bims-cemest 2025-02-16 papers

bims-cemest

Biomed News

on Cell metabolism and stress

Issue of 2025–02–16
twenty papers selected by
Jessica Rosarda, Uniformed Services University

Transcriptomics Unveil Canonical and Non-Canonical Heat Shock-Induced Pathways in Human Cell Lines.
Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.
The fundamental role of mitochondria-endoplasmic reticulum contacts in ageing and declining healthspan.
AA147 Alleviates Symptoms in a Mouse Model of Multiple Sclerosis by Reducing Oligodendrocyte Loss.
Dysregulation of Retinal and Photoreceptor Structural Integrity Genes in ATF6-/- Retinal Organoids.
Transcriptomic Analysis Uncovers an Unfolded Protein Response in ADNP Syndrome.
Initiation of ERAD by the bifunctional complex of Mnl1/Htm1 mannosidase and protein disulfide isomerase.
Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19.
Redox imbalance and hypoxia-inducible factors: a multifaceted crosstalk.
Maturation and detoxification of synphilin-1 inclusion bodies regulated by sphingolipids.
Orchestration of SARS-CoV-2 Nsp4 and host-cell ESCRT proteins induces morphological changes of the endoplasmic reticulum.
The Supramolecular Architecture of Mitochondrial Complex I in the Rat Brain Is Altered by Alzheimer's-Like Cerebral Amyloidosis.
Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.
Development of a highly sensitive method to detect translational infidelity.
More than Just Protein Folding: The Epichaperome, Mastermind of the Cancer Cell.
Regulation of the structural dynamics, aggregation, and pathogenicity of polyQ-expanded Huntingtin by osmolytes.
FKBP51 functions in regulation of circadian rhythm and Alzheimer's disease.
ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress.
Dynamic global acetylation remodeling during the yeast heat shock response.
A microscopy-based screen identifies cellular kinases modulating mitochondrial translation.

Int J Mol Sci. 2025 Jan 26. pii: 1057. [Epub ahead of print]26(3):

Transcriptomics Unveil Canonical and Non-Canonical Heat Shock-Induced Pathways in Human Cell Lines.

Andrew Reinschmidt, Luis Solano, Yonny Chavez, William Drew Hulsy, Nikolas Nikolaidis.

  The cellular stress response (CSR) is a conserved mechanism that protects cells from -environmental and physiological stressors. The heat shock response (HSR), a critical component of the CSR, utilizes molecular chaperones to mitigate proteotoxic stress caused by elevated temperatures. We hypothesized that while the canonical HSR pathways are conserved across cell types, specific cell lines may exhibit unique transcriptional responses to heat shock. To test this, we compared the transcriptomic responses of HEK293, HepG2, and HeLa cells under control conditions immediately following heat shock and after an 8-h recovery period. RNA sequencing revealed the conserved activation of canonical HSR pathways, including the unfolded protein response, alongside the -enrichment of the non-canonical "Receptor Ligand Activity" pathway across all cell lines. Cell-line-specific variations were observed, with HepG2 cells exhibiting significantly higher ex-pression levels of certain genes compared to other cell lines under stress conditions, as well as greater fold changes in gene expression relative to its control conditions. Validation by qPCR confirmed the activation of key genes within the "Receptor Ligand Activity" pathway across time points. These findings provide insights into conserved and context-specific aspects of the HSR, contributing to a more comprehensive understanding of stress response mechanisms across mammalian cells.

Keywords:  RNA sequencing; cellular stress response (CSR); heat shock response (HSR)

DOI:  https://doi.org/10.3390/ijms26031057
Elife. 2025 Feb 12. pii: RP100541. [Epub ahead of print]13

Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.

Kelsey R Baron, Samantha Oviedo, Sophia Krasny, Mashiat Zaman, Rama Aldakhlallah, Prerona Bora, Prakhyat Mathur, Gerald Pfeffer, Michael J Bollong, Timothy E Shutt, Danielle A Grotjahn, R Luke Wiseman.

  Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions, including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Keywords:  cell biology; human; integrated stress response; mitochondrial fragmentation; mitochondrial morphology; mouse; stress signaling

DOI:  https://doi.org/10.7554/eLife.100541
Open Biol. 2025 Feb;15(2): 240287

The fundamental role of mitochondria-endoplasmic reticulum contacts in ageing and declining healthspan.

Richard M Monaghan.

