bims-raghud 2025-10-19 papers

Issue of 2025–10–19
six papers selected by
Irene Sambri, TIGEM

Proc Natl Acad Sci U S A. 2025 Oct 21. 122(42): e2517050122

Global conformation of the Rag GTPase heterodimer governs eukaryotic amino acid sensing.

The Rag GTPase heterodimer is a central mediator of amino acid sensing in eukaryotic cells. When amino acids are abundant, it binds to the mechanistic target of rapamycin complex 1 to activate cellular programs for growth and proliferation. In its functional cycle, besides local conformational changes near the nucleotides that are commonly observed in monomeric signaling GTPases, the relative positioning of the two Rag subunits, i.e., the global conformation, is unique due to the heterodimeric architecture. Although various global conformations have been captured in static structural models, dynamic transitions between these conformations and their biological relevance remain unclear. Here, we visualize the global conformation of the Rag GTPase heterodimer using single-molecule Förster resonance energy transfer. By tracking the movement of individual protein molecules, we found that the two subunits explore a wide conformational space, which is strictly dictated by the bound nucleotides, regulators, and mutations. Additionally, we demonstrate that proper modulation of the global conformation is crucial for correctly interpreting amino acid signals. Our results defined a checkpoint of amino acid sensing in eukaryotic cells.

Keywords: Rag GTPase; amino acid sensing; mTORC1; protein conformation; single-molecule FRET

DOI: https://doi.org/10.1073/pnas.2517050122

Methods Mol Biol. 2026 ;2976 47-60

Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.

Jennifer E Palmer, David C Rubinsztein.

Autophagy is a conserved lysosomal degradation pathway that recycles protein aggregates and damaged organelles to maintain cytoplasmic quality control. Measuring the amount of the lipid-conjugated autophagic protein LC3B-II is a useful way to test whether a particular perturbation affects autophagy. However, the level of LC3B-II is affected by factors that alter either the rate of autophagosome biogenesis or degradation. Consequently, the same steady-state LC3B-II level can be reached by opposing autophagic fluxes. It is thus essential when measuring LC3B-II to perform the assay both in the absence and presence of a lysosomal inhibitor, enabling measurement of the rate of synthesis independent of its degradation. LC3B-II is also a small protein that can be challenging to detect by western blotting. In this chapter, we will provide a method for the efficient western blotting of LC3B-II and guidance as to the interpretation of the results.

Keywords: ATG8; Autophagy; Bafilomycin A1; LC3B; SDS-PAGE; Western blotting

DOI: https://doi.org/10.1007/978-1-0716-4844-5_5

Autophagy Rep. 2025 ;4(1): 2568537

Autophagy in the lung: guardian of homeostasis or driver of disease.

Hyungsin Kim, Wenping Wang, Ioana Dobrescu, Joel Lee, Joshua Martorelli, Samuel Wang, Jessie Yanxiang Guo.

Autophagy is a lysosome-directed recycling program that preserves lung homeostasis yet, when dysregulated, can cause disease. This review organizes current evidence by lung compartment and disease phase, proposing that autophagy polarity is determined by cell identity, micro-niche, and timing along the injury-repair continuum. In chronic obstructive pulmonary disease, epithelial autophagy is initially cytoprotective, but chronic smoke exposure reveals a lysosome bottleneck and stalled flux, while alveolar macrophages show impaired xenophagy and poor acidification. In idiopathic pulmonary fibrosis, autophagy is suppressed in type II epithelial cells and fibroblasts downstream of transforming growth factor beta (TGF-β) and mTORC1, which promotes epithelial stress programs and collagen translation. In acute lung injury and respiratory distress syndrome, timely autophagy activation limits cGAS-STING and NLRP3 signaling, preserves barrier integrity, and supports recovery. In asthma, autophagy supports mucin biogenesis in epithelial cells but is reduced in antigen-presenting cells, while eosinophil and mast cell effector functions rely on autophagy. In infection, xenophagy clears microbes but is actively subverted by bacteria and respiratory viruses. In non-small cell lung cancer (NSCLC), tumor-intrinsic autophagy maintains energy metabolism, redox balance, and enables immune evasion, whereas host autophagy can alternately support antitumor immunity or supply nutrients. We summarize small-molecule modulators, delivery strategies, and flux-aware tools that enable precise, cell- and phase-resolved modulation of autophagy to guide patient selection and improve therapy in respiratory disease.

Keywords: Autophagy; lung disease; lung disease treatment; lung homeostasis; metabolism

DOI: https://doi.org/10.1080/27694127.2025.2568537

Stem Cell Reports. 2025 Oct 16. pii: S2213-6711(25)00284-X. [Epub ahead of print] 102680

mTORC2-mediated cell-cell interactions promote BMP4-induced WNT activation and mesoderm differentiation.

Li Tong, Faiza Batool, Yueh-Ho Chiu, Priscilla Di Wu, Yudong Zhou, Xiaolun Ma, Santosh Atanur, Wei Cui.

