bims-cemest 2025-02-23 papers

bims-cemest

Biomed News

on Cell metabolism and stress

Issue of 2025–02–23
twenty papers selected by
Jessica Rosarda, Uniformed Services University

TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the endoplasmic reticulum and into the Golgi.
Mapping the GDF15 Arm of the Integrated Stress Response in Human Cells and Tissues.
Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.
The integrated stress response engages a cell-autonomous, ligand-independent, DR5-driven apoptosis switch.
A privileged ER compartment for post-translational heteromeric assembly of an ion channel.
Sephin1 suppresses ER stress-induced cell death by inhibiting the formation of PP2A holoenzyme.
The integrated stress response in neurodegenerative diseases.
Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy.
CD40 INDUCES UNFOLDED PROTEIN RESPONSE, UPREGULATION OF VEGF AND VASCULAR LEAKAGE IN DIABETIC RETINOPATHY.
Temperature-Dependent Aggregation of Tau Protein Is Attenuated by Native PLGA Nanoparticles Under in vitro Conditions.
Cross-species transcriptomics translation reveals a role for the unfolded protein response in Mycobacterium tuberculosis infection.
The emerging roles of the endoplasmic reticulum in mechanosensing and mechanotransduction.
CRISPuRe-seq: pooled screening of barcoded ribonucleoprotein reporters reveals regulation of RNA polymerase III transcription by the integrated stress response via mTOR.
The Protective Role of the IRE1α/XBP1 Signaling Cascade in Autophagy During Ischemic Stress and Acute Kidney Injury.
Synergistic effects of mutation and glycosylation on disease progression.
Glycosphingolipids in neurodegeneration - Molecular mechanisms, cellular roles, and therapeutic perspectives.
Altered relaxation and Mitochondria-Endoplasmic Reticulum Contacts Precede Major (Mal)adaptations in Aging Skeletal Muscle and are Prevented by Exercise.
Exploring the proton transport mechanism of the mitochondrial ADP/ATP carrier: FA-cycling hypothesis and beyond.
Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress.
Extracellular Histones as Exosome Membrane Proteins Regulated by Cell Stress.

bioRxiv. 2025 Feb 04. pii: 2024.09.27.615420. [Epub ahead of print]

TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the endoplasmic reticulum 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. By 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/10.1101/2024.09.27.615420
bioRxiv. 2025 Feb 01. pii: 2025.01.31.635929. [Epub ahead of print]

Mapping the GDF15 Arm of the Integrated Stress Response in Human Cells and Tissues.

Janell Lm Smith, Kamaryn Tanner, Jack Devine, Anna S Monzel, Alan A Cohen, Martin Picard.

Mitochondrial stress activates the integrated stress response (ISR) and triggers cell-cell communication through secretion of the metabokine growth differentiation factor 15 (GDF15). However, the gene network underlying the ISR remains poorly defined, particularly across metabolically diverse cellular states and tissues. Using RNAseq data from fibroblasts subjected to metabolic perturbations, we develop an ISR GDF15 index quantifying the GDF15 arm of the ISR activation in human cells. Validation of ISR GDF15 index across 44 postmortem human tissues illustrates how this index can be applied to investigate tissue-specific and age-related ISR activation.

DOI: https://doi.org/10.1101/2025.01.31.635929
Brain. 2025 Feb 21. pii: awae407. [Epub ahead of print]

Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.

Thierry Touvier, Francesca A Veneri, Anke Claessens, Cinzia Ferri, Rosa Mastrangelo, Noémie Sorgiati, Francesca Bianchi, Serena Valenzano, Ubaldo Del Carro, Cristina Rivellini, Phu Duong, Michael E Shy, Jeffery W Kelly, John Svaren, R Luke Wiseman, Maurizio D'Antonio.

  Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves the misfolding and retention of MPZ in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1α/XBP1 pathways. Previous results showed that targeting the PERK UPR pathway mitigates neuropathy in mouse models of CMT1B; however, the contributions of other UPR pathways in disease pathogenesis remains poorly understood. Here, we probe the importance of the IRE1α/XBP1 signalling during normal myelination and in CMT1B. In response to ER stress, IRE1α is activated to stimulate the non-canonical splicing of Xbp1 mRNA to generate spliced Xbp1 (Xbp1s). This results in the increased expression of the adaptive transcription factor XBP1s, which regulates the expression of genes involved in diverse pathways including ER proteostasis. We generated mouse models where Xbp1 is deleted specifically in Schwann cells, preventing XBP1s activation in these cells. We observed that Xbp1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury. However, Xbp1 deletion dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters observed in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1s exerts its adaptive function in CMT1B mouse models in large part via the induction of ER proteostasis genes. Accordingly, the exacerbation of the neuropathy in Xbp1 deficient mice was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling in Schwann cells, suggesting that the activation of XBP1s via IRE1 plays a critical role in limiting mutant protein toxicity and that this toxicity cannot be compensated by other stress responses. Schwann cell specific overexpression of XBP1s partially re-established Schwann cell proteostasis and attenuated CMT1B severity in both the S63del and R98C mouse models. In addition, the selective, pharmacologic activation of IRE1α/XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants. Collectively, these data show that XBP1 has an essential adaptive role in different models of proteotoxic CMT1B neuropathy and suggest that activation of the IRE1α/XBP1 pathway may represent a therapeutic avenue in CMT1B and possibly for other neuropathies characterized by UPR activation.

Keywords:  Charcot-Marie-Tooth; Schwann cell; XBP1; demyelinating neuropathy; proteostasis; unfolded protein response

DOI:  https://doi.org/10.1093/brain/awae407
Cell Death Dis. 2025 Feb 15. 16(1): 101

The integrated stress response engages a cell-autonomous, ligand-independent, DR5-driven apoptosis switch.

Francesca Zappa, Nerea L Muniozguren, Julia E Conrad, Diego Acosta-Alvear.

The integrated stress response (ISR) is a fundamental signaling network that leverages the cell's biosynthetic capacity against different stresses to restore homeostasis. However, when homeostasis is unattainable, the ISR switches to drive cell death and eliminate irreparably damaged cells. Previous work has shown that persistent activity of the ISR kinase PERK during unyielding endoplasmic reticulum (ER) stress induces apoptosis downstream of death receptor 5 (DR5) [1]. ER stress provides activating signals that engage the ectodomain (ED) of DR5 to drive its unconventional activation in the Golgi apparatus [1, 2]. Here, using chemical genetics to uncouple stress sensing from ISR activation, we found that DR5 signaling from the Golgi apparatus is integral to the ISR and not specific to ER stress. Furthermore, we show that DR5 activation can be driven solely by increased expression and does not require its ED. These findings indicate that a general ISR kill switch eliminates irreversibly injured cells.

DOI: https://doi.org/10.1038/s41419-025-07403-8
bioRxiv. 2025 Jan 31. pii: 2025.01.30.635714. [Epub ahead of print]

A privileged ER compartment for post-translational heteromeric assembly of an ion channel.

Sudharsan Kannan, William Kasberg, Liliana R Ernandez, Anjon Audhya, Gail A Robertson.

Mechanisms underlying heterotypic subunit assembly of ion channels and other oligomeric assemblies are poorly understood. In the human heart, heteromeric assembly of two isoforms encoded by the human ether-à-go-go related gene ( hERG ) is essential for the normal function of cardiac I Kr in ventricular repolarization, with loss of hERG1b contributing to arrhythmias associated with long QT-syndrome. While hERG1a homomers traffic efficiently to the plasma membrane, hERG1b homomers are retained in the endoplasmic reticulum (ER). When expressed together, the two subunits avidly associate during biogenesis. Seeking rules specifying heteromeric association, we characterized the fate of hERG1b proteins using confocal and superresolution imaging in fixed and live HeLa cells. We found hERG1b sequestered in punctate intracellular structures when expressed alone in HeLa cells. These puncta, driven by an N-terminal "RXR" ER retention signal and phase separation, are distinct from other membranous compartments and proteasomal degradation pathways. The puncta represent a privileged ER sub-compartment distinct from that of ER-retained, type 2 (hERG-based) LQTS mutant proteins, which were rapidly degraded by the proteasome. Introducing hERG1a to cells with preformed hERG1b puncta dissolved these puncta by rescuing extant hERG1b. Rescue occurs by association of fully translated hERG1b with 1a, a surprising finding given previous studies demonstrating cotranslational heteromeric association. We propose that sequestration limits potentially deleterious surface expression of hERG1b homomeric channels while preserving hERG1b for an alternative mode of heteromeric hERG1a/1b channel assembly post-translationally. These findings reveal a surprising versatility of biosynthetic pathways promoting heteromeric assembly.