  This open question research article highlights mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), which have emerged as crucial cellular structures that challenge our traditional understanding of organelle function. This review highlights the critical importance of MAMs as a frontier in cell biology with far-reaching implications for health, disease and ageing. MAMs serve as dynamic communication hubs between the ER and mitochondria, orchestrating essential processes such as calcium signalling, lipid metabolism and cellular stress responses. Recent research has implicated MAM dysfunction in a wide array of conditions, including neurodegenerative diseases, metabolic disorders, cardiovascular diseases and cancer. The significant lack of biological knowledge behind MAM function emphasizes the need to study these enigmatic subcellular sites in greater detail. Key open questions include the mechanisms controlling MAM formation and disassembly, the full complement of MAM-associated proteins and how MAMs contribute to cellular decision-making and ageing processes. Advancing our understanding of MAMs through interdisciplinary approaches and cutting-edge technologies promises to reveal new insights into fundamental cellular signalling pathways and potentially lead to innovative therapeutic strategies for a range of diseases. As such, MAM research represents a critical open question in biology with the potential to transform our understanding of cellular life and human health.

Keywords:  ageing; endoplasmic reticulum; healthspan; membrane contact sites; metabolism; mitochondria

DOI:  https://doi.org/10.1098/rsob.240287
Glia. 2025 Feb 10.

AA147 Alleviates Symptoms in a Mouse Model of Multiple Sclerosis by Reducing Oligodendrocyte Loss.

Metin Aksu, Kevin Kaschke, Joseph R Podojil, MingYi Chiang, Ian Steckler, Kody Bruce, Andrew C Cogswell, Gwen Schulz, Jeffery W Kelly, R Luke Wiseman, Stephen D Miller, Brian Popko, Yanan Chen.

  Inflammation-induced oligodendrocyte death and CNS demyelination are key features of multiple sclerosis (MS). Inflammation-triggered endoplasmic reticulum (ER) stress and oxidative stress promote tissue damage in MS and in its preclinical animal model, experimental autoimmune encephalitis (EAE). Compound AA147 is a potent activator of the ATF6 signaling arm of the unfolded protein response (UPR) that can also induce antioxidant signaling through activation of the NRF2 pathway in neuronal cells. Previous work showed that AA147 protects multiple tissues against ischemia/reperfusion damage through ATF6 and/or NRF2 activation; however, its therapeutic potential in neuroinflammatory disorders remains unexplored. Here, we demonstrate that AA147 ameliorated the clinical symptoms of EAE and reduced ER stress, oligodendrocyte loss, and demyelination. Additionally, AA147 suppressed T cells in the CNS without altering the peripheral immune response. Importantly, AA147 significantly increased the expressions of Grp78, an ATF6 target gene, in oligodendrocytes, while enhancing levels of Grp78 as well as Ho-1, an NRF2 target gene, in microglia. In cultured oligodendrocytes, AA147 promoted nuclear translocation of ATF6, but not NRF2. Intriguingly, AA147 altered the microglia activation profile, possibly by triggering the NRF2 pathway. AA147 was not therapeutically beneficial during the acute EAE stage in mice lacking ATF6 in oligodendrocytes, indicating that protection primarily involves ATF6 activation in these cells. Overall, our results suggest AA147 as a potential therapeutic opportunity for MS by promoting oligodendrocyte survival and regulating microglia status through distinct mechanisms.

Keywords:  AA147; ATF6; EAE; UPR; oligodendrocyte

DOI:  https://doi.org/10.1002/glia.70001
Adv Exp Med Biol. 2025 ;1468 401-407

Dysregulation of Retinal and Photoreceptor Structural Integrity Genes in ATF6-/- Retinal Organoids.

Allyssa Bradley, Masako Le, Soyeon Park, Heike Kroeger, R Luke Wiseman, Eun-Jin Lee, Jonathan H Lin.

  ATF6 is a key regulator of the unfolded protein response (UPR) pathway that maintains cellular homeostasis during ER stress. In people, loss of ATF6 function causes cone dysfunction, manifesting as achromatopsia (ACHM). Previously, we generated ACHM retinal organoids (ROs) from patient induced pluripotent stem cells (iPSCs) carrying mutant ATF6 variants and gene-edited ATF6-knockout (KO) human embryonic stem cells (hESCs). ACHM and ATF6-KO ROs both showed severe stunting of cone inner and outer segments. RNA-Seq analysis of ACHM 290-day-old ROs showed downregulated cone gene expression and dysregulated mitochondria and ER stress gene expression. Here, we analyzed RNA-Seq analysis of 203-day-old ATF6-KO ROs. In younger ROs, we found dysregulation of genes involved in retinal and photoreceptor structural integrity, including CRB1, EGFLAM, and VTN. In addition, we found dysregulation of ATF6 and UPR-regulated transcriptional signatures. Dysregulation of retinal and photoreceptor structural integrity genes may underlie the observed stunting of cone inner/outer segments in ATF6-achromatopsia patients.

