bims-cemest 2025-04-13 papers

bims-cemest

Biomed News

on Cell metabolism and stress

Issue of 2025–04–13
24 papers selected by
Jessica Rosarda, Uniformed Services University

Cytosolic and endoplasmic reticulum chaperones inhibit wt-p53 to increase cancer cells' survival by refluxing ER-proteins to the cytosol.
TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the ER and into the Golgi.
Hsp70 chaperones, Ssa1 and Ssa2, limit poly(A) binding protein aggregation.
Role of saturated fatty acid metabolism in posttranslational modifications of the Tau protein.
Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry.
MiR-204-5p mediates PERK inhibition to suppress growth and induce apoptosis in ovarian cancer through the eIF2α/ATF-4/CHOP pathway.
Selective Translation Under Heat Shock: Integrating HSP70 mRNA Regulation with Cellular Stress Responses in Yeast and Mammals.
Quiescent cell re-entry is limited by macroautophagy-induced lysosomal damage.
Unraveling the crucial role of UPR pathway in rat brain development.
Unravelling the role of protein kinase R (PKR) in neurodegenerative disease: a review.
Multi-omic Evaluations Nominate an ER-Mitochondrial Axis and Inflammatory Macrophage as Drivers of Right Atrial Dysfunction.
HRI protein kinase in cytoplasmic heme sensing and mitochondrial stress response: relevance to hematological and mitochondrial diseases.
ADP ribosylation factor-like GTPase 6-interacting protein 5 (Arl6IP5) is an ER membrane-shaping protein that modulates ER-phagy.
Targeting the ER stress sensor IRE1 protects the liver from fibrosis through the downregulation of the proteostasis factor P4HB/PDIA1.
Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.
PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.
GPAT4 sustains endoplasmic reticulum homeostasis in endocardial cells and safeguards heart development.
Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway.
Scaling up neuronal Ca2+, signaling on ER ladders.
Differential neuronal vulnerability to C9orf72 repeat expansion driven by Xbp1-induced endoplasmic reticulum-associated degradation.
Structure of a Gcn2 dimer in complex with the large 60S ribosomal subunit.
Hsp70: A Multifunctional Chaperone in Maintaining Proteostasis and Its Implications in Human Disease.
Alternative start codon selection shapes mitochondrial function during evolution, homeostasis, and disease.
The selenocysteine-containing protein SELENOT maintains dopamine signaling in the midbrain to protect mice from hyperactivity disorder.

Elife. 2025 Apr 09. pii: e102658. [Epub ahead of print]14

Cytosolic and endoplasmic reticulum chaperones inhibit wt-p53 to increase cancer cells' survival by refluxing ER-proteins to the cytosol.

Salam Dabsan, Gali Zur, Naim Abu-Freha, Shahar Sofer, Iris Grossman-Haham, Ayelet Gilad, Aeid Igbaria.

  The endoplasmic reticulum (ER) is an essential sensing organelle responsible for the folding and secretion of almost one-third of eukaryotic cells' total proteins. However, environmental, chemical, and genetic insults often lead to protein misfolding in the ER, accumulating misfolded proteins, and causing ER stress. To solve this, several mechanisms were reported to relieve ER stress by decreasing the ER protein load. Recently, we reported a novel ER surveillance mechanism by which proteins from the secretory pathway are refluxed to the cytosol to relieve the ER of its content. The refluxed proteins gain new prosurvival functions in cancer cells, thereby increasing cancer cell fitness. We termed this phenomenon ER to CYtosol Signaling (or 'ERCYS'). Here, we found that in mammalian cells, ERCYS is regulated by DNAJB12, DNAJB14, and the HSC70 cochaperone SGTA. Mechanistically, DNAJB12 and DNAJB14 bind HSC70 and SGTA - through their cytosolically localized J-domains to facilitate ER-protein reflux. DNAJB12 is necessary and sufficient to drive this phenomenon to increase AGR2 reflux and inhibit wt-p53 during ER stress. Mutations in DNAJB12/14 J-domain prevent the inhibitory interaction between AGR2-wt-p53. Thus, targeting the DNAJB12/14-HSC70/SGTA axis is a promising strategy to inhibit ERCYS and impair cancer cell fitness.

Keywords:  DNAJB12; ER stress; ERCYS; UPR; cancer biology; cell biology; chaperones; none; reflux

DOI:  https://doi.org/10.7554/eLife.102658
PLoS Biol. 2025 Apr 09. 23(4): e3003084

TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the ER and into the Golgi.

Elsa Ronzier, Prasanna Satpute-Krishnan.

The p24-family member, TMED9, has recently emerged as a player in secretory pathway protein quality control (PQC) that influences the trafficking and degradation of misfolded proteins. Here, we show that TMED9 plays a central role in the PQC of GPI-anchored proteins (GPI-APs). Typically, upon release from the endoplasmic reticulum (ER)-resident chaperone calnexin, misfolded GPI-APs traffic to the Golgi by an ER-export pathway called Rapid ER stress-induced Export (RESET). From the Golgi, they access the plasma membrane where they are rapidly internalized for lysosomal degradation. We used biochemical and imaging approaches in cultured cells to demonstrate that at steady-state, the majority of misfolded GPI-APs reside in the ER in association with calnexin and TMED9. During RESET, they dissociate from calnexin and increase their association with TMED9. Inhibition of TMED9's function through siRNA-induced depletion or chemical inhibitor, BRD4780, blocked ER-export of misfolded GPI-APs. In contrast, TMED9-inhibition did not prevent ER-export of wild-type GPI-APs, indicating a specific role for TMED9 in GPI-AP PQC. Intriguingly, we discovered that acute treatment with BRD4780 induced a shift in TMED9 localization away from the ER to the downstream Golgi cisternae and blocked the RESET pathway. Upon removal of BRD4780 following acute treatment, TMED9 regained access to the ER where TMED9 was able to associate with the RESET substrate and restore the RESET pathway. These results suggest that TMED9 plays a requisite role in RESET by capturing misfolded GPI-APs that are released by calnexin within the ER and conveying them to the Golgi.

