bims-lypmec 2025-08-03 papers

EMBO J. 2025 Jul 29.

ATXN3 regulates lysosome regeneration after damage by targeting K48-K63-branched ubiquitin chains.

Maike Reinders, Bojana Kravic, Pinki Gahlot, Sandra Koska, Johannes van den Boom, Nina Schulze, Sophie Levantovsky, Stefan Kleine, Markus Kaiser, Yogesh Kulathu, Christian Behrends, Hemmo Meyer.

The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is unclear. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 is required for lysophagic flux after lysosomal damage but is not involved in the initial phagophore formation on terminally damaged lysosomes. Instead, ATXN3 is recruited to a distinct subset of lysosomes that are decorated with phosphatidylinositol-(4,5)-bisphosphate and that are not yet fully reacidified. There, ATXN3, along with its partner VCP/p97, targets and turns over K48-K63-branched ubiquitin conjugates. ATXN3 thus facilitates degradation of a fraction of LAMP2 via microautophagy to regenerate the lysosomal membrane and to thereby reestablish degradative capacity needed also for completion of lysophagy. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.

Keywords: Autophagy; Lysosome; Membrane; Stress Response; Ubiquitin

DOI: https://doi.org/10.1038/s44318-025-00517-x

Traffic. 2025 Jul-Sep;26(7-9):26(7-9): e70013

FGF Signaling Promotes Lysosome Biogenesis in Chondrocytes via the Mannose Phosphate Receptor Pathway.

Laura Cinque, Maria Iavazzo, Gennaro Di Bonito, Elena Polishchuk, Rossella De Cegli, Carmine Settembre.

The mannose 6-phosphate (M6P) pathway is critical for lysosome biogenesis, facilitating the trafficking of hydrolases to lysosomes to ensure cellular degradative capacity. Fibroblast Growth Factor (FGF) signaling, a key regulator of skeletogenesis, has been linked to the autophagy-lysosomal pathway in chondrocytes, but its role in lysosome biogenesis remains poorly characterized. Here, using mass spectrometry, lysosome immune-purification, and functional assays, we reveal that RCS (Swarm rat chondrosarcoma cells) lacking FGF receptors 3 and 4 exhibit dysregulations of the M6P pathway, resulting in hypersecretion of lysosomal enzymes and impaired lysosomal function. We found that FGF receptors control the expression of M6P receptor genes in response to FGF stimulation and during cell cycle via the activation of the transcription factors TFEB and TFE3. Notably, restoring M6P pathway-either through gene expression or activation of TFEB-significantly rescues lysosomal defects in FGFR3;4-deficient RCS. These findings uncover a novel mechanism by which FGF signaling regulates lysosomal function, offering insights into the control of chondrocyte catabolism and the understanding of FGF-related human diseases.

Keywords: FGF signaling; MPR trafficking pathway; TFEB; chondrocytes; lysosome; mannose phosphate receptors

DOI: https://doi.org/10.1111/tra.70013

Cell Rep. 2025 Jul 24. pii: S2211-1247(25)00824-1. [Epub ahead of print]44(8): 116053

RNF13 mediates pH- and Ca2+-dependent regulation of lysosomal positioning.

Lan Thi Trinh, Anh Van Vu, Seunghye Shin, Chanhaeng Lee, Sang-Hee Park, Eun-Bee Cho, Yunseok Heo, Hyun Jin Kim, Sungjoo Kim, Jong-Bok Yoon.

Environmental factors such as extracellular pH (pHe) and nutrition status affect lysosomal localization and autophagy, but how pHe, intracellular pH (pHi), and Ca2+ regulate lysosome transport is not well understood. Here, we identify RNF13 as a key regulator of lysosomal positioning via pHi- and Ca2+-dependent degradation of ARL8B. Ca2+-activated apoptosis-linked gene 2 (ALG-2) promotes retrograde lysosomal transport while increasing pHi and decreasing lysosomal pH (pHlys). Elevated pHi deprotonates RNF13 at His332, enabling its interaction with Ca2+-bound ALG-2 and inhibition of ARL8B-mediated anterograde transport. Alkaline pHe elevates pHlys and activates the lysosomal Ca2+ channel TRPML3, enhancing RNF13 activity and driving lysosomes toward a perinuclear position. Thus, starvation or alkaline pHe induces ALG-2 activation and pHi elevation, facilitating RNF13-mediated ARL8B degradation. In contrast, acidic pHi suppresses RNF13, keeping ARL8B levels high even when ALG-2 is active. These findings reveal a coordinated mechanism involving Ca2+ signaling and pH dynamics in regulating lysosomal positioning.

Keywords: ARL8B; CP: Cell biology; RNF13; TRPML1; TRPML3; autophagy; intracellular Ca(2+); intracellular pH; lysosomal positioning; lysosome; protein degradation

DOI: https://doi.org/10.1016/j.celrep.2025.116053

Nat Commun. 2025 Jul 29. 16(1): 6617

Senescence-associated lysosomal dysfunction impairs cystine deprivation-induced lipid peroxidation and ferroptosis.

Tze Mun Loo, Xiangyu Zhou, Yoko Tanaka, Sho Sugawara, Shota Yamauchi, Hiroko Kawasaki, Yuta Matsuoka, Yuki Sugiura, Shinya Sakuma, Yoko Yamanishi, Satoshi Yotsumoto, Kosuke Dodo, Yoshitaka Shirasaki, Takashi Kamatani, Akiko Takahashi.