DOI: https://doi.org/10.1101/2025.01.30.635714
Cell Death Dis. 2025 Feb 19. 16(1): 117

Sephin1 suppresses ER stress-induced cell death by inhibiting the formation of PP2A holoenzyme.

Satoshi Gojo, Daisuke Kami, Arata Sano, Fumiya Teruyama, Takehiro Ogata, Satoaki Matoba.

Sephin1 was discovered as a protein phosphatase inhibitor, and its efficacy against neurodegenerative diseases has been confirmed. There are conflicting reports on whether inhibition of eIF2α dephosphorylation by PP1 holoenzyme with the protein phosphatase 1 regulatory subunit 15 A is the mechanism of action of Sephin1. In the present study, we found that Sephin1 significantly suppressed renal tubular cell death in an animal model of ER stress administered with tunicamycin. CHOP, which plays a central role in the ER stress-induced cell death pathway, requires nuclear translocation to act as a transcription factor to increase the expression of cell death-related genes. Sephin1 markedly suppressed this nuclear translocation of CHOP. To elucidate the molecular mechanism underlying the cell death suppressive effect of Sephin1, we used human renal tubular epithelial cells under ER stress with tunicamycin. Sephin1 reduced intracellular CHOP levels by promoting CHOP phosphorylation at Ser30, which led to protein degradation in UPS. Phosphorylated CHOP is generated by Thr172-phosphorylated activated AMPK, and Sephin1 increased phosphorylated AMPK. Phosphorylated AMPK is inactivated by PP2A through dephosphorylation of its Thr172, and Sephin1 inhibits the formation of the PP2A holoenzyme with the PP2A subunit B isoform delta. These results indicate that inhibition of PP2A holoenzyme formation is the molecular target of Sephin1 in this experimental system.

DOI: https://doi.org/10.1038/s41419-025-07450-1
Mol Neurodegener. 2025 Feb 19. 20(1): 20

The integrated stress response in neurodegenerative diseases.

Maria Astrid Bravo-Jimenez, Shivangi Sharma, Soheila Karimi-Abdolrezaee.

The integrated stress response (ISR) is a conserved network in eukaryotic cells that mediates adaptive responses to diverse stressors. The ISR pathway ensures cell survival and homeostasis by regulating protein synthesis in response to internal or external stresses. In recent years, the ISR has emerged as an important regulator of the central nervous system (CNS) development, homeostasis and pathology. Dysregulation of ISR signaling has been linked to several neurodegenerative diseases. Intriguingly, while acute ISR provide neuroprotection through the activation of cell survival mechanisms, prolonged ISR can promote neurodegeneration through protein misfolding, oxidative stress, and mitochondrial dysfunction. Understanding the molecular mechanisms and dynamics of the ISR in neurodegenerative diseases aids in the development of effective therapies. Here, we will provide a timely review on the cellular and molecular mechanisms of the ISR in neurodegenerative diseases. We will highlight the current knowledge on the dual role that ISR plays as a protective or disease worsening pathway and will discuss recent advances on the therapeutic approaches that have been developed to target ISR activity in neurodegenerative diseases.

DOI: https://doi.org/10.1186/s13024-025-00811-6
J Vis Exp. 2025 Jan 31.

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy.

Carolina Lagos, Diego Tapia, Cristina Silva, Jorge Cancino.