DOI: https://doi.org/10.1371/journal.pbio.3003084
Mol Biol Cell. 2025 Apr 09. mbcE25010027

Hsp70 chaperones, Ssa1 and Ssa2, limit poly(A) binding protein aggregation.

Hannah E Buchholz, Sean A Martin, Jane E Dorweiler, Claire M Radtke, Adam S Knier, Natalia B Beans, Anita L Manogaran.

Molecular chaperones play a central role in maintaining protein homeostasis. The highly conserved Hsp70 family of chaperones have major functions in folding of nascent peptides, protein refolding, and protein aggregate disassembly. In yeast, loss of two Hsp70 proteins, Ssa1 and Ssa2, is associated with decreased cellular growth and shortened lifespan. While heterologous or mutant temperature sensitive proteins form anomalous large cytoplasmic inclusions in ssa1Δssa2Δ strains, it is unclear how endogenous wildtype proteins behave and are regulated in the presence of limiting Hsp70s. Using the wildtype yeast Poly A binding protein (Pab1), which is involved in mRNA binding and forms stress granules (SGs) upon heat shock, Pab1 forms large inclusions in approximately half of ssa1Δssa2Δ cells in the absence of stress. Overexpression of Ssa1, Hsp104, and Sis1 almost completely limits the formation of these large inclusions in ssa1Δssa2Δ, suggesting that excess Ssa1, Hsp104 and Sis1 can each compensate for the lower levels of Ssa proteins. Upon heat shock, SGs also form in cells whether large Pab1 inclusions are present or not. Surprisingly, cells containing only SGs disassemble faster than wildtype, whereas cells with both large inclusions disassemble slower albeit completely. We suspect that disassembly of these large inclusions is linked to the elevated heat shock response and elevated Hsp104 and Sis1 levels in ssa1Δssa2Δ strains. We also observed that wildtype cultures grown to saturation also form large Pab1-GFP inclusions. These inclusions can be partially rescued by overexpression of Ssa1. Taken together, our data suggests that Hsp70 not only plays a role in limiting unwanted protein aggregation in normal cells, but as cells age, the depletion of active Hsp70 possibly underlies the age-related aggregation of endogenous proteins.

DOI: https://doi.org/10.1091/mbc.E25-01-0027
Mol Cell Biochem. 2025 Apr 10.

Role of saturated fatty acid metabolism in posttranslational modifications of the Tau protein.

Valeria Melissa García-Cruz, Roberto Coria, Clorinda Arias.

  The relationship between metabolic alterations induced by the consumption of a high-fat diet (HFD) and the risk of developing neurodegenerative diseases such as Alzheimer's disease (AD) has been extensively studied. In particular, the induction of neuronal insulin resistance, endoplasmic reticulum stress, and the production of reactive oxygen species by chronic exposure to high concentrations of saturated fatty acids (sFAs), such as palmitic acid (PA), have been proposed as the cellular and molecular mechanisms underlying cognitive decline. Lipid metabolism affects many processes critical for cellular homeostasis. However, questions remain as to whether neuronal exposure to high sFA levels contributes to the onset and progression of AD features, and how their metabolism plays a role in this process. Therefore, the aim of this work is to review the accumulated evidence for the potential mechanisms by which the neuronal metabolism of sFAs affects signaling pathways that may induce biochemical changes in the AD hallmark protein Tau, ultimately promoting its aggregation and the subsequent generation of neurofibrillary tangles. In particular, the data presented here provide evidence that PA-dependent metabolic stress results in an imbalance in the activities of protein kinases and deacetylases that potentially contribute to the post-translational modifications (PTMs) of Tau.

Keywords:  Deacetylases; Energy metabolism; Protein kinases; Saturated fatty acids; Tau

DOI:  https://doi.org/10.1007/s11010-025-05275-2
bioRxiv. 2025 Mar 24. pii: 2025.03.20.644425. [Epub ahead of print]

Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry.

Lea A Barny, Jake N Hermanson, Sarah K Garcia, Philip E Stauffer, Lars Plate.

Cells rely on the Unfolded Protein Response (UPR) to maintain ER protein homeostasis (proteostasis) when faced with elevated levels of misfolded and aggregated proteins. The UPR is comprised of three main branches-ATF6, IRE1, and PERK-that coordinate the synthesis of proteins involved in folding, trafficking, and degradation of nascent proteins to restore ER function. Dysregulation of the UPR is linked to numerous diseases, including neurodegenerative disorders, cancer, and diabetes. Despite its importance, identifying UPR targets has been challenging due to their heterogeneous induction, which varies by cell type and tissue. Additionally, defining the magnitude and range of UPR-regulated genes is difficult because of intricate temporal regulation, feedback between UPR branches, and extensive cross-talk with other stress-signaling pathways. To comprehensively identify UPR-regulated proteins and determine their branch specificity, we developed a data-independent acquisition (DIA) liquid-chromatography mass spectrometry (LC-MS) pipeline. Our optimized workflow improved identifications of low-abundant UPR proteins and leveraged an automated SP3-based protocol on the Biomek i5 liquid handler for label-free peptide preparation. Using engineered stable cell lines that enable selective pharmacological activation of each UPR branch without triggering global UPR activation, we identified branch-specific UPR proteomic targets. These targets were subsequently applied to investigate proteomic changes in multiple patient-derived BRAF-mutant melanoma cell lines treated with a BRAF inhibitor (PLX4720, i.e., vemurafenib). Our findings revealed differential regulation of the XBP1s branch of the UPR in the BRAF-mutant melanoma cell lines after PLX4720 treatment, likely due to calcium activation, suggesting that the UPR plays a role as a non-genetic mechanism of drug tolerance in melanoma. In conclusion, the validated branch-specific UPR proteomic targets identified in this study provide a robust framework for investigating this pathway across different cell types, drug treatments, and disease conditions in a high-throughput manner.

