bims-cemest 2025-08-17 papers

bims-cemest

Biomed News

on Cell metabolism and stress

Issue of 2025–08–17
eighteen papers selected by
Jessica Rosarda, Uniformed Services University

A multichaperone condensate enhances protein folding in the endoplasmic reticulum.
Secreted Protein Production is Improved by Controlling Endoplasmic Reticulum Stress Associated Protein Degradation.
Principles of cotranslational mitochondrial protein import.
RNA triggers chronic stress during neuronal aging.
Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy.
The role of lysine acetylation in metabolic sensing and proteostasis.
Global profiling of N-terminal cysteine-dependent degradation mechanisms.
Defective lipid droplet biogenesis exacerbates oleic acid-induced cellular homeostasis disruption and ferroptosis in mouse cardiac endothelial cells.
Mitochondrial dysfunction and lipid dysregulation in yeast lacking phosphatidylserine.
GCN2 regulates paclitaxel-induced neuropathic pain.
Oxidative and Glycolytic Metabolism: Their Reciprocal Regulation and Dysregulation in Cancer.
Co-translational ribosome pairing enables native assembly of misfolding-prone subunits.
Targeted Protein Acetylation Through Chemically Induced Proximity.
Protein diversity in store-operated calcium entry components and their related variants.
TCDD Induces Hepatocytes Lipid Deposition via Downregulation of MFN2.
SCOPE: Revealing Hidden Mechanisms in Phenotypic Screens Through Target and Pathway Enrichment.
Small-Molecule-Induced Protein Polymerization: Mechanisms and Therapeutic Implications.
AMPylation regulates 5'-3' exonuclease PLD3 processing.

Nat Cell Biol. 2025 Aug 11.

A multichaperone condensate enhances protein folding in the endoplasmic reticulum.

Anna Leder, Guillaume Mas, Viktória Szentgyörgyi, Roman P Jakob, Timm Maier, Anne Spang, Sebastian Hiller.

Protein folding in the endoplasmic reticulum (ER) relies on a network of molecular chaperones that facilitates the folding and maturation of client proteins. How the ER chaperones organize in a supramolecular manner to exert their cooperativity has, however, remained unclear. Here we report the discovery of a multichaperone condensate in the ER lumen, which is formed around the chaperone PDIA6 during protein folding homeostasis. The condensates form in a Ca2+-dependent manner and we resolve the underlying mechanism at the atomic and cellular levels. The PDIA6 condensates recruit further chaperones-Hsp70 BiP, J-domain protein ERdj3, disulfide isomerase PDIA1 and Hsp90 Grp94-which constitute some of the essential components of the early folding machinery. The chaperone condensates enhance folding of proteins, such as proinsulin, and prevent protein misfolding in the ER lumen. The PDIA6-scaffolded chaperone condensates hence provide the functional basis for spatial and temporal coordination of the dynamic ER chaperone network.

DOI: https://doi.org/10.1038/s41556-025-01730-w
bioRxiv. 2025 Aug 07. pii: 2025.08.05.666879. [Epub ahead of print]

Secreted Protein Production is Improved by Controlling Endoplasmic Reticulum Stress Associated Protein Degradation.

Ryan Chauncey Splichal, Christina Chan, S Patrick Walton.

Therapeutic proteins are produced frequently by mammalian cells in large-scale bioreactors. As a result, producer cells are exposed to a chemically (nutrients, gas exchange, target protein overexpression) and physically (shear due to mixing) stressful environment, which can lead to loss of proteostasis and endoplasmic reticulum (ER) stress. In response, cells activate the unfolded protein response (UPR). The UPR includes activation of autophagy and proteasomes, both of which target unfolded/misfolded proteins for degradation. To investigate the impacts of autophagy and proteasome activity on secreted protein production in ER-stressed cells, we used HeLa and MDA-MB-231 cells transfected to express Gaussia luciferase (as a model for therapeutic protein production) and exposed to tunicamycin (TM) (to activate ER stress). As expected, TM exposure decreased protein production and secretion. Inhibiting autophagy improved secretion in stressed cells as expected. However, counterintuitively, increasing proteasomal degradation improved secretion while inhibiting proteasomal activity decreased secretion, that is proteasomal activity was directly correlated to secretion. Taken together, our results demonstrate that protein secretion can be improved through control of autophagy and proteasomal activity, providing insight into strategies for improving yield from protein production bioprocesses.