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

DOI: https://doi.org/10.3791/67287
Diabetes. 2025 Feb 20. pii: db230538. [Epub ahead of print]

CD40 INDUCES UNFOLDED PROTEIN RESPONSE, UPREGULATION OF VEGF AND VASCULAR LEAKAGE IN DIABETIC RETINOPATHY.

Sarah Vos, Jose-Andres C Portillo, Alyssa Hubal, Reena Bapputty, Amelia Pfaff, Rachel Aaron, Matthew Weng, Da Sun, Zheng-Rong Lu, Jin-Sang Yu, Carlos S Subauste.

The unfolded protein response (UPR) drives events that promote diabetic retinopathy including VEGF upregulation in Müller cells. How UPR is activated in vivo in the diabetic retina is not well understood. CD40 is required for development of diabetic retinopathy but whether CD40 mediates activation of UPR sensors is unknown. CD40 ligation in Müller cells caused phospholipase Cγ1 (PLCγ1)-dependent activation of UPR sensors (PERK, IRE1α and ATF6α), and VEGF production dependent on PLCγ1 and UPR sensors. Diabetic Cd40-/- mice did not exhibit UPR activation and VEGF upregulation in the retina. These responses were restored in diabetic Cd40-/- mice rescued to express WT CD40 in Müller cells but not in mice rescued to express a CD40 mutant unable to recruit TRAF2,3. Intravitreal administration of a cell-permeable CD40-TRAF2,3 disrupting peptide reduced UPR activation, VEGF upregulation and vascular leakage in diabetic mice. CD40 and TRAF2 in Müller cells from patients with diabetic retinopathy co-localized with activated UPR sensors and VEGF. Our studies indicate that CD40 (via TRAF2,3 signaling) is an inducer of UPR activation that triggers VEGF production in Müller cells. This work uncovered inhibition of CD40-TRAF2,3 signaling as a potential approach to impair UPR activation, VEGF upregulation and vascular leakage in diabetic retinopathy.

DOI: https://doi.org/10.2337/db23-0538
Int J Nanomedicine. 2025 ;20 1999-2019

Temperature-Dependent Aggregation of Tau Protein Is Attenuated by Native PLGA Nanoparticles Under in vitro Conditions.

Pallabi Sil Paul, Mallesh Rathnam, Aria Khalili, Leonardo M Cortez, Mahalashmi Srinivasan, Emmanuel Planel, Jae-Young Cho, Holger Wille, Valerie L Sim, Sue-Ann Mok, Satyabrata Kar.

   Introduction: Hyperphosphorylation and aggregation of the microtubule-associated tau protein, which plays a critical role in many neurodegenerative diseases (ie, tauopathies) including Alzheimer's disease (AD), are known to be regulated by a variety of environmental factors including temperature. In this study we evaluated the effects of FDA-approved poly (D,L-lactide-co-glycolic) acid (PLGA) nanoparticles, which can inhibit amyloid-β aggregation/toxicity in cellular/animal models of AD, on temperature-dependent aggregation of 0N4R tau isoforms in vitro.
Methods: We have used a variety of biophysical (Thioflavin T kinetics, dynamic light scattering and asymmetric-flow field-flow fractionation), structural (fluorescence imaging and transmission electron microscopy) and biochemical (Filter-trap assay and detection of soluble protein) approaches, to evaluate the effects of native PLGA nanoparticles on the temperature-dependent tau aggregation.
Results: Our results show that the aggregation propensity of 0N4R tau increases significantly in a dose-dependent manner with a rise in temperature from 27°C to 40°C, as measured by lag time and aggregation rate. Additionally, the aggregation of 2N4R tau increases in a dose-dependent manner. Native PLGA significantly inhibits tau aggregation at all temperatures in a concentration-dependent manner, possibly by interacting with the aggregation-prone hydrophobic hexapeptide motifs of tau. Additionally, native PLGA is able to trigger disassembly of preformed 0N4R tau aggregates as a function of temperature from 27°C to 40°C.
Conclusion: These results, taken together, suggest that native PLGA nanoparticles can not only attenuate temperature-dependent tau aggregation but also promote disassembly of preformed aggregates, which increased with a rise of temperature. Given the evidence that temperature can influence tau pathology, we believe that native PLGA may have a unique potential to regulate tau abnormalities associated with AD-related pathology.