DOI: https://doi.org/10.1101/2025.03.20.644425
Sci Rep. 2025 Apr 11. 15(1): 12435

MiR-204-5p mediates PERK inhibition to suppress growth and induce apoptosis in ovarian cancer through the eIF2α/ATF-4/CHOP pathway.

Faranak Fallahian, Seyedeh Sara Ghorbanhosseini, Shekufe Rezghi Barez, Mahmoud Aghaei.

  The unfolded protein response (UPR) is crucial in maintaining cell survival during stressful conditions, but prolonged ER stress can lead to apoptosis. Based on the evidence acquired, it has been suggested that inhibiting the protein kinase RNA-like ER kinase (PERK) pathway, which constitutes an adaptive branch of UPR, may represent a viable approach for impeding the proliferation of neoplastic cells. This study assesses the influence of PERK inhibition mediated by miR-204-5p on the growth of ovarian cancer cell lines, OVCAR3 and SKOV3. We demonstrated that miR-204-5p significantly downregulated the expression of PERK at the RNA and protein levels. The suppression of PERK, mediated by miR-204-5p, significantly diminished cellular viability and enhanced apoptotic cell death in cells exposed to Tunicamycin (Tm). We ascertained that the inhibition of PERK by miR-204-5p decreased eukaryotic initiation factor 2alpha (eIF2α) phosphorylation. Moreover, activating transcription factor 4 (ATF4) and CCAAT-enhancer-binding homologous protein (CHOP) expression levels were notably elevated in response to miR-204-5p. The expression of Bax and caspase-12 was found to be upregulated, while the expression of Bcl-2 was reduced. This study is the first to demonstrate that silencing the PERK gene through miR-204-5p significantly inhibits cell growth and promotes ER-stress-induced apoptosis in ovarian cancer cells.

Keywords:  Apoptosis; ER stress; MiR-204-5p; MicroRNA; Ovarian cancer; PERK

DOI:  https://doi.org/10.1038/s41598-025-95883-1
Mol Biol Cell. 2025 May 01. 36(5): re2

Selective Translation Under Heat Shock: Integrating HSP70 mRNA Regulation with Cellular Stress Responses in Yeast and Mammals.

Talar Ghadanian, Shruti Iyer, Luca Lazzari, Maria Vera.

Under stress, cells orchestrate a complex regulatory response to maintain protein homeostasis, leveraging differential translational regulation for constitutively expressed mRNAs and the transcriptionally induced heat shock protein HSP70 transcripts. Constitutive mRNAs typically experience partial translational suppression, consistent with their partitioning into stress-induced phase-separated condensates and the global reduction in protein synthesis. In contrast, inducible HSP70 mRNAs bypass this repression to remain in the cytosol where they recruit the available components of the translational machinery to ensure the rapid synthesis of HSP70. Although the components involved in the preferential translation of HSP70 mRNA during heat stress have not been fully elucidated, differences in the mRNA and translation factors between yeast and mammals suggest organism-specific mechanisms of HSP70 mRNA translation. In this review, we consider these differences to discuss the current knowledge on heat shock regulation of translation. We extend the discussion to go beyond the cytosolic needs of HSP70 to ponder the important interplay between the cytosol and mitochondria in activating HSP70 accumulation, which becomes vital for preserving intercompartmental proteostasis and cell survival.

DOI: https://doi.org/10.1091/mbc.E24-12-0564
Cell. 2025 Apr 04. pii: S0092-8674(25)00282-X. [Epub ahead of print]

Quiescent cell re-entry is limited by macroautophagy-induced lysosomal damage.

Andrew Murley, Ann Catherine Popovici, Xiwen Sophie Hu, Anina Lund, Kevin Wickham, Jenni Durieux, Larry Joe, Etai Koronyo, Hanlin Zhang, Naomi R Genuth, Andrew Dillin.

  To maintain tissue homeostasis, many cells reside in a quiescent state until prompted to divide. The reactivation of quiescent cells is perturbed with aging and may underlie declining tissue homeostasis and resiliency. The unfolded protein response regulators IRE-1 and XBP-1 are required for the reactivation of quiescent cells in developmentally L1-arrested C. elegans. Utilizing a forward genetic screen in C. elegans, we discovered that macroautophagy targets protein aggregates to lysosomes in quiescent cells, leading to lysosome damage. Genetic inhibition of macroautophagy and stimulation of lysosomes via the overexpression of HLH-30 (TFEB/TFE3) synergistically reduces lysosome damage. Damaged lysosomes require IRE-1/XBP-1 for their repair following prolonged L1 arrest. Protein aggregates are also targeted to lysosomes by macroautophagy in quiescent cultured mammalian cells and are associated with lysosome damage. Thus, lysosome damage is a hallmark of quiescent cells, and limiting lysosome damage by restraining macroautophagy can stimulate their reactivation.

Keywords:  aging; endoplasmic reticulum; lysosome; mTOR; macroautophagy; protein aggregates; quiescence

DOI:  https://doi.org/10.1016/j.cell.2025.03.009
Mol Biol Rep. 2025 Apr 10. 52(1): 382

Unraveling the crucial role of UPR pathway in rat brain development.