DOI: https://doi.org/10.1101/2025.08.05.666879
Cell. 2025 Aug 07. pii: S0092-8674(25)00811-6. [Epub ahead of print]

Principles of cotranslational mitochondrial protein import.

Zikun Zhu, Saurav Mallik, Taylor A Stevens, Riming Huang, Emmanuel D Levy, Shu-Ou Shan.

  Nearly all mitochondrial proteins are translated on cytosolic ribosomes. How these proteins are subsequently delivered to mitochondria remains poorly understood. Using selective ribosome profiling, we show that nearly 20% of mitochondrial proteins can be imported cotranslationally in human cells. Cotranslational import requires an N-terminal presequence on the nascent protein and contributes to localized translation at the mitochondrial surface. This pathway does not favor membrane proteins but instead prioritizes large, multi-domain, topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. In contrast to the early onset of cotranslational protein targeting to the endoplasmic reticulum (ER), the presequence on mitochondrial proteins is inhibited from initiating targeting early during translation until a large globular domain emerges from the ribosome. Our findings reveal a multi-layered protein sorting strategy that controls the timing and specificity of mitochondrial protein targeting.

Keywords:  NAC; TOM complex; cotranslational protein import; localized translation; mitochondria; mitochondrial targeting sequence; nascent polypeptide-associated complex; protein folding; protein targeting; ribosome profiling

DOI:  https://doi.org/10.1016/j.cell.2025.07.021
bioRxiv. 2025 Aug 05. pii: 2025.08.04.668575. [Epub ahead of print]

RNA triggers chronic stress during neuronal aging.

Kevin Rhine, Elle Epstein, Natasha M Carlson, Xuezhen Ge, Orel Mizrahi, Anika Kamat, Anita Hermann, William R Brothers, John Ravits, Eric J Bennett, Gülçin Pekkurnaz, Gene W Yeo.

Neurodegenerative diseases are linked with dysregulation of the integrated stress response (ISR), which coordinates cellular homeostasis during and after stress events. Cellular stress can arise from several sources, but there is significant disagreement about which stress might contribute to aging and neurodegeneration. Here, we leverage directed transdifferentiation of human fibroblasts into aged neurons to determine the source of ISR activation. We demonstrate that increased accumulation of cytoplasmic double-stranded RNA (dsRNA) activates the eIF2α kinase PKR, which in turn triggers the ISR in aged neurons and leads to sequestration of dsRNA in stress granules. Aged neurons accumulate endogenous mitochondria-derived dsRNA that directly binds to PKR. This mitochondrial dsRNA leaks through damaged mitochondrial membranes and forms cytoplasmic foci in aged neurons. Finally, we demonstrate that PKR inhibition leads to the cessation of stress, resumption of cellular translation, and restoration of RNA-binding protein expression. Together, our results identify a source of RNA stress that destabilizes aged neurons and may contribute to neurodegeneration.

DOI: https://doi.org/10.1101/2025.08.04.668575
Nat Commun. 2025 Aug 09. 16(1): 7367

Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy.

Bishal Basak, Erika L F Holzbaur.