Keywords:  PLGA nanoparticles; protein aggregation; tau pathology; tau protein

DOI:  https://doi.org/10.2147/IJN.S494104
NPJ Syst Biol Appl. 2025 Feb 15. 11(1): 19

Cross-species transcriptomics translation reveals a role for the unfolded protein response in Mycobacterium tuberculosis infection.

Krista M Pullen, Ryan Finethy, Seung-Hyun B Ko, Charlotte J Reames, Christopher M Sassetti, Douglas A Lauffenburger.

Numerous studies have identified similarities in blood transcriptomic signatures of tuberculosis (TB) phenotypes between mice and humans, including type 1 interferon production and innate immune cell activation. However, murine infection pathophysiology is distinct from human disease. We hypothesized that this is partly due to differences in the relative importance of biological pathways across species. To address this animal-to-human gap, we applied a systems modeling framework, Translatable Components Regression, to identify the axes of variation in the preclinical data most relevant to human TB disease state. Among the pathways our cross-species model pinpointed as highly predictive of human TB phenotype was the infection-induced unfolded protein response. To validate this mechanism, we confirmed that this cellular stress pathway modulates immune functions in Mycobacterium tuberculosis-infected mouse macrophages. Our work demonstrates how systems-level computational models enhance the value of animal studies for elucidating complex human pathophysiology.

DOI: https://doi.org/10.1038/s41540-024-00487-6
J Cell Sci. 2025 Feb 15. pii: JCS263503. [Epub ahead of print]138(4):

The emerging roles of the endoplasmic reticulum in mechanosensing and mechanotransduction.

Jonathan Townson, Cinzia Progida.

  Cells are continuously subjected to physical and chemical cues from the extracellular environment, and sense and respond to mechanical cues via mechanosensation and mechanotransduction. Although the role of the cytoskeleton in these processes is well known, the contribution of intracellular membranes has been long neglected. Recently, it has become evident that various organelles play active roles in both mechanosensing and mechanotransduction. In this Review, we focus on mechanosensitive roles of the endoplasmic reticulum (ER), the functions of which are crucial for maintaining cell homeostasis. We discuss the effects of mechanical stimuli on interactions between the ER, the cytoskeleton and other organelles; the role of the ER in intracellular Ca2+ signalling via mechanosensitive channels; and how the unfolded protein response and lipid homeostasis contribute to mechanosensing. The expansive structure of the ER positions it as a key intracellular communication hub, and we additionally explore how this may be leveraged to transduce mechanical signals around the cell. By synthesising current knowledge, we aim to shed light on the emerging roles of the ER in cellular mechanosensing and mechanotransduction.

Keywords:  Endoplasmic reticulum; Mechanosensing; Mechanotransduction; Membrane contact sites

DOI:  https://doi.org/10.1242/jcs.263503
Nucleic Acids Res. 2025 Feb 08. pii: gkaf062. [Epub ahead of print]53(4):

CRISPuRe-seq: pooled screening of barcoded ribonucleoprotein reporters reveals regulation of RNA polymerase III transcription by the integrated stress response via mTOR.

David T Harris, Calvin H Jan.

Genetic screens using CRISPR (Clustered Regularly Interspaced Palindromic Repeats) provide valuable information about gene function. Nearly all pooled screening technologies rely on the cell to link genotype to phenotype, making it challenging to assay mechanistically informative, biochemically defined phenotypes. Here, we present CRISPuRe-seq (CRISPR PuRification), a novel pooled screening strategy that expands the universe of accessible phenotypes through the purification of ribonucleoprotein complexes that link genotypes to expressed RNA barcodes. While screening for regulators of the integrated stress response (ISR), we serendipitously discovered that the ISR represses transfer RNA (tRNA) production under conditions of reduced protein synthesis. This regulation is mediated through inhibition of mTORC1 and corresponding activation of the RNA polymerase III inhibitor MAF1. These data demonstrate that coherent downregulation of tRNA expression and protein synthesis is achieved through cross-talk between the ISR and mTOR, two master integrators of cell state.