Yan Jiang, Ajun Wang, Nian Zou, Huimin Dai, Ran Shan, Siyu Ren, Tongcui Jiang, Nan Li.

  The development of the central nervous system (CNS) is essential for the growth of an organism. The unfolded protein response (UPR) plays an important role in the occurrence and progression of CNS. Hence, to identify the changing pattern of UPR signaling molecules and their related target molecules is extremely important. Immunohistochemical (IHC) analysis was conducted to assess the expression of NeuN, a neuronal marker, within the M1 region of the cerebral cortex of wild-type (WT) rats aged different weeks. The results showed a progressive increase in the expression of NeuN in correlation with the weekly age progression of the rats. Concurrently, in the CA3 region of the hippocampus and the S1Tr area of the cerebral cortex, the levels of UPR key molecules, GRP78 and XBP1s, exhibited an upward trend in accordance with the rats' weekly age. Western blot analysis revealed an inclination toward an increase in the expression of endoplasmic reticulum (ER) stress -associated key molecules, including GRP78, ATF6, XBP1, eIF-2α, CHOP and phosphorylated eIF-2α (p-eIF-2α). Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) demonstrated a positive correlation between the expression of CHOP and the weekly age of the rats. These findings demonstrate that UPR signaling pathway has a certain pattern of change in the development of rat brain, suggesting that the UPR signaling pathway may play an important role in the development of the CNS, and suggesting that an applicability of XBP1s as a therapeutic candidate for childhood autism.

Keywords:  ER stress; Rat brain development; Unfolded protein response; XBP1s

DOI:  https://doi.org/10.1007/s11033-025-10483-6
Mol Biol Rep. 2025 Apr 09. 52(1): 377

Unravelling the role of protein kinase R (PKR) in neurodegenerative disease: a review.

Maneesh Mohan, Ashi Mannan, Thakur Gurjeet Singh.

  Protein Kinase R is an essential regulator of many cell activities and belongs to one of the largest and most functionally complex gene families. These are found all over the body, and by adding phosphate groups to the substrate proteins, they regulate their activity and coordinate the action of almost all cellular processes. Recent research has illuminated the involvement of PKR in the pathogenesis of neurodegenerative disorders (NDs), thereby expanding our understanding of intricate molecular mechanisms underlying disease progression. Through their inhibition or activation, they hold potential therapeutic targets for the pathogenesis or protection of NDs. In the case of AD (AD), PKR contributes to the protection or elevation of Aβ accumulation, neuroinflammation, synaptic plasticity alterations, and neuronal excitability. Similarly, in Parkinson's disease (PD), PKR again has a dual role in dopaminergic neuronal loss, gene mutations, and mitochondrial dysfunction via various pathways. Notably, neuronal excitotoxicity, as well as genetic mutations, have been linked to ALS. In Huntington's disease (HD), PKR is associated with decreased or increased mutated genes, striatal neuron degeneration, neuroinflammation, and excitotoxicity. This review emphasizes strategies that target PKR for the treatment of neurodegenerative disorders. Doing so offers valuable insights that can guide future research endeavors and the development of innovative therapeutic approaches.

Keywords:  Excitotoxicity; Neurodegeneration; Neuroinflammation; Protein kinases; Signaling pathways

DOI:  https://doi.org/10.1007/s11033-025-10484-5
bioRxiv. 2025 Mar 25. pii: 2025.03.22.644722. [Epub ahead of print]

Multi-omic Evaluations Nominate an ER-Mitochondrial Axis and Inflammatory Macrophage as Drivers of Right Atrial Dysfunction.

Jenna B Mendelson, Jacob D Sternbach, Jeffrey C Blake, Minwoo Kim, Ryan A Moon, Rashmi M Raveendran, Lynn M Hartweck, Walt Tollison, Matthew Lahiri, John P Carney, Todd Markowski, LeeAnn Higgins, Cutler T Lewandowski, Felipe Kazmirczak, Gaurav Choudhary, Kurt W Prins.

Background: Right atrial (RA) dysfunction is an emerging risk factor for poor outcomes in pulmonary arterial hypertension, however the mechanisms underlying compromised RA function are understudied.
Objectives: Multi-omic analyses defined the cellular and molecular mediators associated with RA dysfunction in pulmonary artery banded (PAB) swine.
Methods: 4-week-old castrated male Yorkshire pigs were subjected to PAB and aged six weeks to induce right heart failure. Cardiac MRI evaluated RA size and function. snRNAseq defined the cell-specific alterations in RA tissue. Mitochondrial proteomics and metabolomics analyses examined the metabolic alterations in RA samples. Inducible pluripotent stem cell-derived atrial cardiomyocytes (iPSC-ACM) were treated with tunicamycin to induce endoplasmic reticulum (ER) stress and mitochondrial structure and function were probed.
Results: PAB induced RA dilation/dysfunction and atrial cardiomyocyte hypertrophy. snRNAseq demonstrated PAB altered the cellular composition of the RA defined by increased inflammatory macrophages and an alteration of cardiomyocyte subpopulations. RA cardiomyocytes exhibited ER stress and mitochondrial metabolic enzyme dysregulation. PAB RAs, but not PAB right ventricles, had downregulation of branched chain amino acid degrading enzymes. Metabolomics profiling revealed BCAA and fatty acid metabolism were impaired in the dysfunctional RA. Tunicamycin-induced ER stress disrupted mitochondrial structure/function in iPSC-ACMs.
Conclusions: Multi-omic evaluations demonstrate RA dysfunction is characterized by cardiomyocyte metabolic derangements due to ER dysregulation and an accumulation of pro-inflammatory macrophages.