Mutations that disrupt the clearance of damaged mitochondria via mitophagy are causative for neurological disorders including Parkinson's. Here, we identify a Mitophagic Stress Response (MitoSR) activated by mitochondrial damage in neurons and operating in parallel to canonical Pink1/Parkin-dependent mitophagy. Increasing levels of mitochondrial stress trigger a graded response that induces the concerted degradation of negative regulators of autophagy including Myotubularin-related phosphatase (MTMR)5, MTMR2 and Rubicon via the ubiquitin-proteasome pathway and selective proteolysis. MTMR5/MTMR2 inhibit autophagosome biogenesis; consistent with this, mitochondrial engulfment by autophagosomes is enhanced upon MTMR2 depletion. Rubicon inhibits lysosomal function, blocking later steps of neuronal autophagy; Rubicon depletion relieves this inhibition. Targeted depletion of both MTMR2 and Rubicon is sufficient to enhance mitophagy, promoting autophagosome biogenesis and facilitating mitophagosome-lysosome fusion. Together, these findings suggest that therapeutic activation of MitoSR to induce the selective degradation of negative regulators of autophagy may enhance mitochondrial quality control in stressed neurons.

DOI: https://doi.org/10.1038/s41467-025-62379-5
Pharmacol Ther. 2025 Aug 07. pii: S0163-7258(25)00120-2. [Epub ahead of print] 108908

The role of lysine acetylation in metabolic sensing and proteostasis.

Aziz Eftekhari, Sabir Usman, Takhar Kasumov.

  Post-translational acetylation of lysine residues is a dynamic and reversible modification that plays a pivotal role in regulating protein structure, function, and interactions. This modification is mediated by central metabolite acetyl-CoA and is tightly controlled by the opposing actions of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), including the NAD+-dependent sirtuins. As a nutrient-sensing post-translational modification (PTM), acetylation is essential for maintaining cellular homeostasis, particularly by modulating proteostasis and metabolic flexibility-the ability of cells to rewire metabolic pathways in response to fluctuating energy demands and nutrient availability. Dysregulation of acetylation has been implicated in the pathogenesis of metabolic disorders, neurodegenerative diseases, and cancer. Emerging evidence suggests that targeting acetylation-regulating enzymes with small-molecule inhibitors or activators hold promise for elucidating the role of acetylation in metabolic sensing and protein homeostasis, also known as proteostasis. This review examines the regulation of acetylation across various metabolic states, its impact on metabolic adaptability, and its intricate interplay with proteostasis mechanisms. Additionally, it highlights the role of site-specific acetylation dynamics and sirtuin biology shaping metabolic regulation, providing key insights into the mechanisms underlying metabolic disorders and their progression. Understanding the regulatory mechanisms governing acetylation-dependent metabolic sensing could facilitate the development of precision therapeutics to restore metabolic homeostasis.

Keywords:  Acetylation; Autophagy; Deacetylation; Metabolic flexibility; Proteastasis; Sirtuins

DOI:  https://doi.org/10.1016/j.pharmthera.2025.108908
Proc Natl Acad Sci U S A. 2025 Aug 19. 122(33): e2501681122

Global profiling of N-terminal cysteine-dependent degradation mechanisms.

Aizat Bekturova, Yaara Makaros, Shahar Ben-David, Itay Koren.

  Hypoxia, a condition characterized by insufficient oxygen supply, challenges cellular homeostasis and energy production, triggering adaptive responses to promote survival under these stressful conditions. One key strategy involves enzymatic oxidation of N-terminal cysteine residues coupled with proteolysis through the Cys-Arg/N-degron pathway. Despite hundreds of human proteins possessing N-terminal cysteine, very few have been identified as substrates of this pathway, and its substrate selectivity remains unclear. Moreover, the biological role of this pathway in the cellular response to hypoxia is not well defined. Here, by systematically screening protein stability using an N-terminome library, we reveal a broad set of cysteine-initiating proteins regulated by this pathway. Mutagenesis experiments further revealed the specificity of Cys-Arg/N-degron pathway, showing a preference for hydrophobic and positively charged residues following cysteine. Additionally, we uncovered full-length substrates that are regulated by this pathway during hypoxia, including IP6K1. Loss of IP6K1 impaired glucose uptake, glycolytic ATP production, and overall mitochondrial function. Consequently, IP6K1-deficient cells exhibited disrupted metabolic adaptation under hypoxic conditions and reduced survival under stress. These findings underscore the importance of the Cys-Arg/N-degron pathway in regulating metabolic responses and highlight its potential importance in hypoxia-related disorders.

