic ductal adenocarcinoma; protein disulfide isomerase

DOI:  https://doi.org/10.3390/ijms242216467
Int J Mol Sci. 2023 Nov 11. pii: 16200. [Epub ahead of print]24(22):

The Unfolded Protein Response in a Murine Model of Alzheimer's Disease: Looking for Predictors.

Giulia Sita, Agnese Graziosi, Camilla Corrieri, Luca Ghelli, Sabrina Angelini, Pietro Cortelli, Patrizia Hrelia, Fabiana Morroni.

  Alzheimer's disease (AD) represents the most frequent type of dementia worldwide, and aging is the most important risk factor for the sporadic form of the pathology. The endoplasmic reticulum (ER), the main cellular actor involved in proteostasis, appears significantly compromised in AD due to the accumulation of the β-amyloid (Aβ) protein and the phosphorylated Tau protein. Increasing protein misfolding activates a specific cellular response known as Unfolded Protein Response (UPR), which orchestrates the recovery of ER function. The aim of the present study was to investigate the role of UPR in a murine model of AD induced by intracerebroventricular (i.c.v.) injection of Aβ1-42 oligomers at 3 or 18 months. The oligomer injection in aged animals induced memory impairment, oxidative stress, and the depletion of glutathione reserve. Furthermore, the RNA sequencing and the bioinformatic analysis performed showed the enrichment of several pathways involved in neurodegeneration and protein regulations. The analysis highlighted the significant dysregulation of the protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF-6). In turn, ER stress affected the PI3K/Akt/Gsk3β and MAPK/ERK pathways, highlighting Mapkapk5 as a potential marker, whose regulation could lead to the definition of new pharmacological and neuroprotective strategies to counteract AD.

Keywords:  Alzheimer’s disease; Aβ oligomers; UPR; aging; neurodegeneration

DOI:  https://doi.org/10.3390/ijms242216200
Drug Deliv. 2023 Dec;30(1): 2284684

Intricate subcellular journey of nanoparticles to the enigmatic domains of endoplasmic reticulum.

Koyeli Girigoswami, Pragya Pallavi, Agnishwar Girigoswami.

  It is evident that site-specific systemic drug delivery can reduce side effects, systemic toxicity, and minimal dosage requirements predominantly by delivering drugs to particular pathological sites, cells, and even subcellular structures. The endoplasmic reticulum (ER) and associated cell organelles play a vital role in several essential cellular functions and activities, such as the synthesis of lipids, steroids, membrane-associated proteins along with intracellular transport, signaling of Ca2+, and specific response to stress. Therefore, the dysfunction of ER is correlated with numerous diseases where cancer, neurodegenerative disorders, diabetes mellitus, hepatic disorder, etc., are very common. To achieve satisfactory therapeutic results in certain diseases, it is essential to engineer delivery systems that can effectively enter the cells and target ER. Nanoparticles are highly biocompatible, contain a variety of cargos or payloads, and can be modified in a pliable manner to achieve therapeutic effectiveness at the subcellular level when delivered to specific organelles. Passive targeting drug delivery vehicles, or active targeting drug delivery systems, reduce the nonselective accumulation of drugs while reducing side effects by modifying them with small molecular compounds, antibodies, polypeptides, or isolated bio-membranes. The targeting of ER and closely associated organelles in cells using nanoparticles, however, is still unsymmetrically understood. Therefore, here we summarized the pathophysiological prospect of ER stress, involvement of ER and mitochondrial response, disease related to ER dysfunctions, essential therapeutics, and nanoenabled modulation of their delivery to optimize therapy.

Keywords:  ER stress; cancer; drug delivery; hepatic disorder; nanoformulations; neurodegenerative disorder

DOI:  https://doi.org/10.1080/10717544.2023.2284684
Sci Rep. 2023 Nov 22. 13(1): 20467

ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation.

Audrey M G Ragagnin, Vinod Sundaramoorthy, Fabiha Farzana, Shashi Gautam, Sayanthooran Saravanabavan, Zeinab Takalloo, Prachi Mehta, Dzung Do-Ha, Sonam Parakh, Sina Shadfar, Julie Hunter, Marta Vidal, Cyril J Jagaraj, Mariana Brocardo, Anna Konopka, Shu Yang, Stephanie L Rayner, Kelly L Williams, Ian P Blair, Roger S Chung, Albert Lee, Lezanne Ooi, Julie D Atkin.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.