Keywords:  ATF6; Achromatopsia; Brush border (inner segment/outer segment); Cones; Outer limiting membrane (external limiting membrane); Photoreceptor structure; Retinal organoid; UPR

DOI:  https://doi.org/10.1007/978-3-031-76550-6_66
Mol Cell Biol. 2025 Feb 14. 1-11

Transcriptomic Analysis Uncovers an Unfolded Protein Response in ADNP Syndrome.

Anna Bieluszewska, Phillip Wulfridge, Kuo-Chen Fang, Yan Hong, Tomoyo Sawada, Jennifer Erwin, Hongjun Song, Guo-Li Ming, Kavitha Sarma.

  Chromatin regulators are frequently mutated in autism spectrum disorders, but in most cases how they cause disease is unclear. Mutations in the activity dependent neuroprotective protein (ADNP) causes ADNP syndrome, which is characterized by intellectual deficiency and developmental delays. To identify mechanisms that contribute to ADNP syndrome, we used induced pluripotent stem cells derived from ADNP syndrome patients as a model to test the effects of syndromic ADNP mutations on gene expression and neurodifferentiation. We found that some ADNP mutations result in truncated ADNP proteins, which displayed aberrant subcellular localization. Gene expression analyses revealed widespread transcriptional deregulation in all tested mutants. Interestingly, mutants that show presence of ADNP fragments show ER stress as evidenced by activation of the unfolded protein response (UPR). The mutants showing the greatest UPR pathway activation associated with the most severe neurodifferentiation and survival defects. Our results reveal the potential to explore UPR activation as a new biomarker for ADNP syndrome severity and perhaps also in other ASDs where mutations result in presence of truncated proteins.

Keywords:  ADNP syndrome; neurodifferentiation; patient-derived induced pluripotent stem cells; transcriptomics; unfolded protein response

DOI:  https://doi.org/10.1080/10985549.2025.2463892
Nat Struct Mol Biol. 2025 Feb 10.

Initiation of ERAD by the bifunctional complex of Mnl1/Htm1 mannosidase and protein disulfide isomerase.

Dan Zhao, Xudong Wu, Tom A Rapoport.

Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). In Saccharomyces cerevisiae, the glycan of these proteins is trimmed by the luminal mannosidase Mnl1 (Htm1) to generate a degradation signal. Interestingly, Mnl1 is associated with protein disulfide isomerase (Pdi1). Here we used cryo-electron microscopy, biochemical and in vivo experiments to elucidate how this complex initiates ERAD. The Mnl1-Pdi1 complex first demannosylates misfolded, globular proteins that are recognized through the C-terminal domain (CTD) of Mnl1; Pdi1 causes the CTD to ignore completely unfolded polypeptides. The disulfides of these globular proteins are then reduced by the Pdi1 component of the complex. Mnl1 blocks the canonical oxidative function of Pdi1, allowing it to function as a disulfide reductase in ERAD. The generated unfolded polypeptides can then be translocated across the membrane into the cytosol.

DOI: https://doi.org/10.1038/s41594-025-01491-y
Cell Mol Life Sci. 2025 Feb 13. 82(1): 76

Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19.

Flavien Picard, Takashi Nonaka, Edwige Belotti, Alexis Osseni, Elisabeth Errazuriz-Cerda, Coline Jost-Mousseau, Emilien Bernard, Agnès Conjard-Duplany, Delphine Bohl, Masato Hasegawa, Cédric Raoul, Thierry Galli, Laurent Schaeffer, Pascal Leblanc.