Keywords:  E3 ligases; N-degron; cysteine; hypoxia; protein degradation

DOI:  https://doi.org/10.1073/pnas.2501681122
Cell Death Discov. 2025 Aug 09. 11(1): 374

Defective lipid droplet biogenesis exacerbates oleic acid-induced cellular homeostasis disruption and ferroptosis in mouse cardiac endothelial cells.

Yun-Ting Wang, Alexandra K Moura, Rui Zuo, Zhengchao Wang, Kiana Roudbari, Jenny Z Hu, Mi Wang, Pin-Lan Li, Yang Zhang, Xiang Li.

Endothelial dysfunction is a hallmark of various metabolic disorders and plays a pivotal role in the progression of cardiovascular diseases, including coronary microvascular dysfunction and myocardial ischemia. Lipid droplets (LDs) have emerged as key regulators of fatty acid metabolism in endothelial cells (ECs), but their functional role in lipotoxicity-induced EC damage in the context of coronary microvascular dysfunction remains unclear. Here, we examined the contribution of LD biogenesis to oleic acid-induced lipotoxic effects in mouse cardiac ECs (MCECs). Our findings reveal that oleic acid markedly increases LD biogenesis in MCECs via a diacylglycerol O-acyltransferase 1 (DGAT1)-dependent pathway. This process is accompanied by substantial disruptions in cellular homeostasis, including elevated endoplasmic reticulum (ER) stress, impaired mitochondrial respiration, reduced ATP production, and heightened hypoxic responses. Furthermore, oleic acid-induced lipotoxicity is primarily mediated by ferroptosis-a form of lipid peroxide-dependent, caspase-independent cell death. Notably, pharmacological inhibition or genetic knockdown of DGAT1, which diminishes LD biogenesis, exacerbates oleic acid-induced cellular stress, mitochondrial dysfunction, and ferroptosis in MCECs. These results suggest that LD biogenesis plays a protective role in mitigating lipotoxicity, preserving mitochondrial function, and preventing lipid peroxide accumulation and ferroptosis, thereby safeguarding cardiac microvascular endothelial function in the context of metabolic disorders.

DOI: https://doi.org/10.1038/s41420-025-02669-5
Mol Biol Cell. 2025 Aug 13. mbcE25030128

Mitochondrial dysfunction and lipid dysregulation in yeast lacking phosphatidylserine.

Alaumy Joshi, Zakery N Baker, Rachel A Stanfield, Dimitris T Kalafatis, David J Pagliarini, Vishal M Gohil.

Mitochondrial membrane phospholipids impact mitochondrial structure and function by influencing the assembly and activity of membrane proteins. While the specific roles of the three most abundant mitochondrial phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL), have been extensively studied, the precise function of less abundant phosphatidylserine (PS) is not yet determined. Here, we used genetic and nutritional manipulation to engineer a set of yeast mutants, including a mutant completely devoid of PS, to assess its role in mitochondrial bioenergetics and lipid homeostasis. To circumvent the confounding effect of downstream PS products, PE and PC, we exogenously supplied ethanolamine that allows their biosynthesis via an alternate pathway. Using this system, we demonstrate that PS does not impact the abundance or the assembly of mitochondrial respiratory chain complexes; however, mitochondrial respiration is impaired. PS-lacking mitochondria cannot maintain mitochondrial membrane potential and exhibit leaky membranes. A mass spectrometry-based analysis of the cellular and mitochondrial lipidomes revealed an unexpected increase in odd-chain fatty acid-containing lipids in PS-lacking cells that may impact mitochondrial bioenergetics. Our study uncovers novel roles of PS in mitochondrial membrane biogenesis and bioenergetics and provides a viable eukaryotic system to unravel the cellular functions of PS.