DOI: https://doi.org/10.1101/2025.03.22.644722
J Biol Chem. 2025 Apr 08. pii: S0021-9258(25)00343-6. [Epub ahead of print] 108494

HRI protein kinase in cytoplasmic heme sensing and mitochondrial stress response: relevance to hematological and mitochondrial diseases.

Jane-Jane Chen.

  Most iron in humans is bound in heme used as a prosthetic group for hemoglobin. Heme-regulated inhibitor (HRI) is responsible for coordinating heme availability and protein synthesis. Originally characterized in rabbit reticulocyte lysates, HRI was shown in 1976 to phosphorylate the α-subunit of eIF2, revealing a new molecular mechanism for regulating protein synthesis. Since then, HRI research has mostly been focused on the biochemistry of heme inhibition through direct binding, and heme sensing in balancing heme and globin synthesis to prevent proteotoxicity in erythroid cells. Beyond inhibiting translation of highly translated mRNAs, eIF2α phosphorylation also selectively increases translation of certain poorly translated mRNAs, notably ATF4 mRNA, for reprogramming of gene expression to mitigate stress, known as the integrated stress response (ISR). In recent years, there have been novel mechanistic insights of HRI-ISR in oxidative stress, mitochondrial function and erythroid differentiation during heme deficiency. Furthermore, HRI-ISR is activated upon mitochondrial stress in several cell types, establishing the bifunctional nature of HRI protein. The role of HRI and ISR in cancer development and vulnerability is also emerging. Excitingly, the UBR4 ubiquitin ligase complex has been demonstrated to silence the HRI-ISR by degradation of activated HRI proteins, suggesting additional regulatory processes. Together, these recent advancements indicate that the HRI-ISR mechanistic axis is a target for new therapies for hematological and mitochondrial diseases, as well as oncology. This review covers the historical overview of HRI biology, the biochemical mechanisms of regulating HRI, and the biological impacts of the HRI-ISR pathway in human diseases.

Keywords:  ATF4; E3 ubiquitin ligase; Erythropoiesis; Heme; Mitochondrial stress; Protein kinase; Protein synthesis; Proteostasis; Stress response; eIF2

DOI:  https://doi.org/10.1016/j.jbc.2025.108494
J Biol Chem. 2025 Apr 08. pii: S0021-9258(25)00342-4. [Epub ahead of print] 108493

ADP ribosylation factor-like GTPase 6-interacting protein 5 (Arl6IP5) is an ER membrane-shaping protein that modulates ER-phagy.

Yasunori Yamamoto, Toshiaki Sakisaka.

  The endoplasmic reticulum (ER) is the membrane-bound organelle characterized by the reticular network of tubules. It is well established that the ER tubules are shaped by ER membrane proteins containing the conserved reticulon-homology domain (RHD). Membrane shaping by the RHD-containing proteins is also involved in regulation of ER-phagy, selective autophagy of the ER. However, it remains unclear whether there exists ER membrane-shaping proteins other than the RHD-containing proteins. In this study, we characterize Arl6IP5, an ER membrane protein containing the conserved PRA1 domain, as an ER membrane-shaping protein. Upon overexpression, Arl6IP5 induces the extensive network of the ER tubules, and constricts the ER membrane as judged by exclusion of a luminal ER enzyme from the ER tubules. The membrane constriction by Arl6IP5 allows the cells to maintain the tubular ER network in the absence of microtubules. siRNA-mediated knockdown of Arl6IP5 impairs the ER morphology, and the phenotype of the Arl6IP5 knockdown cells is rescued by exogenous expression of Arl6IP1, an RHD-containing protein. Furthermore, exogenous expression of Arl6IP5 rescues the phenotype of Arl6IP1 knockdown cells, and the PRA1 domain is sufficient to rescue it. Upon disruption of the possible short hairpin structures of the PRA1 domain, Arl6IP5 abolishes membrane constriction. The siRNA-mediated knockdown of Arl6IP5 impairs flux of the ER-phagy mediated by FAM134B. These results indicate that Arl6IP5 acts as an ER membrane-shaping protein involved in regulation of ER-phagy, implying that the PRA1 domain may serve as a general membrane-shaping unit other than the RHD.

Keywords:  autophagy; endoplasmic reticulum (ER); membrane protein; membrane structure; protein domain

DOI:  https://doi.org/10.1016/j.jbc.2025.108493
Hepatology. 2025 Apr 09.

Targeting the ER stress sensor IRE1 protects the liver from fibrosis through the downregulation of the proteostasis factor P4HB/PDIA1.

Younis Hazari, Lama Habbouche, Valeria A Garcia Lopez, Hery Urra, Javier Diaz, Giovanni Tamburini, Mateus Milani, Sylvere Durand, Fanny Aprahamian, Reese Baxter, Menghao Huang, X Charlie Dong, Luis Gonzalez-Rojas, Juan Francisco Silva, Ignacio Tapia-Dufey, Helena Vihinen, Vlad Ratziu, Fabienne Foufelle, Jan G Hengstler, Eija Jokitalo, Jessica L Maiers, Lars Plate, Guido Kroemer, Beatrice Bailly-Maitre, Claudio Hetz.

  Collagen is the main cargo of the secretory pathway, contributing to hepatic fibrogenesis due to extensive accumulation of extracellular matrix. An excess of collagen deposition is a characteristic feature of several chronic liver diseases. Collagen overproduction imposes pressure on the secretory pathway, altering endoplasmic reticulum (ER) proteostasis. Here we investigated the possible contribution of the unfolded protein response UPR, the main adaptive pathway that monitors and adjusts protein production capacity at the ER, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 in the liver using conditional knockout mice reduced liver damage and collagen deposition in models of fibrosis, steatosis, and acute hepatotoxicity. Proteomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1) as a major IRE1-regulated gene, a critical factor involved in collagen maturation. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER, reducing its secretion, and this phenotype is rescued by P4HB/PDIA1 overexpression. Analyses of human MASH samples revealed a positive correlation between IRE1 signaling and P4HB/PDIA1 expression as well as the severity of the disease. Altogether, our results establish a role of the IRE1/P4HB axis in the regulation of collagen production and support its implication in the pathogenesis of liver fibrosis.