DOI: https://doi.org/10.1093/nar/gkaf062
Cell Stress Chaperones. 2025 Feb 19. pii: S1355-8145(25)00010-0. [Epub ahead of print]

The Protective Role of the IRE1α/XBP1 Signaling Cascade in Autophagy During Ischemic Stress and Acute Kidney Injury.

Ting Liu, Lu Li, Meixia Meng, Ming Gao, Jinhua Zhang, Yuan Zhang, Yukun Gan, Yangjie Dang, Limin Liu.

  Acute kidney injury (AKI) is a common and serious complication resulting from ischemia and hypoxia, leading to significant morbidity and mortality. Autophagy, a cellular process for degrading damaged components, plays a crucial role in kidney protection. The unfolded protein response (UPR) pathway, particularly the IRE1α/XBP1 signaling cascade, is implicated in regulating autophagy during renal stress. To elucidate the role of the IRE1α/XBP1 pathway in autophagy during hypoxia/reoxygenation (H/R) and ischemia/reperfusion (I/R) injury, renal tubular epithelial cells (TECs) were subjected to H/R conditions, and I/R injury was induced in mice. The expression of autophagy-related and ER stress markers (IRE1α, XBP1, GRP78, Beclin1, LC3I/II, and P62) was assessed using immunoblotting and immunofluorescence. Additionally, the impacts of IRE1α overexpression and pharmacological agents, IXA6 (IRE1α agonist) and STF083010 (IRE1α inhibitor), were evaluated on autophagy regulation. H/R injury significantly increased mitochondrial damage and the formation of autophagic vesicles in TECs. Key markers of autophagy were elevated in response to H/R and I/R injury, with activation of the IRE1α/XBP1 pathway enhancing autophagic processes. IXA6 treatment improved renal function and reduced injury in I/R models, while STF083010 exacerbated kidney damage. The IRE1α/XBP1 pathway is a critical regulator of autophagy in renal TECs during ischemic stress, suggesting that pharmacological modulation of this pathway may offer therapeutic avenues for preventing or mitigating AKI. Enhanced understanding of these mechanisms may lead to novel strategies for kidney disease management.

Keywords:  Autophagy; Endoplasmic reticulum stress; Ischemia-reperfusion; XBP1s; chronic kidney disease

DOI:  https://doi.org/10.1016/j.cstres.2025.02.004
Front Mol Biosci. 2025 ;12 1550815

Synergistic effects of mutation and glycosylation on disease progression.

Shodai Suzuki, Motoyuki Itoh.

  Glycosylation, a post-translational modification, plays a crucial role in proper localization and function of proteins. It is regulated by multiple glycosyltransferases and can be influenced by various factors. Inherited missense mutations in glycosylated proteins such as NOTCH3, Low-density lipoprotein receptor (LDLR), and Amyloid precursor protein (APP) could affect their glycosylation states, leading to cerebral small vessel disease, hypercholesterolemia, and Alzheimer's disease, respectively. Additionally, physiological states and aging-related conditions can affect the expression levels of glycosyltransferases. However, the interplay between mutations in glycosylated proteins and changes in their glycosylation levels remains poorly understood. This mini-review summarizes the effects of glycosylation on transmembrane proteins with pathogenic mutations, including NOTCH3, LDLR, and APP. We highlight the synergistic contributions of missense amino acids in the mutant proteins and alterations in their glycosylation states to their molecular pathogenesis.

Keywords:  APP; CADASIL; LDLR; Notch3; aging; familial hypercholesterolemia; glycosylation; mutation

DOI:  https://doi.org/10.3389/fmolb.2025.1550815
Neurobiol Dis. 2025 Feb 18. pii: S0969-9961(25)00067-1. [Epub ahead of print] 106851

Glycosphingolipids in neurodegeneration - Molecular mechanisms, cellular roles, and therapeutic perspectives.

Andreas J Hülsmeier.