DOI: https://doi.org/10.1091/mbc.E25-03-0128
Br J Pharmacol. 2025 Aug 13.

GCN2 regulates paclitaxel-induced neuropathic pain.

Alexander R Mikesell, Angela R Meyer, Guadalupe García, Luke R Frietze, Cheryl L Stucky, Tao Pan, Zachary T Campbell.

   BACKGROUND AND PURPOSE: Neuropathic pain is debilitating and pervasive. Chemotherapeutic agents commonly induce chronic neuropathic pain. Paclitaxel is a prototypical example, causing painful peripheral neuropathy in a majority of patients. Paclitaxel triggers persistent changes in the excitability of sensory neurons resulting in hypersensitivity to sensory cues. The molecular mechanisms underlying paclitaxel-induced maladaptive plasticity are unclear. Here, we demonstrate a role for the Integrated Stress Response (ISR)-a key translational control mechanism-and its activating kinase, general control non-derepressible 2 kinase (GCN2), in paclitaxel-induced neuropathic pain (PINP).
EXPERIMENTAL APPROACH: We used genetic and pharmacological techniques, including sensory neuron-specific GCN2 conditional knockout mice and the selective GCN2 inhibitor GCN2-IN-7. Behavioural assays assessed mechanical and cold hypersensitivity, while primary DRG neuron cultures were used to evaluate neuronal excitability via calcium imaging and protein translation by puromycin incorporation (surface sensing of translation, SUnSET). tRNA charging and abundance were measured using MSR-seq.
KEY RESULTS: Paclitaxel robustly activated the ISR via GCN2 in mouse DRG sensory neurons, shown by increased eIF2α phosphorylation, elevated ATF4 levels and reduced global translation rates. Genetic deletion or pharmacological inhibition of GCN2 blocked paclitaxel-induced sensory neuron sensitisation and significantly attenuated mechanical and cold hypersensitivity in vivo. Mechanistically, paclitaxel reduced global tRNA charging and abundance in DRGs, providing a molecular basis for GCN2 activation.
CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that GCN2-dependent ISR activation is critical for PINP. Targeting GCN2 may represent a promising therapeutic strategy for preventing or alleviating chemotherapy-induced peripheral neuropathy, potentially improving patient quality of life and chemotherapy tolerance.

Keywords:  GCN2; ISR; PINP; eIF2

DOI:  https://doi.org/10.1111/bph.70154
Cells. 2025 Jul 30. pii: 1177. [Epub ahead of print]14(15):

Oxidative and Glycolytic Metabolism: Their Reciprocal Regulation and Dysregulation in Cancer.

Marco Cordani, Cristiano Rumio, Giulio Bontempi, Raffaele Strippoli, Fabrizio Marcucci.

  Oxidative and glycolytic metabolism produce energy in the form of ATP and produce intermediates for biomass production. Oxidative metabolism predominates under normoxic conditions and in quiescent or slowly proliferating cells. On the other hand, under hypoxic or pseudohypoxic conditions and in rapidly proliferating cells, glycolysis becomes the predominant pathway. The balance between oxidative and glycolytic metabolism is finely tuned in physiological conditions and becomes dysregulated in many pathological conditions, most notably cancer. In this article we summarize the evidence that has been gathered over the last few years on the mechanisms underlying this balance and the consequences of their dysregulation. We discuss first the non-metabolic factors (mitochondria, cell cycle, cell type, tissue type), then molecules that are at the intersection between glycolytic and oxidative metabolism and those molecules that are inherent to oxidative or glycolytic metabolism that affect the equilibrium between the two energy-producing pathways. Eventually, we discuss pharmacologic or genetic means that allow manipulating this equilibrium. As will be seen, lactic acidosis has taken center stage in this field and lactate has been shown to fuel oxidative metabolism. This suggests that if glycolytic metabolism predominates, as has often been shown in cancer, mechanisms come into work that reestablish a metabolic heterogeneity. Thus, while one pathway may be predominant over the other, it seems as if fail-safe mechanisms are at work that avoid the possibility that it becomes the only energy-producing pathway. Eventually, we discuss possible therapeutic consequences that may derive from this expanding knowledge, in particular, as regards tumor therapy.