The mechanistic target of rapamycin complex 2 (mTORC2) is essential for embryonic development, but its underlying molecular mechanisms remain unclear. Here, we show that disruption of mTORC2 in human embryonic stem cells (hESCs) considerably alters the Rho/Rac signaling dynamics and reduces E-cadherin expression and cell adhesion. Despite this, mTORC2-deficient hESCs maintain self-renewal and expression of pluripotent markers when cultured in mouse embryonic fibroblast conditioned medium (MEF-CM) supplemented with bFGF. However, these hESCs exhibit significantly impaired mesoderm and endoderm differentiation in response to BMP4 and Activin treatment, respectively, possibly due to reduced WNT activation mediated by cell-cell interactions. Direct activation of the WNT pathway using a GSK3 inhibitor restores mesendoderm differentiation in mTORC2-deficient hESCs. Our study uncovers a novel mechanism by which mTORC2 regulates cell fate determination and highlights a critical link between the intercellular adhesion and the activation of canonical WNT genes.

Keywords: BMP4; WNT; cell adhesion; cell-cell interactions; gastrulation; human embryonic stem cells; mTORC2; mesoderm/endoderm differentiation

DOI: https://doi.org/10.1016/j.stemcr.2025.102680

J Diabetes. 2025 Oct;17(10): e70159

Diabetic Kidney Disease-Associated Pathological Angiogenesis: The Role of Aquaporin-1.

Fengyi Zhang, Jiayi Zhang, Xin Wang, Yaxin Chen, Ziyang Cheng, Jingjing Pan, Yufeng Zhang, Yujie Li, Wenbo Wang, Linhua Zhao.

Diabetic kidney disease (DKD) is recognized as one of the leading causes of chronic kidney disease (CKD) and end-stage kidney disease (ESKD) worldwide, representing a rapidly growing global public health concern. Despite significant advances in understanding the complex pathophysiological mechanisms of DKD, curative treatments are currently unavailable, and the reversal of established renal injury remains an elusive goal in clinical practice. Among various pathological features, aberrant angiogenesis has been closely associated with glomerular injury and the early development of proteinuria in DKD, playing a crucial role in driving disease progression. However, the molecular mechanisms underlying this pathological angiogenesis in DKD remain incompletely understood and warrant further elucidation. Recent research has increasingly implicated aquaporins (AQPs), a family of transmembrane water channel proteins, in the pathogenesis of both acute and chronic kidney disorders, including DKD. In particular, aquaporin-1 (AQP1), which is highly expressed in renal tissues, has emerged as a potential modulator of angiogenic activity within the kidney microenvironment. Although AQP1 and aberrant angiogenesis have been individually explored in the context of DKD, no comprehensive review has systematically examined their interrelationship. This review consolidates current evidence regarding the role of AQP1 in pathological angiogenesis during DKD progression, highlighting its potential significance and identifying gaps that warrant further investigation.

Keywords: Aquaporin‐1; angiogenesis; diabetic kidney disease; review

DOI: https://doi.org/10.1111/1753-0407.70159

Circ Arrhythm Electrophysiol. 2025 Oct 14. e013964

Lysosomal Ca2+ Release Through TRPML1 Governs Ventricular Arrhythmia After Myocardial Infarction.

An Xie, Gyeoung-Jin Kang, Hong Liu, Eun Ji Kim, Feng Feng, Dobromir Dobrev, Samuel C Dudley.

BACKGROUND: In nonischemic cardiomyopathy, mitochondrial Ca2+ handling is involved in arrhythmogenesis by modulating diastolic sarcoplasmic reticulum (SR) Ca2+ release. Recently, it has been reported that lysosomal Ca2+ release can trigger an SR Ca2+ release. We investigated whether lysosomal Ca2+ flux through the TRPML1 (transient receptor potential mucolipin 1) channel could contribute to ischemic cardiomyopathy-related arrhythmia by causing diastolic SR Ca2+ release.
METHODS: Ischemic cardiomyopathy was induced in wild-type C57BL/6J and TRPML1 heterozygous knockdown (TRPML1±) mice by ligating the left anterior descending coronary artery. Mice were studied at 3 weeks after myocardial infarction (MI).
RESULTS: After MI, the lysosomal-restricted TRPML1 Ca2+ release channel was significantly increased in human patients with ischemic or nonischemic cardiomyopathy. TRPML1, but not the TPC2 (2-pore channel 2), was significantly upregulated by 85% in the mouse MI border zone and by 55% in a remote zone. Lysosomal number and approximation to the SR were increased after MI. Lysosomal Ca2+ release was substantially upregulated in MI mouse cardiomyocytes compared with sham cardiomyocytes. The action potential duration was prolonged, and arrhythmogenic diastolic SR Ca2+ release was increased in the cardiomyocytes isolated from MI mice. Blocking TRPML1 reduced action potential duration prolongation and depressed early and delayed afterdepolarizations in cardiomyocytes isolated from MI mice, while the TRPML1 agonist increased TRPML1-dependent cellular triggered activity. A TRPML1 antagonist could inhibit induced ventricular fibrillation in MI mice. Consistent with that result, genetic knockdown of TRPML1 could inhibit arrhythmic risk after MI. The effects of TRPML1-targeted drugs were not seen in control cardiomyocytes.
CONCLUSIONS: Lysosomes contribute to arrhythmic risk after MI because of increased number, proximity to the SR, and induction of diastolic SR Ca2+ release mediated by TRPML1-dependent lysosomal Ca2+ release.

Keywords: arrhythmias, cardiac; calcium; lysosomes; myocardial infarction; myocytes, cardiac

DOI: https://doi.org/10.1161/CIRCEP.125.013964