  Neurodegenerative diseases, including Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), and amyotrophic lateral sclerosis (ALS), are characterized by progressive neuronal loss and pose significant global health challenges. Glycosphingolipids (GSLs), critical components of neuronal membranes, regulate signal transduction, membrane organization, neuroinflammation, and lipid raft functionality. This review explores GSL roles in neural development, differentiation, and neurogenesis, along with their dysregulation in neurodegenerative diseases. Aberrations in GSL metabolism drive key pathological features such as protein aggregation, neuroinflammation, and impaired signaling. Specific GSLs, such as GM1, GD3, and GM3, influence amyloid-beta aggregation in AD, α-synuclein stability in PD, and mutant huntingtin toxicity in HD. Therapeutic strategies targeting GSL metabolism, such as GM1 supplementation and enzyme modulation, have demonstrated potential to mitigate disease progression. Further studies using advanced lipidomics and glycomics may support biomarker identification and therapeutic advancements. This work aims to highlight the translational potential of GSL research for diagnosing and managing devastating neurodegenerative conditions.

Keywords:  Cellular signaling; Gangliosides; Glycosphingolipids; Lipid rafts; Neurodgeneration

DOI:  https://doi.org/10.1016/j.nbd.2025.106851
bioRxiv. 2025 Feb 05. pii: 2025.01.14.633043. [Epub ahead of print]

Altered relaxation and Mitochondria-Endoplasmic Reticulum Contacts Precede Major (Mal)adaptations in Aging Skeletal Muscle and are Prevented by Exercise.

Ryan J Allen, Ana Kronemberger, Qian Shi, Marshall Pope, Elizabeth Cuadra-Muñoz, Wangkuk Son, Long-Sheng Song, Ethan J Anderson, Renata O Pereira, Vitor A Lira.

Sarcopenia, or age-related muscle dysfunction, contributes to morbidity and mortality. Besides decreases in muscle force, sarcopenia is associated with atrophy and fast-to-slow fiber type switching, which is typically secondary to denervation in humans and rodents. However, very little is known about cellular changes preceding these important (mal)adaptations. To this matter, mitochondria and the sarcoplasmic reticulum are critical for tension generation in myofibers. They physically interact at the boundaries of sarcomeres forming subcellular hubs called mitochondria-endo/sarcoplasmic reticulum contacts (MERCs). Yet, whether changes at MERCs ultrastructure and proteome occur early in aging is unknown. Here, studying young adult and older mice we reveal that aging slows muscle relaxation leading to longer excitation-contraction-relaxation (ECR) cycles before maximal force decreases and fast-to-slow fiber switching takes place. We reveal that muscle MERC ultrastructure and mitochondria-associated ER membrane (MAM) protein composition are also affected early in aging and are closely associated with rate of muscle relaxation. Additionally, we demonstrate that regular exercise preserves muscle relaxation rate and MERC ultrastructure in early aging. Finally, we profile a set of muscle MAM proteins involved in energy metabolism, protein quality control, Ca 2+ homeostasis, cytoskeleton integrity and redox balance that are inversely regulated early in aging and by exercise. These may represent new targets to preserve muscle function in aging individuals.

DOI: https://doi.org/10.1101/2025.01.14.633043
Protein Sci. 2025 Mar;34(3): e70047

Exploring the proton transport mechanism of the mitochondrial ADP/ATP carrier: FA-cycling hypothesis and beyond.

Elena E Pohl, Mario Vazdar, Jürgen Kreiter.

  The mitochondrial ADP/ATP carrier (AAC, ANT), a member of the SLC25 family of solute carriers, plays a critical role in transporting purine nucleotides (ATP and ADP) as well as protons across the inner mitochondrial membrane. However, the precise mechanism and physiological significance of proton transport by ADP/ATP carrier remain unclear. Notably, the presence of uncouplers-such as long-chain fatty acids (FA) or artificial compounds like dinitrophenol (DNP)-is essential for this process. We explore two potential mechanisms that describe ADP/ATP carrier as either (i) a proton carrier that functions in the presence of FA or DNP, or (ii) an anion transporter (FA- or DNP). In the latter case, the proton is translocated by the neutral form of FA, which carries it from the matrix to the intermembrane space (FA-cycling hypothesis). Our recent results support this hypothesis. We describe a four-step mechanism for the "sliding" of the FA anion from the matrix to the mitochondrial intermembrane space and discuss a possible generalization of this mechanism to other SLC25 carriers.