  Proteinopathies, such as amyotrophic lateral sclerosis (ALS), are marked by the accumulation of misfolded proteins that disrupt cellular processes. Eukaryotic cells have developed protein quality control systems to eliminate these aberrant proteins, but these systems often fail to differentiate between normal and misfolded proteins. In ALS, pathological inclusions primarily composed of misfolded TDP-43 are a hallmark of the disease. Recently, a novel unconventional secretion process called misfolding-associated protein secretion (MAPS) has been discovered to selectively export misfolded proteins. USP19, an Endoplasmic Reticulum-associated ubiquitin peptidase, plays a crucial role in this process. In this study, we investigated the impact of ER-anchored USP19 on the secretion of misfolded TDP-43. Here we found that USP19 overexpression significantly promotes the secretion of soluble and aggregated misfolded TDP-43, requiring both ER anchoring and ubiquitin peptidase activity. Characterization of the cellular and molecular mechanisms involved in this process highlighted the importance of early autophagosomal and late endosomal/amphisomal compartments, while lysosomes did not play a key role. By using dominant-negative mutants and small interfering RNAs, we identified that USP19-mediated secretion of misfolded TDP-43 is modulated by key factors involved in cellular trafficking and secretion pathways, such as ATG7, the ESCRT-O HGS/HRS, the Rab GTPases RAB11A, RAB8A, and RAB27A, and the v-SNARE VAMP7. We also confirmed the crucial role of the DNAJC5/CSPα cochaperone. Overall, this study provides new insights into how cells manage the secretion of misfolded TDP-43 proteins and potentially opens new avenues for therapeutic interventions in ALS and related disorders.

Keywords:  ALS; Aggregates; Autophagosomes; Autophagy; Endosomes; Misfolding; Release/secretion; TDP-43; USP19; Ubiquitin peptidase

DOI:  https://doi.org/10.1007/s00018-025-05589-w
FEBS J. 2025 Feb 11.

Redox imbalance and hypoxia-inducible factors: a multifaceted crosstalk.

Ravi, Jogender Singh.

  Redox homeostasis, the delicate balance between oxidative and reductive processes, is crucial for cellular function and overall organismal health. At the molecular level, cells need to maintain a fine balance between the levels of reactive oxygen species (ROS) and reducing equivalents such as glutathione and nicotinamide adenine dinucleotide phosphate. The perturbation of redox homeostasis due to excessive ROS production leads to oxidative stress that can damage lipids, proteins, and nucleic acids. Conversely, an overly reduced cellular environment due to overabundant reducing equivalents results in reductive stress, which also interferes with important cellular signaling and physiological processes. Disrupted redox homeostasis is linked to various pathological conditions, including neurodegenerative diseases, inflammatory diseases, cancer, and cardiovascular diseases. Cells employ diverse mechanisms to manage redox imbalance. The hypoxia response pathway, mediated by hypoxia-inducible factors and responsible for sensing and defending against low oxygen levels, plays a vital role in maintaining redox homeostasis. In this review, we highlight the complex and multifaceted crosstalk between hypoxia-inducible factors and redox homeostasis and discuss avenues for future research. Understanding the molecular mechanisms that link hypoxia-inducible factors to oxidative and reductive stresses is essential for comprehending several pathological conditions associated with hypoxia and redox imbalance.

Keywords:  HIF‐1; antioxidants; oxidative stress; reactive oxygen species; reductive stress

DOI:  https://doi.org/10.1111/febs.70013
Elife. 2025 Feb 10. pii: RP92180. [Epub ahead of print]12

Maturation and detoxification of synphilin-1 inclusion bodies regulated by sphingolipids.

Xiuling Cao, Xiang Wu, Lei Zhao, Ju Zheng, Xuejiao Jin, Xinxin Hao, Joris Winderickx, Shenkui Liu, Lihua Chen, Beidong Liu.

  Due to proteostasis stress induced by aging or disease, misfolded proteins can form toxic intermediate species of aggregates and eventually mature into less toxic inclusion bodies (IBs). Here, using a yeast imaging-based screen, we identified 84 potential synphilin-1 (SY1) IB regulators and isolated the conserved sphingolipid metabolic components in the most enriched groups. Furthermore, we show that, in both yeast cells and mammalian cells, SY1 IBs are associated with mitochondria. Pharmacological inhibition of the sphingolipid metabolism pathway or knockout of its key genes results in a delayed IB maturation and increased SY1 cytotoxicity. We postulate that SY1 IB matures by association with the mitochondrion membrane, and that sphingolipids stimulate the maturation via their membrane-modulating function and thereby protecting cells from SY1 cytotoxicity. Our findings identify a conserved cellular component essential for IB maturation and suggest a mechanism by which cells may detoxify the pathogenic protein aggregates through forming mitochondrion-associated IBs.

