Myocardial ischemia-reperfusion (IR) injury, a prevalent heart disease, disrupts mitochondrial homeostasis, affecting heart energy metabolism. The role of mitochondrial protein Sirt5 in protecting the heart from IR injury is explored in mice. Reduced Sirt5 levels in IR-injured hearts correlate with increased protein lactylation and impaired mitochondrial function. Sirt5 overexpression mitigates, while its knockout worsens, mitochondrial and cardiac damage. Sirt5 interacts with ANT2, reducing its lysine lactylation to enhance its association with VDAC1. This interaction improves cardiac function post-injury. Targeting ANT2 lactylation may offer a therapeutic approach for cardiac injury.

Biogenesis of UsnRNPs occurs in distinct steps in the nucleus and the cytoplasm. Sequential cytoplasmic actions of CLNS1A in the PRMT5 complex and of the SMN complex assemble the Sm core structure consisting of RNA and proteins. Nuclear SMN, condensed in Cajal Bodies, promotes late maturation steps. Whether cytoplasmic SMN undergoes condensation, and how this contributes to UsnRNP biogenesis or homeostasis, is poorly defined. Here, we show that molecular crowding stress induces rapid, reversible condensation of cytoplasmic SMN into droplets and filamentous assemblies, S-bodies, that sequester mislocalized cytoplasmic UsnRNPs along microtubules. During stress recovery, S-bodies undergo microtubule-dependent reorganization into split SMN-CLNS1A condensates, Janus bodies, that promote clearance of mislocalized UsnRNPs. Strikingly, cellular models of the SMN-associated disease spinal muscular atrophy fail to assemble S-bodies and to clear cytoplasmic UsnRNPs. Our findings identify stress-induced SMN condensation as a mechanism to buffer and resolve cytoplasmic UsnRNP mislocalization and reveal impaired tolerance to molecular crowding as a hallmark of spinal muscular atrophy.

Integrins are cell-surface adhesion receptors that mediate bidirectional signaling governing migration, proliferation, and survival. Emerging evidence reveals that integrin function is finely controlled by an interconnected network of post-translational modifications (PTMs), spanning classical modifications such as glycosylation and phosphorylation, as well as novel metabolite-driven alterations including carbamoylation and cysteine carboxyethylation. These PTMs collectively orchestrate integrin stability, conformation, trafficking, and downstream signaling, with profound implications for tumor progression, metastasis, fibrosis, and therapy resistance. We further highlight critical cross‑talk among distinct modification types and demonstrate how environmental cues (e.g., metabolic byproducts, gut microbiota metabolites) directly modify integrins. Finally, we explore emerging therapeutic strategies inspired by these insights, including targeted enzyme inhibitors, proteolysis targeting chimeras (PROTACs), deubiquitinase-targeting chimeras (DUBTACs), and PTM-editing tools such as dCasRx-based m6A editors. Deciphering the integrin PTM code transforms our understanding of cell adhesion biology and opens new frontiers for diagnostic biomarkers and precision therapy.

Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical of keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild-type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.

Aberrant expression of EphA2 (Ephrin receptor A2) in gastric cancer (GC) promotes chemoresistance by suppressing ferroptosis, yet the underlying mechanism remains poorly defined. In this study, we show that EphA2 confers resistance to platinum-based chemotherapy by suppressing ferroptosis through redox homeostasis regulation. Mechanistically, EphA2 directly phosphorylates and activates the deubiquitinase USP38, which in turn stabilizes SLC7A11 by preventing its proteasomal degradation. Elevated SLC7A11 sustains intracellular glutathione homeostasis, attenuates lipid peroxidation, and limits reactive oxygen species accumulation, thereby enabling GC cells to evade ferroptotic cell death under chemotherapeutic stress. Importantly, genetic or pharmacological disruption of the EphA2-USP38 interaction leads to SLC7A11 destabilization, excessive ROS accumulation, ferroptosis induction, and restoration of chemosensitivity. Together, these findings identify the EphA2-USP38/ROS axis as a key determinant of oxaliplatin resistance in GC and highlight a promising therapeutic target for overcoming chemotherapy resistance by reactivating ferroptosis.