Keywords:  IRE1; PDIA1/ P4HB; UPR; collagen; endoplasmic reticulum; liver fibrosis

DOI:  https://doi.org/10.1097/HEP.0000000000001335
STAR Protoc. 2025 Apr 03. pii: S2666-1667(25)00133-9. [Epub ahead of print]6(2): 103727

Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.

Ritika Chaudhary, Vineet Choudhary.

  Here, we present a protocol to determine the topology of Fld1 (few lipid droplets), an integral endoplasmic reticulum (ER) membrane protein in Saccharomyces cerevisiae. We describe steps to generate functional N-terminal GFP and C-terminal mCherry fusion with Fld1. We detail the strategy to perform subcellular fractionation to isolate ER-derived microsomes that were subjected to salt detergent extraction analysis. We then provide procedures to determine the topology of Fld1 using proteinase K treatment.

Keywords:  cell biology; microscopy; molecular biology

DOI:  https://doi.org/10.1016/j.xpro.2025.103727
J Cell Biol. 2025 May 05. pii: e202407166. [Epub ahead of print]224(5):

PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

Kunyou Park, Sungeun Ju, Hyewon Choi, Peng Gao, Geul Bang, Jung Hoon Choi, Jiwon Jang, Andrew J Morris, Byung-Ho Kang, Victor W Hsu, Seung-Yeol Park.

Intracellular transport among organellar compartments occurs in two general ways: by membrane-bound carriers and membrane contacts. Specific circumstances that involve the coordination of these two modes of transport remain to be defined. By studying coat protein I (COPI) transport, we find that phosphatidylcholine with short acyl chains (sPC) is delivered through membrane contact from the endoplasmic reticulum (ER) to sites of COPI vesicle formation at the Golgi to support the fission stage. Phosphatidylinositol transfer protein beta (PITPβ) plays a key role in this process, with the elucidation of this role shedding new insights into how PITPβ acts, providing a mechanistic understanding of a specific circumstance when vesicular transport requires membrane contact and contributing to the general understanding of how intracellular transport carriers are formed.

DOI: https://doi.org/10.1083/jcb.202407166
Nat Commun. 2025 Apr 08. 16(1): 3345

GPAT4 sustains endoplasmic reticulum homeostasis in endocardial cells and safeguards heart development.

Tianyang Zhao, Kuipei Jin, Xiaodong Wang, Xiong Su, Youjun Wang, Mingming Gao, Wen Luo, Hongyuan Yang, Zhongzhou Yang.

The endocardium plays a pivotal role in governing myocardial development, and understanding the intrinsic regulatory insights will help apprehend pathological cardiomyopathy. Glycerol-3-phosphate acyltransferase 4 (GPAT4) is an endoplasmic reticulum (ER) membrane anchored protein. While the role of GPAT4 in glycerophospholipid biosynthesis is well established, its function in the ER is less explored. Here, we generate Gpat4 global and tissue-specific knockout mice and identify the essential role of GPAT4 in endocardial development. Deficiency of GPAT4 provokes endocardial ER stress response and enhances ER-mitochondrial (ER-mito) communications, leading to mitochondrial DNA (mtDNA) escape. As a result, the cGAS-STING pathway is triggered to stimulate type-I-interferon response, which affects heart development. Finally, abolishment of the cGAS-STING-type-I-interferon pathway rescues the heart defects of Gpat4 deletion mice. These findings uncover the pivotal role of GPAT4 in the maintenance of ER homeostasis during endocardial and heart development. Meanwhile, this study highlights the importance of the cGAS-STING pathway in cardiac organogenesis.

DOI: https://doi.org/10.1038/s41467-025-58722-5
Liver Res. 2025 Mar;9(1): 49-56

Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway.

Minghong Li, Mengyi Duan, Ying Yang, Xingdao Li, Dan Li, Wenting Gao, Xiaotong Ji, Jianying Bai.

   Background and aims: Hepatocellular carcinoma (HCC) is a malignant tumor with a high mortality rate, but there are still no effective treatments. The aim of this study was to investigate the anticancer potential of the combined use of brefeldin A (BFA) and tunicamycin (TM) in HepG2 cells, as well as the underlying mechanisms.
Methods: HepG2 cells were treated with different concentrations of BFA (0.1-2.5 mg/L) and TM (1-5 mg/L) for 24 h. DMSO (0.1 %, v/v) was used as a vehicle control. Cell viability and cell migration were measured using MTT assay and scratch wound assay, respectively. Apoptosis was detected using flow cytometry and acridine orange (AO) staining. The protein and mRNA levels of various factors involved in apoptosis (poly (ADP-ribose) polymerase-1 (PARP-1), caspase-12, caspase-3, and stearoyl-CoA desaturase 1) and endoplasmic reticulum (ER) stress (binding immunoglobulin protein (BiP), protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, phosphorylation of eukaryotic translation initiation factor 2alpha (p-eIF2α), activating transcription factor (ATF) 4, and C/EBP homologous protein (CHOP)) were measured using Western blotting and qRT-PCR, respectively.
Results: Both BFA and TM alone significantly reduced the viability of HepG2 cells in a dose-dependent way. The co-incubation with TM (1 mg/L) further significantly reduced the viability of HepG2 cells treated with BFA (0.25 mg/L) alone (P < 0.05). BFA significantly increased the protein and mRNA levels of caspase-3 and PARP-1 (P < 0.05) compared to control and DMSO-treated cells, indicating that BFA induced apoptosis in HepG2 cells by increasing the expression of caspase-3 and PARP-1. The induction of apoptosis by BFA could be further significantly enhanced by co-incubation with TM. In addition, BFA significantly increased the mRNA levels of BiP, PERK and ATF4 (P < 0.05) compared to control and DMSO-treated cells. After co-incubation of BFA and TM, the protein levels of BiP, p-PERK, p-eIF2α and CHOP were significantly increased, indicating that TM could enhance BFA-induced ER stress in HepG2 cells through the PERK-eIF2α-ATF4-CHOP pathway.
Conclusions: BFA could induce apoptosis and ER stress, and TM could enhance the ability of BFA to induce apoptosis and ER stress in HepG2 cells through the PERK-eIF2ɑ-ATF4-CHOP pathway. The findings highlight the therapeutic potential of the combined use of BFA and TM in treating HCC.