Keywords:  S. cerevisiae; genetics; genomics; imaging-based screen; inclusion bodies; mammalian cells; mitochondrion; sphingolipids; synphilin-1

DOI:  https://doi.org/10.7554/eLife.92180
Mol Biol Cell. 2025 Feb 12. mbcE24120542

Orchestration of SARS-CoV-2 Nsp4 and host-cell ESCRT proteins induces morphological changes of the endoplasmic reticulum.

Allison Kifer, Franciso Pina, Nicholas Codallos, Anita Hermann, Lauren Ziegler, Maho Niwa.

Upon entry into the host cell, the non-structural proteins 3, 4, and 6 (Nsp3, Nsp 4, and Nsp6) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate the formation of double-membrane vesicles (DMVs) through extensive rearrangement of the host cell endoplasmic reticulum (ER) to replicate the viral genome and translate viral proteins. To dissect the functional roles of each Nsp and the molecular mechanisms underlying the ER changes, we exploited both yeast S. cerevisiae and human cell experimental systems. Our results demonstrate that Nsp4 alone is sufficient to induce ER structural changes. Nsp4 expression led to robust activation of both the unfolded protein response (UPR) and the ER surveillance (ERSU) cell cycle checkpoint, resulting in cortical ER inheritance block and septin ring mislocalization. Interestingly, these ER morphological changes occurred independently of the canonical UPR and ERSU components but were mediated by the endosomal sorting complex for transport (ESCRT) proteins Vps4 and Vps24 in yeast. Similarly, ER structural changes occurred in human cells upon Nsp4 expression, providing a basis for a minimal experimental system for testing the involvement of human ESCRT proteins and ultimately advancing our understanding of DMV formation.

DOI: https://doi.org/10.1091/mbc.E24-12-0542
J Neurochem. 2025 Feb;169(2): e70017

The Supramolecular Architecture of Mitochondrial Complex I in the Rat Brain Is Altered by Alzheimer's-Like Cerebral Amyloidosis.

Gisela V Novack, Pablo Galeano, Lucas A Defelipe, Lorenzo Campanelli, Karen S Campuzano, Cecilia Rotondaro, Eduardo M Castaño, Sonia Do Carmo, A Claudio Cuello, María M García-Alai, Laura Morelli.

  Mitochondrial respiratory complexes are organized into supercomplexes (SC) to regulate electron flow and mitigate oxidative stress. Alterations in SC organization in the brain may affect energy expenditure, oxidative stress, and neuronal survival. In this report, we investigated the amount, activity and organization of mitochondrial complex I (CI) in the hippocampus of 12-month-old McGill-R-Thy1-APP transgenic (Tg) rats, an animal model of Alzheimer's-like cerebral amyloidosis. By means of BN-PAGE, we found that the organization of SC did not differ between genotypes, but a lower abundance of SC was detected in Tg compared to wild-type (WT) rats. Using a more sensitive technique (2-D electrophoresis followed by Western blot), higher levels of free CI and a decrease in the relative abundance of assembled CI in SC (I-III2 and I-III2-IV) were observed in Tg rats. In-gel activity assays showed that the total activity of CI (CI + SC-CI) is lower in Tg compared to WT, while Tg samples show a significant decrease in SC-CI-associated activity. This alteration in CI assembly was associated with nitro-oxidative stress and changes in mitochondrial fusion-fission parameters. To assess CI composition, we applied LC-MS/MS to the isolated CI from BN-PAGE and found that 11 of 45 subunits described in mammals were found to be less abundant in Tg. We examined the levels of the nuclear-derived NDUFA9 subunit, which is critical for CI assembly, and found higher levels in the cytoplasmic fraction and lower levels in the mitochondrial fraction in Tg, suggesting that brain amyloidosis affects the import of CI subunits from the cytosol to the mitochondria, destabilizing the SC. This is the first report to characterize the types, abundance and activity of SC in the hippocampus of an animal model of cerebral amyloidosis, providing additional experimental evidence for the molecular mechanisms underlying the brain bioenergetic deficit characteristic of Alzheimer's disease.

Keywords:  OXPHOS; mitochondria; native electrophoresis; neurodegeneration; nitroxidative stress; supercomplexes

DOI:  https://doi.org/10.1111/jnc.70017
Nat Commun. 2025 Feb 10. 16(1): 1501

Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.