Mitochondria are well known to produce ATP for cell energy requirements and thermogenesis simultaneously by the respiratory chain, with a potential optimum temperature of 50 °C, while deleterious overheating should be prevented by a yet unknown system. Here, we report the identification of a human Transient Receptor Potential Vanilloid 1 (mitoTRPV1) alternative isoform located in the mitochondrial inner membrane, which acts as a thermostat regulating mitochondrial maximal temperature. The ubiquitously expressed mitoTRPV1 open reading frame overlaps TRPV1 exons 1 and 2 and intron 2 in a + 1 frame, encoding for an effective 150 amino-acid N-terminal mitochondrial targeting sequence (MTS) conserved amongst mammalian species, followed by the 687 amino acids of the TRPV1 C-terminal sequence. Pharmacological activation of mitoTRPV1 induces a low cytoplasmic Ca[2+] increase and mitochondrial cooling without modification of mitochondrial respiration and ATP production. We conclude that, in our experimental conditions, mitoTRPV1 acts as a thermostat to promote mitochondrial thermolysis without impairing respiration and ATP production. They point to the potential implication of mitoTRPV1 in human diseases related to temperature dysregulation, and should be further studied in physiological conditions in cellular and animal models expressing mitoTRPV1 endogenously.

Skeletal muscle differentiation requires coordinated cytoskeletal remodeling and transcriptional reprogramming. Members of the actin-depolymerizing factor/Cofilin family regulate actin filament turnover, yet the isoform-specific functions of Cofilin-1 and Cofilin-2 during myogenesis remain incompletely understood. Murine C2C12 myoblasts were used to investigate the expression and function of Cofilin isoforms during differentiation. CRISPR/Cas9-mediated knockout, shRNA-mediated knockdown, immunofluorescence, gene expression analyses, MRTF reporter assays, and pharmacological inhibition of LIM kinase were used to assess myogenic progression and signaling. Myogenic differentiation was accompanied by a pronounced isoform transition characterized by progressive downregulation of Cofilin-1 and Actin-Depolymerizing Factor together with increased expression of Cofilin-2. Loss of Cofilin-1 resulted in marked morphological abnormalities, impaired cell-cycle exit, elevated MRTF activity, and reduced myotube formation, whereas Cofilin-2 deficiency produced comparatively mild effects during early differentiation. Importantly, partial reduction of Cofilin-1 enhanced myoblast fusion, while stronger depletion impaired differentiation, demonstrating a dosage-sensitive requirement for Cofilin-1. In addition, Cofilin-1 activity was dynamically regulated by transient LIM kinase-mediated phosphorylation during early differentiation, and inhibition of this pathway disrupted myogenic progression. Analyses of mRNA and protein stability indicated that isoform switching is primarily regulated at the level of gene expression. Cofilin-1 functions as a dosage-sensitive regulator of myogenic progression that links cytoskeletal remodeling to signaling pathways controlling proliferation and differentiation. Coordinated regulation of Cofilin-1 expression and activity, together with the transition to Cofilin-2, is required for efficient myoblast fusion and muscle formation.

Type I interferon (IFN) signaling plays an essential role in restricting viral infection. Accumulating evidence indicates that microRNAs (miRNAs) serve as key regulators of the type I IFN signaling in mammals. However, whether the miRNA-mediated regulation is involved in type I IFN signaling in teleost fish remains unknown. In large yellow croaker (Larimichthys crocea, Lc), we found that miR-7132a induced by poly(I: C) or type I IFN LcIFNi promotes type I IFN responses in vitro and in vivo. Mechanistically, miR-7132a directly targets the 3'UTR of LcSOCS1 and inhibits LcSOCS1 expression by reducing its mRNA stability and repressing its protein translation. LcSOCS1 promotes K48-linked polyubiquitination at K382 residue of LcSTAT2 via its E3 ubiquitin ligase activity, leading to proteasomal degradation of LcSTAT2. By reducing LcSOCS1-mediated degradation of LcSTAT2, miR-7132a promotes LcSTAT2-dependent type I IFN antiviral responses. Notably, miR-7132a-mediated inhibition of SOCS1 is also present in other teleost species. Collectively, these findings reveal a novel positive regulatory axis by which miR-7132a promotes type I IFN antiviral responses by suppressing SOCS1-mediated STAT2 degradation. Thus, this study provides valuable insights into miRNA-mediated regulatory axis of type I IFN responses in teleost fish.