Keywords:  Apoptosis; Brefeldin A (BFA); Caspase-3; Endoplasmic reticulum stress (ERS); Hepatocellular carcinoma (HCC); Tunicamycin (TM)

DOI:  https://doi.org/10.1016/j.livres.2025.01.004
Cell Calcium. 2025 Apr 01. pii: S0143-4160(25)00025-9. [Epub ahead of print]127 103016

Scaling up neuronal Ca2+, signaling on ER ladders.

Khaled Machaca.

Dendritic Ca2+ signaling is critical for neural transmission and signal processing, however the detailed molecular mechanisms have not been elucidated. Using elegant and complementary imaging approaches Benedetti et al. discover a distinctive ER ladder architecture in dendrites and show that precise localization of Ca2+ signaling proteins at endoplasmic reticulum (ER)-plasma membrane (PM) junctions supports integration of Ca2+ signaling along the dendrite.

DOI: https://doi.org/10.1016/j.ceca.2025.103016
Cell Rep. 2025 Mar 23. pii: S2211-1247(25)00230-X. [Epub ahead of print] 115459

Differential neuronal vulnerability to C9orf72 repeat expansion driven by Xbp1-induced endoplasmic reticulum-associated degradation.

Dunxin Shen, Alec Vincent, Evan Udine, Yazead Buhidma, Sharifah Anoar, Elli Tsintzas, Marie Maeland, Dongwei Xu, Mireia Carcolé, David Osumi-Sutherland, Benjamin Aleyakpo, Alexander Hull, Guillermo Martínez Corrales, Nathan Woodling, Rosa Rademakers, Adrian M Isaacs, Carlo Frigerio, Marka van Blitterswijk, Tammaryn Lashley, Teresa Niccoli.

  Neurodegenerative diseases are characterized by the localized loss of neurons. Why cell death is triggered only in specific neuronal populations and whether it is the response to toxic insults or the initial cellular state that determines their vulnerability is unknown. To understand individual cell responses to disease, we profiled their transcriptional signatures throughout disease development in a Drosophila model of C9orf72 (G4C2) repeat expansion (C9), the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. We identified neuronal populations specifically vulnerable or resistant to C9 expression and found an upregulation of protein homeostasis pathways in resistant neurons at baseline. Overexpression of Xbp1s, a key regulator of the unfolded protein response and a central node in the resistance network, rescues C9 toxicity. This study shows that neuronal vulnerability depends on the intrinsic transcriptional state of neurons and that leveraging resistant neurons' properties can boost resistance in vulnerable neurons.

Keywords:  ALS; C9; C9orf72; CP: Molecular biology; CP: Neuroscience; Drosophila; ERAD; FTD; Xbp1; neuronal vulnerability; protein homeostasis; unfolded protein response

DOI:  https://doi.org/10.1016/j.celrep.2025.115459
Proc Natl Acad Sci U S A. 2025 Apr 15. 122(15): e2415807122

Structure of a Gcn2 dimer in complex with the large 60S ribosomal subunit.

Helge Paternoga, Lu Xia, Lyudmila Dimitrova-Paternoga, Sihan Li, Liewei L Yan, Malte Oestereich, Sergo Kasvandik, Ankanahalli N Nanjaraj Urs, Bertrand Beckert, Tanel Tenson, Hani Zaher, Toshifumi Inada, Daniel N Wilson.

  The integrated stress response (ISR) is a central signaling network that enables eukaryotic cells to respond to a variety of different environmental stresses. Such stresses cause ribosome collisions that lead to activation of the kinase Gcn2, resulting in the phosphorylation and inactivation of eukaryotic initiation factor 2 and thereby promoting selective translation of mRNAs to restore homeostasis. Despite the importance of the ISR and intensive study over the past decades, structural insight into how Gcn2 interacts with ribosomal particles has been lacking. Using ex vivo affinity purification approaches, we have obtained a cryoelectron microscopy structure of a yeast Gcn2 dimer in complex with the ribosomal 60S subunit. The Gcn2 dimer is formed by dimerization of the histidine tRNA synthetase-like domains, which establish extensive interactions with the stalk-base and sarcin-ricin loop of the 60S subunit. The C-terminal domain of Gcn2 is also dimerized and occupies the A- and P-site tRNA binding sites at the peptidyl-transferase center of the 60S subunit. Complementary functional studies indicate that binding of Gcn2 to the 60S subunit does not require the coactivators Gcn1 or Gcn20, nor does it lead to phosphorylation of eIF2α. Instead, upon stress, we observe a shift of Gcn2 from the 60S subunit into the colliding ribosome fraction, suggesting that the Gcn2-60S complex represents an inactive stand-by state to enable a rapid redistribution to collided ribosomes, and thereby facilitating a quick and efficient response to stress.