Keywords:  cancer; glycolysis; lactic acidosis; oxidative metabolism; regulation

DOI:  https://doi.org/10.3390/cells14151177
Nat Commun. 2025 Aug 15. 16(1): 7626

Co-translational ribosome pairing enables native assembly of misfolding-prone subunits.

Florian Wruck, Jaro Schmitt, Katharina Till, Kai Fenzl, Matilde Bertolini, Frank Tippmann, Alexandros Katranidis, Bernd Bukau, Günter Kramer, Sander J Tans.

Protein complexes are pivotal to most cellular processes. Emerging evidence indicating dimer assembly by pairs of ribosomes suggests yet unknown folding mechanisms involving two nascent chains. Here, we show that co-translational ribosome pairing allows their nascent chains to 'chaperone each other', thus enabling the formation of coiled-coil homodimers from subunits that misfold individually. We developed an integrated single-molecule fluorescence and force spectroscopy approach to probe the folding and assembly of two nascent chains extending from nearby ribosomes, using the intermediate filament lamin as a model system. Ribosome proximity during early translation stages is found to be critical: when interactions between nascent chains are inhibited or delayed, they become trapped in stable misfolded states that are no longer assembly-competent. Conversely, early interactions allow the two nascent chains to nucleate native-like quaternary structures that grow in size and stability as translation advances. We conjecture that protein folding mechanisms enabled by ribosome cooperation are more broadly relevant to intermediate filaments and other protein classes.

DOI: https://doi.org/10.1038/s41467-025-61500-y
Acc Chem Res. 2025 Aug 13.

Targeted Protein Acetylation Through Chemically Induced Proximity.

Wesley W Wang, Soumya Jyoti Singha Roy, Christopher G Parker.

ConspectusProtein acetylation is a pervasive and reversible post-translational modification (PTM) that impacts various protein features including stability, localization, and interactions and regulates diverse cellular functions, including transcription, signal transduction, and metabolism. This process is orchestrated by "writer" lysine acetyltransferases (KATs) and "eraser" deacetylases (KDACs), and its dysregulation is implicated in a broad spectrum of diseases including cancer, metabolic syndromes, and immune disorders. However, dissecting the roles of specific acetylation events in live cells remains a challenge due to the lack of tools that enable precise, rapid, and reversible acetylation at defined protein sites.To begin addressing these challenges, we recently developed AceTAG (acetylation tagging), a chemically induced proximity (CIP) platform for targeted protein acetylation in live cells. AceTAG molecules are heterobifunctional ligands that recruit endogenous KATs─such as p300/CBP or PCAF/GCN5─to a tagged protein of interest, enabling selective, tunable, and dynamic acetylation. We demonstrated the utility of AceTAG across diverse proteins, including histone H3.3, p65/RelA, and p53. We further show that chemically induced acetylation of p53, including multiple hotspot p53 mutants, leads to enhanced stability and transcriptional activation, underscoring the potential of AceTAG for functional investigations and the potential for therapeutic exploration.In this Account, we provide an overview of protein acetylation and survey chemical biology technologies for its manipulation, with a focus on AceTAG. We describe the conceptual motivation of AceTAG, applications, technical considerations, and recent efforts to expand this concept to endogenous proteins. Finally, we offer a forward-looking perspective of targeted acetylation as a chemical tool to investigate the biology of this PTM, as well as its potential as a therapeutic modality.