Keywords:  MD simulations; bilayer lipid membranes; membrane proteins; mitochondrial transporter; reconstituted protein; uncoupling protein

DOI:  https://doi.org/10.1002/pro.70047
bioRxiv. 2025 Feb 05. pii: 2024.05.02.592277. [Epub ahead of print]

Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress.

Shannon E Dougherty, Géssica C Barros, Matthew W Foster, Guoshou Teo, Hyungwon Choi, Gustavo M Silva.

Cellular exposure to stress is known to activate several translational control pathways through ribosome ubiquitination. However, how unique patterns of ribosome ubiquitination act at the site-specific level to drive distinct modes of translation regulation remains unclear. To further understand the complexity of these ubiquitin signals, we developed a new targeted proteomics approach to quantify site-specific ubiquitin modification across the ribosome. This method increased the sensitivity and throughput of current approaches and allowed us to systematically measure the ubiquitin status of 78 ribosome peptides and ubiquitin linkages in response to stress. Using this method, we were able to detect the ubiquitination of several ribosome sites even in steady-state conditions, and to show that their modification increases non-stoichiometrically in a dynamic range of >4 orders of magnitude in response to hydrogen peroxide. Besides demonstrating new patterns of global ribosome ubiquitination, our study also revealed an unexpected increase of ubiquitination of ribosomal protein uS10/Rps20 and uS3/Rps3 independent of the canonical E3 ubiquitin ligase Hel2. Furthermore, we show that unique and mixed patterns of ribosome ubiquitination occur in a stress specific manner, depending on the nature of stressor and the enzymes involved. Finally, we showed that while deletion of HEL2 further induces the integrated stress response in response to the nucleotide alkylating agent 4-NQO, deletion of the E2 conjugase RAD6 leads to sustained translation only in response to H 2 O 2 . Our findings contribute to deciphering the complexity of the stress response at the translational level, revealing the induction of dynamic and selective ubiquitin codes, which shed light on the integration of important quality control pathways during cellular response to stress.

DOI: https://doi.org/10.1101/2024.05.02.592277
J Extracell Vesicles. 2025 Feb;14(2): e70042

Extracellular Histones as Exosome Membrane Proteins Regulated by Cell Stress.

Birendra Singh, Marcus Fredriksson Sundbom, Uma Muthukrishnan, Balasubramanian Natarajan, Stephanie Stransky, André Görgens, Joel Z Nordin, Oscar P B Wiklander, Linda Sandblad, Simone Sidoli, Samir El Andaloussi, Michael Haney, Jonathan D Gilthorpe.

  Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals, and when elevated, may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin. We analysed cell culture models under normal and stressed conditions to identify pathways of histone secretion. We report that core and linker histones localize to extracellular vesicles (EVs) and are secreted via the multivesicular body/exosome pathway. Upregulation of EV histone secretion occurs in response to cellular stress, with enhanced vesicle secretion and a shift towards a population of smaller EVs. Most histones were membrane associated with the outer surface of EVs. Degradation of EV-DNA did not impact significantly on EV-histone association. Individual histones and histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. Histones colocalized to tetraspanin positive EVs but using genetic or pharmacological intervention, we found that all known pathways of exosome biogenesis acted positively on histone secretion. Inhibition of autophagy and lysosomal degradation had a strong positive effect on EV histone release. Unexpectedly, EV-associated histones lacked the extensive post-translational modification of their nuclear counterparts, suggesting loss of PTMs may be involved in their trafficking or secretion. Our data does not support a significant role for EV-histones existing as nucleosomes. We show for the first time that histones are secreted from cells as membrane proteins via EVs/exosomes. This fundamental discovery provides support for further investigation of the biological activity of exosome associated histones and their role in disease.

Keywords:  cellular stress; exosome; extracellular vesicles; histone; membrane associated proteins; posttranslational modification

DOI:  https://doi.org/10.1002/jev2.70042