Proteostasis is essential for maintaining neuronal function, and its dysregulation is a hallmark of aging and neurodegeneration. The ubiquitin-proteasome system (UPS) and macroautophagy are the two major proteolytic pathways responsible for protein degradation. However, their capacity and regulation differ between cell types and across aging. To elucidate the activity of both proteolytic pathways with aging, we performed a comparative analysis of the activity of UPS and macroautophagy in distinct neuronal subcellular compartments, in the soma and at synaptic terminals, across aging in neurons of Mus musculus (mouse) and Caenorhabditis elegans (nematode). In mice, our results identified differences between brain areas. While the cortical proteasomal activity declined with aging in both the somatic as well as synaptic-enriched neuronal subcompartments, the cerebellar proteasomal activity decreased only in the somatic-enriched compartments with aging. In C. elegans, we detected a decrease of proteasomal activity in both somatic and synaptic compartments of neurons. Interestingly, we observed an age-dependent change in several markers of autophagy in different fractions and brain areas of mice and a reduction of autophagosomes and autolysosomes with aging in C. elegans. Thus, we uncovered neuron-specific and subcompartmental-specific proteolytic capacities with aging that could manifest in different neuronal vulnerabilities for proteotoxic challenges with aging.

The SM-protein Munc18 and its binding partner syntaxin are crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the release site, and priming reactions that prepare docked granules for Ca[2+] dependent fusion. Both proteins cluster simultaneously at the docking site seconds after a granule arrives at the plasma membrane. To study the mechanisms of these separate Munc18 functions, we generated a Munc18-1 knockout (1KO) and a double knockout of both Munc18-1 and Munc18-2 (DKO) in insulin-secreting cells, and quantified granule docking, exocytosis, and protein clustering at the docking site by TIRF imaging and capacitance measurements. In the 1KO, priming and exocytosis were lost, but granule docking remained intact. In the DKO, both priming and granule docking were impaired. Expression of either Munc18-1 or -2 rescued these defects, as did expression of mutants with weakened binding to syntaxin (EA and EK mutants). Munc18 clustered at granule docking sites, with apparent affinities decreasing in the order Munc18-1 > Munc18-1EA > Munc18-1EK > Munc18-2. Clustering of syntaxin-1 and - 3 at granules was impaired in both knockout lines. Finally, single molecule and imaging fluorescence recovery after photobleaching (FRAP) revealed that Syntaxin and Munc18 molecules were bound to each other, regardless of whether the proteins were clustered at a granule or not. We conclude that that Munc18-1 and - 2 molecules co-cluster with syntaxin at the release site and support docking and priming, and different affinities of the isoforms for the granule docking site contribute to the observed functional preferences.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer a powerful platform for disease modeling, drug discovery, and regenerative therapies. However, their clinical and research utility remains limited by their immature, fetal-like phenotype. In the human heart, postnatal metabolic maturation involves a critical switch from glycolysis to fatty acid β-oxidation, enabling efficient ATP production via oxidative phosphorylation. In this study, we investigated whether mimicking this metabolic shift in vitro by culturing hiPSC-CMs in a fatty acid-based maturation medium (FAM) could enhance their structural and functional development compared to a conventional glucose-based medium (GLM). hiPSC-CMs cultured in FAM for two weeks exhibited significant improvements in morphological, metabolic, and mechanical maturation markers. Morphologically, FAM-cultured CMs exhibited enhanced sarcomeric organization, increased cellular alignment, and a more elongated and rod-like shape, which are characteristics typically associated with mature CMs. Ultrastructural analysis further confirmed improved maturation, revealing more organized sarcomeres and densely packed mitochondria compared to GLM-cultured CMs. Metabolically, FAM-cultured CMs demonstrated a clear shift from glycolytic to oxidative metabolism, as evidenced by higher mitochondrial membrane potential, increased oxidative phosphorylation capacity, elevated ATP production, and reduced glycolytic activity. These metabolic adaptations indicate a more adult-like energy profile, consistent with enhanced fatty acid β-oxidation. Mechanically, FAM-cultured cardiomyocytes exhibited enhanced functional maturity, as evidenced by faster calcium transients and greater contraction amplitude, indicating improvements in specific electrophysiological properties. In conclusion, fatty acid supplementation effectively promotes the structural, metabolic, and mechanical maturation of hiPSC-CMs, resulting in a more adult-like phenotype. This strategy provides a robust and straightforward approach to enhance the physiological relevance of hiPSC-CMs for preclinical applications in disease modeling, drug testing, and regenerative medicine.