Mariana Joaquim, Selver Altin, Maria-Bianca Bulimaga, Tânia Simões, Hendrik Nolte, Verian Bader, Camilla Aurora Franchino, Solenn Plouzennec, Karolina Szczepanowska, Elena Marchesan, Kay Hofmann, Marcus Krüger, Elena Ziviani, Aleksandra Trifunovic, Arnaud Chevrollier, Konstanze F Winklhofer, Elisa Motori, Margarete Odenthal, Mafalda Escobar-Henriques.

Mitochondria are essential organelles and their functional state dictates cellular proteostasis. However, little is known about the molecular gatekeepers involved, especially in absence of external stress. Here we identify a role of MFN2 in quality control independent of its function in organellar shape remodeling. MFN2 ablation alters the cellular proteome, marked for example by decreased levels of the import machinery and accumulation of the kinase PINK1. Moreover, MFN2 interacts with the proteasome and cytosolic chaperones, thereby preventing aggregation of newly translated proteins. Similarly to MFN2-KO cells, patient fibroblasts with MFN2-disease variants recapitulate excessive protein aggregation defects. Restoring MFN2 levels re-establishes proteostasis in MFN2-KO cells and rescues fusion defects of MFN1-KO cells. In contrast, MFN1 loss or mitochondrial shape alterations do not alter protein aggregation, consistent with a fusion-independent role of MFN2 in cellular homeostasis. In sum, our findings open new possibilities for therapeutic strategies by modulation of MFN2 levels.

DOI: https://doi.org/10.1038/s41467-025-56673-5
Biol Methods Protoc. 2025 ;10(1): bpaf008

Development of a highly sensitive method to detect translational infidelity.

Max Hartmann, Lisa Neher, Benjamin Grupp, Zhouli Cao, Chloe Chiew, Sebastian Iben.

  Protein homeostasis (proteostasis) is the balance of protein synthesis, protein maintenance, and degradation. Loss of proteostasis contributes to the aging process and characterizes neurodegenerative diseases. It is well established that the processes of protein maintenance and degradation are declining with aging; however, the contribution of a declining quality of protein synthesis to the loss of proteostasis is less well understood. In fact, protein synthesis at the ribosome is an error-prone process and challenges the cell with misfolded proteins. Here, we present the development of a highly sensitive and reproducible reporter assay for the detection of translational errors and the measurement of translational fidelity. Using Nano-luciferase, an enzyme 3 times smaller and 50 times more sensitive than the hitherto used Firefly-luciferase, we introduced stop-codon and amino-acid exchanges that inactivate the enzyme. Erroneous re-activation of luciferase activity indicates ribosomal inaccuracy and translational infidelity. This highly sensitive and reproducible method has broad applications for studying the molecular mechanisms underlying diseases associated with defective protein synthesis and can be used for drug screening to modulate translational fidelity.

Keywords:  nanoluciferase; ribosome; translational fidelity

DOI:  https://doi.org/10.1093/biomethods/bpaf008
Cells. 2025 Jan 30. pii: 204. [Epub ahead of print]14(3):

More than Just Protein Folding: The Epichaperome, Mastermind of the Cancer Cell.

Haneef Ahmed Amissah, Maxwell Hubert Antwi, Tawfeek Ahmed Amissah, Stephanie E Combs, Maxim Shevtsov.

  The epichaperome, a dynamic and integrated network of chaperone proteins, extends its roles beyond basic protein folding to protein stabilization and intracellular signal transduction to orchestrating a multitude of cellular processes critical for tumor survival. In this review, we explore the multifaceted roles of the epichaperome, delving into its diverse cellular locations, factors that modulate its formation and function, its liquid-liquid phase separation, and the key signaling and crosstalk pathways it regulates, including cellular metabolism and intracellular signal transduction. We further highlight techniques for isolating and identifying epichaperome networks, pitfalls, and opportunities. Further, we review the profound implications of the epichaperome for cancer treatment and therapy design, underscoring the need for strategic engineering that hinges on a comprehensive insight into the comprehensive structure and workings of the epichaperome across the heterogeneous cell subpopulations in the tumor milieu. By presenting a holistic view of the epichaperome's functions and mechanisms, we aim to underscore its potential as a key target for novel anti-cancer strategies, revealing that the epichaperome is not merely a piece of protein folding machinery but a mastermind that facilitates the malignant phenotype.