DOI: https://doi.org/10.1021/acs.accounts.5c00326
Cell Calcium. 2025 Aug 05. pii: S0143-4160(25)00075-2. [Epub ahead of print]131 103066

Protein diversity in store-operated calcium entry components and their related variants.

Mélanie Robitaille.

  Protein diversity is a fundamental biological process that enhances the functional complexity of cellular signaling pathways. This diversity arises through multiple molecular mechanisms such as gene duplication, alternative splicing, and alternative translation initiation, which together expand the proteome landscape. Calcium signaling showcases this diversity, with several channels, pumps, and regulatory proteins expressed as multiple isoforms and variants. Within the store-operated calcium entry pathway, protein diversity is evident in the existence of distinct paralogs of ORAI channels and STIM proteins. The additional presence of numerous isoforms and variants of ORAI and STIM shapes the store-operated calcium entry pathway, providing flexibility to cellular calcium regulation in various contexts. Deciphering how protein diversity modulates store-operated calcium entry function is essential for advancing our understanding of calcium signaling in both health and disease.

Keywords:  Calcium; ORAI; SOCE; STIM; Store-operated calcium entry

DOI:  https://doi.org/10.1016/j.ceca.2025.103066
J Appl Toxicol. 2025 Aug 11.

TCDD Induces Hepatocytes Lipid Deposition via Downregulation of MFN2.

Changming Xing, Yongjun Ai, Yue Wu, Xiping Wang, Haoyu Ding, Chunxi Wei, Guangfei Xu, Wenxing Sun.

  The persistent environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been implicated in hepatic lipid metabolism disorders and steatosis. However, the precise mechanisms underlying TCDD-induced hepatic lipid deposition remain incompletely elucidated. Mitofusin 2 (MFN2), a key mitochondrial dynamics protein, plays a critical role in lipid metabolism, as its deficiency leads to metabolic dysregulation. In this study, we investigate the role of MFN2 in TCDD-induced lipid deposition. Our findings demonstrate that TCDD exposure significantly reduces MFN2 protein expression both in vivo and in vitro, while concomitantly decreasing mitochondrial membrane potential and increasing reactive oxygen species (ROS) levels in Huh7 cells. Notably, overexpression of MFN2 effectively mitigates TCDD-induced pathological effects, preventing lipid accumulation, restoring mitochondrial membrane potential, and reducing ROS levels. Mechanistically, although TCDD does not alter the MFN2 mRNA expression, it promotes protein degradation through enhanced ubiquitination in vitro. These findings demonstrate that TCDD induces lipid accumulation in Huh7 cells through ubiquitination-mediated degradation of MFN2. Our study thus identifies MFN2 as a novel target in TCDD-induced hepatic steatosis.

Keywords:  MFN2; ROS; TCDD; mitochondrial membrane potential

DOI:  https://doi.org/10.1002/jat.4890
bioRxiv. 2025 Jul 14. pii: 2025.07.11.664427. [Epub ahead of print]

SCOPE: Revealing Hidden Mechanisms in Phenotypic Screens Through Target and Pathway Enrichment.

Abhijeet Kapoor, Keith Kelleher, Suzanne Underhill, Sankalp Jain, Brandon K Harvey, Mark J Henderson.

Phenotypic screening enables discovery of small molecules without requiring predefined targets, but mechanistic interpretation remains challenging due to polypharmacology and pathway complexity. We developed SCOPE (Screening Compound Ontology for Pathway Enrichment), a KNIME-based computational framework that resolves the molecular drivers of phenotypic activity by linking compound-level screening data to annotated targets and pathways. SCOPE integrates multi-source target annotations and performs statistical enrichment to identify shared mechanisms of action. Applied to a high-throughput screen for modulators of ER-stress induced secretion of endoplasmic reticulum (ER) resident proteins, a process known as exodosis, SCOPE identified calcium signaling as the most enriched KEGG pathway without prior biological context. Target enrichment revealed G protein-coupled receptors (GPCRs) involved in inositol 1,4,5-trisphosphate receptors (IP3Rs)-mediated signaling, with widespread antagonism among hit compounds implicating this pathway in the regulation of exodosis. Notably, SCOPE uncovered a novel role for the histamine receptor HRH1, which was validated by RNAi knockdown and pharmacological inhibition, implicating HRH1 as a potential therapeutic target in ER stress-related disorders. These results highlight SCOPE's potential to deconvolute phenotypic screens and uncover actionable mechanisms in complex cellular systems.