DOI: https://doi.org/10.1038/s41598-023-46802-9
FASEB J. 2023 Dec;37(12): e23318

Higher matrix stiffness promotes VSMC senescence by affecting mitochondria-ER contact sites and mitochondria/ER dysfunction.

Haipeng He, Baozhu Zeng, Xinxiang Wu, Jianfeng Hou, Yannan Wang, Yanheng Wang, Yuqing Lin, Peng Wu, Changyu Zheng, Henghui Yin, Nan Wang.

  Abdominal aortic aneurysm (AAA) is a prevalent condition characterized by the weakening and bulging of the abdominal aorta. This study aimed to investigate the impact of a stiff matrix on vascular smooth muscle cells (VSMCs) in AAA development. Bioinformatics analysis revealed that differentially expressed genes (DEGs) in VSMCs of an AAA mouse model were enriched in cellular senescence and related pathways. To simulate aging-related changes, VSMCs were cultured on stiff matrices, and compared to those on soft matrices, the VSMCs cultured on stiff matrices exhibited cellular senescence. Furthermore, the mutual distance between mitochondria and endoplasmic reticulum (ER) in VSMCs was increased, indicating altered mitochondria-endoplasmic reticulum contacts (MERCs). The observed upregulation of reactive oxygen species (ROS) levels, antioxidant gene expression, and decreased mitochondrial membrane potential suggested the presence of mitochondrial dysfunction in VSMCs cultured on a stiff matrix. Additionally, the induction of ER stress-related genes indicated ER dysfunction. These findings collectively indicated impaired functionality of both mitochondria and ER in VSMCs cultured on a stiff matrix. Moreover, our data revealed that high lipid levels exacerbated the effects of high matrix stiffness on VSMCs senescence, MERC sites, and mitochondria/ER dysfunction. Importantly, treatment with the antilipemic agent CI-981 effectively reversed these detrimental effects. These findings provide insights into the role of matrix stiffness, mitochondrial dysfunction, ER stress, and lipid metabolism in AAA development, suggesting potential therapeutic targets for intervention.

Keywords:  abdominal aortic aneurysm; cellular senescence; lipid; mitochondria-endoplasmic reticulum contact sites

DOI:  https://doi.org/10.1096/fj.202301198RR
Biochem Pharmacol. 2023 Nov 22. pii: S0006-2952(23)00522-1. [Epub ahead of print] 115929

Adaptive changes in tumor cells in response to reductive stress.

Leilei Zhang, Jie Zhang, Zhi-Wei Ye, Aslam Muhammad, Li Li, John W Culpepper, Danyelle M Townsend, Kenneth D Tew.

  Reductive stress is characterized by an excess of cellular electron donors and can be linked with various human pathologies including cancer. We developed melanoma cell lines resistant to reductive stress agents: rotenone (ROTR), n-acetyl-L-cysteine, (NACR), or dithiothreitol (DTTR). Resistant cells divided more rapidly and had intracellular homeostatic redox-couple ratios that were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROTR cells had significant changes in mitochondrial morphology with higher numbers that were more isolated, fragmented and swollen, with greater membrane depolarization and decreased numbers of networks. These changes were accompanied by lower basal oxygen consumption and maximal respiration rates. Whole cell flux analyses and mitochondrial function assays showed that NACR and DTTR preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROTR used ketone body substrates such as D, L-β-hydroxybutyric acid. NACR and DTTR cells had constitutively decreased levels of reactive oxygen species (ROS), although this was accompanied by activation of nuclear factor erythroid 2-related factor 2 (Nrf2), with concomitant increased expression of the downstream gene products such as glutathione S-transferase P (GSTP). Further adaptations included enhanced expression of endoplasmic reticulum proteins controlling the unfolded protein response (UPR). Although expression patterns of these UPR proteins were distinct between the resistant cells, a trend implied that resistance to reductive stress is accompanied by a constitutively increased UPR phenotype in each line. Overall, tumor cells, although tolerant of oxidative stress, can adapt their energy and survival mechanisms in lethal reductive stress conditions.

Keywords:  Glycolysis; Melanoma cell lines; Mitochondrial morphology; Reactive oxygen species; Reductive stress; Unfolded protein response

DOI:  https://doi.org/10.1016/j.bcp.2023.115929