Keywords:  Gcn2; eIF2; integrated stress response; ribosome; translation

DOI:  https://doi.org/10.1073/pnas.2415807122
Cells. 2025 Mar 29. pii: 509. [Epub ahead of print]14(7):

Hsp70: A Multifunctional Chaperone in Maintaining Proteostasis and Its Implications in Human Disease.

Manish Kumar Singh, Sunhee Han, Songhyun Ju, Jyotsna S Ranbhise, Joohun Ha, Seung Geun Yeo, Sung Soo Kim, Insug Kang.

  Hsp70, a 70 kDa molecular chaperone, plays a crucial role in maintaining protein homeostasis. It interacts with the DnaJ family of co-chaperones to modulate the functions of client proteins involved in various cellular processes, including transmembrane transport, extracellular vesicle trafficking, complex formation, and proteasomal degradation. Its presence in multiple cellular organelles enables it to mediate stress responses, apoptosis, and inflammation, highlighting its significance in disease progression. Initially recognized for its essential roles in protein folding, disaggregation, and degradation, later studies have demonstrated its involvement in several human diseases. Notably, Hsp70 is upregulated in multiple cancers, where it promotes tumor proliferation and serves as a tumor immunogen. Additionally, epichaperome networks stabilize protein-protein interactions in large and long-lived assemblies, contributing to both cancer progression and neurodegeneration. However, extracellular Hsp70 (eHsp70) in the tumor microenvironment can activate immune cells, such as natural killer (NK) cells, suggesting its potential in immunotherapeutic interventions, including CAR T-cell therapy. Given its multifaceted roles in cellular physiology and pathology, Hsp70 holds immense potential as both a biomarker and a therapeutic target across multiple human diseases. This review highlights the structural and functional importance of Hsp70, explores its role in disease pathogenesis, and discusses its potential in diagnostic and therapeutic applications.

Keywords:  Hsp70; apoptosis; cancer; epichaperome; inflammation; protein-folding

DOI:  https://doi.org/10.3390/cells14070509
bioRxiv. 2025 Mar 27. pii: 2025.03.27.645657. [Epub ahead of print]

Alternative start codon selection shapes mitochondrial function during evolution, homeostasis, and disease.

Jimmy Ly, Yi Fei Tao, Matteo Di Bernardo, Ekaterina Khalizeva, Christopher J Giuliano, Sebastian Lourido, Mark D Fleming, Iain M Cheeseman.

Mitochondrial endosymbiosis was a pivotal event in eukaryotic evolution, requiring core proteins to adapt to function both within the mitochondria and in the host cell. Here, we systematically profile the localization of protein isoforms generated by alternate start codon selection during translation. We identify hundreds of pairs of differentially-localized protein isoforms, many of which affect mitochondrial targeting and are essential for mitochondrial function. The emergence of dual-localized mitochondrial protein isoforms coincides with mitochondrial acquisition during early eukaryotic evolution. We further reveal that eukaryotes use diverse mechanisms-such as leaky ribosome scanning, alternative transcription, and paralog duplication-to maintain the production of dual-localized isoforms. Finally, we identify multiple isoforms that are specifically dysregulated by rare disease patient mutations and demonstrate how these mutations can help explain unique clinical presentations. Together, our findings illuminate the evolutionary and pathological relevance of alternative translation initiation, offering new insights into the molecular underpinnings of mitochondrial biology.

DOI: https://doi.org/10.1101/2025.03.27.645657
EMBO J. 2025 Apr 07.

The selenocysteine-containing protein SELENOT maintains dopamine signaling in the midbrain to protect mice from hyperactivity disorder.

Qing Guo, Zhao-Feng Li, Dong-Yan Hu, Pei-Jun Li, Kai-Nian Wu, Hui-Hui Fan, Jie Deng, Hong-Mei Wu, Xiong Zhang, Jian-Hong Zhu.

  Dopaminergic neuron dysfunction has been implicated in multiple neurological and psychiatric disorders. SELENOT is a selenocysteine-containing protein of the ER membrane with antioxidant and neuroprotective activities, but its pathophysiological role in dopaminergic neurons remains unclear. In this study we show that male mice with SELENOT-deficient dopaminergic neurons exhibit attention deficit/hyperactivity disorder (ADHD)-like symptoms, including hyperlocomotion, recognition memory deficits, repetitive movements, and impulsivity. Dopamine metabolism, extrasynaptic dopamine levels, spontaneous excitatory postsynaptic currents in the striatum, and electroencephalography theta power are all enhanced in these animals, while dopaminergic neurons in the substantia nigra are slightly reduced but with normal firing and cellular stress levels. Our results also indicate that the expression of dopamine transporter (DAT) is significantly reduced in the absence of SELENOT. Both the development of ADHD-like phenotypes and DAT downregulation are also observed when SELENOT is absent from the whole brain, but not when its conditional knockout is restricted to astrocytes. Mechanistically, we show that SELENOT downregulates DAT expression via interaction with SERCA2 of the ER -but not with IP3R or RYR- to regulate the ER-cytosol Ca2+ flux and, subsequently, the activity of transcription factor NURR1 and the expression levels of DAT. Treatment with amphetamine or methylphenidate, which are commonly used to treat ADHD, reverses the hyperactivity observed in mice with SELENOT-deficient dopaminergic neurons. Our study demonstrates that SELENOT in mouse dopaminergic neurons maintains proper dopamine signaling in the midbrain against the development of ADHD-like behaviors.

Keywords:  Attention Deficit/Hyperactivity Disorder; Calcium; Dopamine Transporter; Dopaminergic Neurons; SELENOT

DOI:  https://doi.org/10.1038/s44318-025-00430-3