Keywords:  cancer; epichaperome; liquid–liquid phase separation; signaling pathways

DOI:  https://doi.org/10.3390/cells14030204
Prog Mol Biol Transl Sci. 2025 ;pii: S1877-1173(24)00174-1. [Epub ahead of print]211 113-143

Regulation of the structural dynamics, aggregation, and pathogenicity of polyQ-expanded Huntingtin by osmolytes.

Alice Y Liu, Amala Mathew, Christopher Karim, Pierre Eshak, Kuang Yu Chen.

  Huntington Disease is an autosomal dominant neurodegenerative disease caused by expansion of the polymorphic trinucleotide CAG repeat of the HTT gene to code for an expanded glutamine track of the mutant Huntingtin protein (mHTT). Like other neurodegenerative diseases, symptomatic presentation of Huntington Disease is age-dependent or age-related. This age-dependent manifestation of an autosomal dominant disease trait underscores important and possibly priming role of age-related changes in cellular physiology that are conducive to disease presentation. Herein, we present studies on the effects of osmolytes on mHTT structuring and aggregation, vis-a-vis pathogenicity. We show that stabilizing polyol osmolytes, by their generic activity in promoting protein structuring and compaction, drive aggregation of the disordered mHTT protein and simultaneously inhibit their binding to and sequestration of key transcription factors for improved homeostasis and cell survival under stress. These and related observations in the literature give strong support to the notion that lower molecular weight and structurally dynamic forms of mHTT contribute importantly to disease pathogenesis. Aging is associated with important changes in the cell environment-disease protein accumulation, reduced hydration, and macromolecular crowding as examples. These changes have significant consequences on the structuring and pathogenicity of the disordered mHTT protein. A crowded and less hydrated aging cell environment is conducive to mHTT binding to and inhibition of cell regulatory protein function on the one hand, and in promoting mHTT aggregation on the other hand, to culminate in Huntington disease presentation.

Keywords:  Aging and Hypohydration; IDP Structuring & Aggregation; Neurodegeneration; Osmolytes; PolyQ-huntingtin

DOI:  https://doi.org/10.1016/bs.pmbts.2024.08.005
Cell Stress Chaperones. 2025 Feb 09. pii: S1355-8145(25)00004-5. [Epub ahead of print]

FKBP51 functions in regulation of circadian rhythm and Alzheimer's disease.

Jill L Johnson.

  The FK506-binding protein 51 (FKBP51) is an important regulator of glucocorticoid receptor activity and an Hsp90 cochaperone. FKBP51 has previously been identified as a drug target due to its roles in stress-related disorders and pain tolerance. Two recent publications in Cell Stress and Chaperones further explore FKBP51 functions. To understand whether FKBP51 plays a role in sleep disturbances linked to stress disorders, Gebru et al. examined the role of FKBP51 in regulating the circadian rhythm. Broadening the range of Hsp90 cochaperone function, Jeanne et al. summarized the role of multiple cochaperones in Alzheimer's disease, discussing how cochaperones affect both Aβ and tau. They emphasize the role of FKBP51 in promoting tau pathogenesis and discuss the small molecule LA1011, which binds Hsp90 and competes with Hsp90-FKBP51 interaction. Further studies with LA1011 may lead to new treatments for Alzheimer's disease and will help clarify the contributions of FKBP51 to human disorders.

Keywords:  Alzheimer’s disease; FKBP51; LA1011; circadian rhythm; cochaperone

DOI:  https://doi.org/10.1016/j.cstres.2025.02.002
Nat Commun. 2025 Feb 10. 16(1): 1508

ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress.

Isshin Shiiba, Naoki Ito, Hijiri Oshio, Yuto Ishikawa, Takahiro Nagao, Hiroki Shimura, Kyu-Wan Oh, Eiki Takasaki, Fuya Yamaguchi, Ryoan Konagaya, Hisae Kadowaki, Hideki Nishitoh, Takehito Tanzawa, Shun Nagashima, Ayumu Sugiura, Yuuta Fujikawa, Keitaro Umezawa, Yasushi Tamura, Byung Il Lee, Yusuke Hirabayashi, Yasushi Okazaki, Tomohiro Sawa, Ryoko Inatome, Shigeru Yanagi.