DOI: https://doi.org/10.1101/2025.07.11.664427
Biomol Ther (Seoul). 2025 Aug 12.

Small-Molecule-Induced Protein Polymerization: Mechanisms and Therapeutic Implications.

Young-Sool Hah, Sun-Young Han.

  Small molecules that induce protein polymerization represent an emerging class of compounds with diverse therapeutic potential. This review provides a comprehensive overview of five such molecules: arsenic trioxide (As2O3), BI-3802, NVS-STG2, paclitaxel, and verteporfin. These compounds target different proteins (PML-RARα, BCL6, STING, β-tubulin, and p62, respectively) and exhibit varied mechanisms of action. Some, like As2O3 and BI-3802, induce polymerization leading to protein degradation, while others, such as NVS-STG2, activate protein function through polymerization. Paclitaxel, distinct from these, induces the stabilization of tubulin polymers. Verteporfin, on the other hand, uniquely causes covalent cross-linking of its target and other cellular proteins. This review explores the molecular mechanisms, structural insights, and therapeutic implications of these compounds, highlighting their potential in targeted protein degradation, cancer treatment, and modulation of cellular processes, such as autophagy and immune response. The diverse effects of these molecules underscore the complexity of protein polymerization in cellular function and disease, opening new avenues for drug discovery and development.

Keywords:  Arsenic trioxide; BI-3802; NVS-STG2; Paclitaxel; Verteporfin

DOI:  https://doi.org/10.4062/biomolther.2024.211
Mol Cell Proteomics. 2025 Aug 13. pii: S1535-9476(25)00150-1. [Epub ahead of print] 101051

AMPylation regulates 5'-3' exonuclease PLD3 processing.

Laura Hoffmann, Eva-Maria Eckl, Marleen Bérouti, Michael Pries, Aron Koller, Charlotte Guhl, Ute A Hellmich, Veit Hornung, Wei Xiang, Lucas T Jae, Pavel Kielkowski.

The 5'-3' exonuclease phospholipase D3 (PLD3) is a single-pass transmembrane protein undergoing sequential post-translational modifications (PTM) by N-glycosylation, AMPylation and proteolytic cleavage. The substrates of PLD3 5'-3' exonuclease activity are single-stranded DNAs and RNAs, which act as ligands for Toll-like receptors (TLRs) and trigger a downstream pro-inflammatory response. Although PLD3 has primarily been studied in immune cells, recent findings indicate its enrichment in neurons, where it plays a role in regulating axonal fitness in Alzheimer's disease (AD). However, the regulatory mechanisms governing the proteolytic processing of PLD3 into its catalytically active soluble form and its functional roles in both immune and neuronal cells remain unclear. Here, we describe the functional implications of PLD3 AMPylation, its direct interaction with the protein adenylyltransferase FICD (FICD), and changes in PLD3 processing in Parkinson's disease (PD) patient-derived neurons. We identified PLD3 AMPylation sites within the proteins' soluble region and show that mutation of these sites hampers PLD3 activation and its catalytic activity. Overexpression of FICD AMP-transferase accelerates PLD3 degradation and induces cellular stress response. Together, our findings demonstrate a critical role of AMPylation in PLD3 processing and regulation of its catalytic activity and provide new insights into the protein's transport and localization to lysosomes. The observation that PLD3 regulation in PD-derived neurons is altered compared to healthy neurons further highlights its role in neurodegenerative diseases.

DOI: https://doi.org/10.1016/j.mcpro.2025.101051