Senescent cells, characterized by irreversible cell cycle arrest and inflammatory factor secretion, promote various age-related pathologies. Senescent cells exhibit resistance to ferroptosis, a form of iron-dependent cell death; however, the underlying mechanisms remain unclear. Here, we discovered that lysosomal acidity was crucial for lipid peroxidation and ferroptosis induction by cystine deprivation. In senescent cells, lysosomal alkalinization causes the aberrant retention of ferrous iron in lysosomes, resulting in resistance to ferroptosis. Treatment with the V-ATPase activator EN6 restored lysosomal acidity and ferroptosis sensitivity in senescent cells. A similar ferroptosis resistance mechanism involving lysosomal alkalinization was observed in pancreatic cancer cell lines. EN6 treatment prevented pancreatic cancer development in xenograft and Kras mutant mouse models. Our findings reveal a link between lysosomal dysfunction and the regulation of ferroptosis, suggesting a therapeutic strategy for the treatment of age-related diseases.

DOI: https://doi.org/10.1038/s41467-025-61894-9

Methods. 2025 Jul 24. pii: S1046-2023(25)00163-X. [Epub ahead of print]

A simple, accurate method for the measurement of lysosomal activity.

Yoshito Iwai, Yuriko Furuya, Yuji Oguro, Keiji Yamamoto.

Lysosomes are responsible for the degradation of intra- and extracellular components and are thus essential for the quality control of proteins and organelles. Lysosomal dysfunction leads to lysosomal storage diseases, and it is therefore important to identify which types of stress cause functional abnormalities. Lysosomal function is generally evaluated by measuring the enzyme activity of lysosomes with fluorescent dyes. However, fluorescence microscopy can lead to different outcomes due to variations in the field of view, the analysis software used, and the parameter settings. We therefore developed a method that uses only a microplate reader and DQ Green BSA, a dye that emits fluorescence upon lysosomal degradation, to ascertain lysosomal activity. HEK293 cells were treated with DQ Green BSA with or without bafilomycin A1 and lysates extracted using radioimmunoprecipitation buffer. Fluorescence intensities and protein concentrations in the cell lysates were then measured using a microplate reader and the bicinchoninic acid method, respectively, and the fluorescence intensity divided by the protein concentration. Results indicated a significant lysosome inhibitor-induced dose-dependent decrease in the lysosomal activity. The Z'-factor of 0.77 obtained using the proposed method is a significant improvement over the - 0.06 obtained using the conventional method. The versatility of the method was evaluated with different cell types, cell lysis buffers, inhibitors, and protease substrates, with results suggesting that the method works regardless of the cells or reagents used, indicating the relative simplicity and accuracy of the proposed method as compared to the currently utilized method.

Keywords: Autophagy-lysosomal pathway; Cell lysate; Experimental design; Fluorescent protein; Lysosomal activity; Microplate reader

DOI: https://doi.org/10.1016/j.ymeth.2025.07.008

J Physiol. 2025 Jul 27.

Metabolite-sensitive cross-bridge models of human atria reveal the impact of diabetes on muscle mechanoenergetics.

Julia H Musgrave, June-Chiew Han, Marie-Louise Ward, Andrew J Taberner, Kenneth Tran.

Type 2 diabetes is associated with a range of adverse health outcomes, including metabolic dysfunction and increased risk of heart failure. Although the interactions between diabetes and heart disease are complex and incompletely understood, they can be more effectively investigated using multiscale, biophysically based models of the underlying physiological processes. In this study, experimental data from non-diabetic and diabetic human atrial muscles were used to develop metabolite-sensitive cross-bridge models representing each group. The parameterisation of these cross-bridge models revealed that reduced muscle stress development and a leftward shift of the complex modulus measured in the diabetic muscles could be attributed to reduced cross-bridge stiffness and slower cross-bridge detachment rates, respectively. These cross-bridge models were also integrated into muscle models to investigate the effects of diabetic cross-bridge function, Ca2+ handling and altered metabolite availability on isometric and physiological work-loop contractions. The diabetic model produced isometric twitches with lower amplitude and prolonged duration and, in response to lowered ATP concentration, the diastolic stress increased notably. In work-loop simulations, the diabetic model exhibited slower shortening, reduced work output and lower power of shortening. However, it was also more efficient and had a less pronounced negative response to increases in Pi concentration. These simulations demonstrate that while experimentally measured differences in diabetic cardiac tissues can lead to impaired function during physiological contractions, they may also offer compensatory advantages. The insights of this study offer clear mechanisms of mechanoenergetic dysfunction in diabetic heart muscle, identifying potential therapeutic targets to improve cardiac outcomes for individuals with diabetes. KEY POINTS: We used multiscale mathematical models to investigate the interaction between mechanical and energetic systems in diabetic cardiomyopathy. Using previously collected data from human atrial tissues, we developed models of non-diabetic and diabetic cross-bridge function. The models confirmed that the mechanisms underlying slower cycling and lower force production in the diabetic tissues were slower cross-bridge detachment rates and lower cross-bridge stiffness. Integrating into muscle models and predicting the behaviour under different types of contraction revealed that these differences lead to longer force twitches with lower amplitude, less work done, slower shortening and lower power generation in the diabetic muscles. This modelling study presents novel human atrial cross-bridge models and offers clear mechanisms of mechanical and energetic dysfunction in diabetic heart muscle with potential pathways for treatment.

Keywords: cardiac energetics; cardiac mechanics; cross‐bridge model; diabetes

DOI: https://doi.org/10.1113/JP288463