The proximal domains of mitochondria and the endoplasmic reticulum (ER) are linked by tethering factors on each membrane, allowing the efficient transport of substances, including lipids and calcium, between them. However, little is known about the regulation and function of mitochondria-ER contacts (MERCs) dynamics under mitochondrial damage. In this study, we apply NanoBiT technology to develop the MERBiT system, which enables the measurement of reversible MERCs formation in living cells. Analysis using this system suggests that induction of mitochondrial ROS increases MERCs formation via RMDN3 (also known as PTPIP51)-VAPB tethering driven by RMDN3 phosphorylation. Disruption of this tethering caused lipid radical accumulation in mitochondria, leading to cell death. The lipid radical transfer activity of the TPR domain in RMDN3, as revealed by an in vitro liposome assay, suggests that RMDN3 transfers lipid radicals from mitochondria to the ER. Our findings suggest a potential role for MERCs in cell survival strategy by facilitating the removal of mitochondrial lipid radicals under mitochondrial damage.

DOI: https://doi.org/10.1038/s41467-025-56666-4
bioRxiv. 2025 Jan 10. pii: 2025.01.10.632339. [Epub ahead of print]

Dynamic global acetylation remodeling during the yeast heat shock response.

Rebecca E Hardman-Kavanaugh, Aaron J Storey, Tara N Stuecker, Stephanie E Hood, Gregory A Barrett-Wilt, Venkata R Krishnamurthi, Yong Wang, Stephanie D Byrum, Samuel G Mackintosh, Rick D Edmondson, Wayne P Wahls, Alan J Tackett, Jeffrey A Lewis.

All organisms experience stress and must rapidly respond to changing conditions. Thus, cells have evolved sophisticated rapid-response mechanisms such as post-translational protein modification to rapidly and reversibly modulate protein activity. One such post-translational modification is reversible lysine acetylation, where proteomic studies have identified thousands of acetylated proteins across diverse organisms. While the sheer size of the 'acetylome' is striking, the function of acetylation for the vast majority of proteins remains largely obscure. Here, we show that global acetylation plays a previously unappreciated role in the heat shock response of Saccharomyces cerevisiae. We find that dysregulated acetylation renders cells heat sensitive, and moreover, that the acetylome is globally remodeled during heat shock over time. Using quantitative acetyl-proteomics, we identified ∼400 high-confidence acetyl marks across ∼200 proteins that significantly change in acetylation when cells are shifted to elevated temperature. Proteins with significant changes in lysine acetylation during heat shock strongly overlap with genes induced or repressed by stress. Thus, we hypothesize that protein acetylation augments the heat shock response by activating induced proteins and inactivating repressed proteins. Intriguingly, we find nearly 40 proteins with at least two acetyl marks that significantly change in the opposite directions. These proteins are strongly enriched for chaperones and ribosomal proteins, suggesting that these two key processes are coordinately regulated by protein acetylation during heat shock. Moreover, we hypothesize that the same type of activating and inactivating marks that exist on histones may be a general feature of proteins regulated by acetylation. Overall, this work has identified a new layer of post-translational regulation that likely augments the classic heat shock response.

DOI: https://doi.org/10.1101/2025.01.10.632339
Cell Rep. 2025 Jan 28. pii: S2211-1247(24)01494-3. [Epub ahead of print]44(1): 115143

A microscopy-based screen identifies cellular kinases modulating mitochondrial translation.

Roya Yousefi, Luis Daniel Cruz-Zaragoza, Anusha Valpadashi, Carina Hansohn, Drishan Dahal, Ricarda Richter-Dennerlein, Silvio Rizzoli, Henning Urlaub, Peter Rehling, David Pacheu-Grau.

  Mitochondrial DNA encodes 13 subunits of the oxidative phosphorylation (OXPHOS) system, which are synthesized inside the organelle and essential for cellular energy supply. How mitochondrial gene expression is regulated and integrated into cellular physiology is little understood. Here, we perform a high-throughput screen combining fluorescent labeling of mitochondrial translation products with small interfering RNA (siRNA)-mediated knockdown to identify cellular kinases regulating translation. As proof of principle, the screen identifies known kinases that affect mitochondrial translation, and it also reveals several kinases not yet linked to this process. Among the latter, we focus on the primarily cytosolic kinase, fructosamine 3 kinase (FN3K), which localizes partially to the mitochondria to support translation. FN3K interacts with the mitochondrial ribosome and modulates its assembly, thereby affecting translation. Overall, our work provides a reliable approach to identify protein functions for mitochondrial gene expression in a high-throughput manner.

Keywords:  CP: Metabolism; CP: Molecular biology; cellular kinases; click chemistry; mito-FUNCAT; mitochondrial translation; siRNA library

DOI:  https://doi.org/10.1016/j.celrep.2024.115143