Cellular and Molecular Life Sciences

The voltage-gated chloride channels ClC-1 and ClC-2 are homodimeric structures essential for maintaining muscle excitability and tissue fluid homeostasis, respectively. Mutations in these channels disrupt protein homeostasis (proteostasis), leading to hereditary disorders such as myotonia congenita and leukodystrophy. Specifically, the substitution of highly conserved alanine residues within the transmembrane helix O, exemplified by ClC-1 (A531V) and ClC-2 (A500V), results in severe proteostatic defects characterized by reduced protein stability and impaired surface trafficking. However, the precise role of helix O in these pathological processes remains poorly understood. In this study, we investigated these conserved residues using biochemical and functional approaches. Our findings demonstrate that even subtle structural alterations at these critical sites significantly interfere with channel stability and membrane expression. This study highlights the critical contribution of helix O to proper CLC channel folding and endoplasmic reticulum (ER) quality control, providing deeper insights into the molecular mechanisms of CLC-related channelopathies.

The endoplasmic reticulum (ER) and its muscle-specialized form, the sarcoplasmic reticulum (SR), are crucial organelles in muscle cells, involved notably in protein synthesis, calcium regulation and muscle contraction. A well-known process involved in ER remodeling and homeostasis is ER-phagy, also called reticulophagy, a selective form of autophagic process in which ER-phagy receptors mediate the delivery of ER portions to lysosomes for degradation. SH3KBP1 is an adaptor protein involved in membrane trafficking. Recently, it was shown to control ER morphology and SR formation in striated skeletal muscle. In this study, we demonstrate that SH3KBP1 can bind to LC3B and CKAP4 proteins, bridging ER to autophagosome membranes, and is degraded by autophagy, in developing muscle fibers. Moreover, SH3KBP1 down-regulation impacts basal autophagy efficiency and ER-phagy stimulation; it also impairs the turnover of numerous ER-resident proteins. Our work highlights a new role for SH3KBP1 as a soluble ER-phagy receptor in striated skeletal muscle.

LIM kinases, LIMK1 and LIMK2, play a crucial role in cytoskeleton dynamics. They are involved in many physiological processes but also in several pathologies such as cancer, neuronal diseases and neurofibromatosis. Although LIM kinases appear as promising therapeutic targets, they remain undruggable. A better understanding of their activity and regulation is thus required to better design efficient targeted therapies. Here, we have shown the impact of a single amino acid on LIMK activity on cofilin, their main substrate in actin filament remodelling. We demonstrated that Y632 and Y630, for LIMK1 and LIMK2 respectively, mediate LIMK dimerization, resulting in their transphosphorylation. This process seems to be a prerequisite for their canonical phosphorylation on their respective T508 and T505 residues within the activation loop. These Tyrosine are not phosphorylated, their aromatic nature is rather critical to ensure proper LIMK activity on cofilin. These results bring new insights into LIMK molecular features.

Inflammation increases with aging and contributes to neurodegeneration, yet the principles that determine how immune effectors target host neural tissue remain poorly understood. Antimicrobial peptides (AMPs) are central components of innate immune defenses strongly induced upon infection and upon aging. Studies have shown that AMPs can exhibit cytotoxicity toward host cells, pointing to a role in neurodegeneration. We show that cationic AMPs selectively bind and damage motoneurons that expose phosphatidylserine (PS), an anionic phospholipid normally restricted to the inner leaflet of the plasma membrane. Both infection and aging increase neuronal PS exposure alongside AMP expression. AMP binding occurs in a PS-dependent manner, leading to synaptic bouton fragmentation, accelerated neuronal aging, and locomotor decline. This toxicity is prevented in Drosophila by Turandot proteins, which reduce AMP-PS interactions on motoneurons. Together, our findings define a molecular mechanism underlying neuronal susceptibility to immunopathology and a set of proteins with neuroprotective potential. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/721952v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@ca817aorg.highwire.dtl.DTLVardef@fa8cb0org.highwire.dtl.DTLVardef@12a8bd4org.highwire.dtl.DTLVardef@423907_HPS_FORMAT_FIGEXP M_FIG This study shows that infection or dysbiosis in Drosophila can simultaneously induce antimicrobial peptide expression while promoting phosphatidylserine (PS) exposure on neurons at the neuromuscular junction. Cationic antimicrobial peptides contribute to neurodegeneration by binding to neurons that expose negatively charged phospholipids such as PS. In Drosophila, a family of secreted peptides, the Turandot proteins, can protect the peripheral nervous system by binding to PS-exposed membranes. Together, these findings reveal a role for antimicrobial peptides, a key component of innate immunity, in promoting neurodegeneration as well as a potential protective mechanism by PS masking agent. C_FIG

Tissue-resident peritoneal macrophages (pM[FE]) programmed by GATA6 are essential players regulating immunity and tissue balance in the peritoneal cavity. How GATA6 regulates global lipid metabolism is currently unknown. Addressing this, in myeloid-restricted Gata6-deficient (Gata6-KOmye) mice, significant changes to the pM[FE] lipidome were found. First, Bodipy staining and anti-Stokes Raman scattering (CARS) microscopy demonstrated significant intracellular lipid accumulation in lipid droplets in Gata6-KOmye. Untargeted and targeted lipidomics revealed this to result from increased levels of multiple sphingolipid (SL) molecular species, including sphingomyelins, ceramides, and glycosphingolipids, along with upregulation of the cysteinyl leukotriene (CysLTs) pathway at both lipidomic and transcriptional levels. Evidencing a functional role for the lipidomic phenotype, Gata6-KOmye showed significant eosinophil accumulation, associated with decreased apoptosis which was exclusively driven by CysLT signalling. In summary, GATA6 is demonstrated as a regulator of sphingolipid accumulation and CysLT generation in pM[FE], with secondary impacts on associated leukocytes through regulation of transcellular CysLT signaling.

Type I Interferons (IFN-I) are cytokines with pleiotropic activities involved in antiviral and antitumor immune responses. They can reduce oncolytic virus replication in tumor cells by inducing expression of interferon stimulated genes (ISG) with antiviral functions. To specifically neutralize the IFN-/-{beta} receptor (IFNAR) on specific cell types, we created novel IFNAR1-targeted antagonists constituted of a high-affinity nanobody targeting a specific cell surface marker conjugated to a low-affinity blocking nanobody targeting IFNAR1. We first show in vitro and in vivo that such an antagonist targeting the mouse CD20 molecule (mCD20) inhibits IFNAR signaling only in B cells among splenocytes. We then showed in vitro that a human CD20 (hCD20)-targeted antagonist blocks IFNAR signaling and induces vesicular stomatitis virus (VSV) oncolytic activity against IFN-11-treated AK7 mesothelioma or B16 melanoma cells only if these cells express hCD20. In vivo, we show that the antagonist binds to hCD20 and enhances VSV replication by inhibiting ISG expression specifically in hCD20+ AK7 mesothelioma tumors. Altogether our results demonstrate the efficient and cell-type specific inhibition of IFNAR signaling through the use of these novel IFNAR1 antagonists, both in vitro and in vivo. These antagonists could have many therapeutic applications given the importance of IFN-I in numerous diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/729496v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@4e76b6org.highwire.dtl.DTLVardef@153c39borg.highwire.dtl.DTLVardef@4f0948org.highwire.dtl.DTLVardef@ea8d85_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisIn this study, we created cell-specific IFNAR antagonists that allow to inhibit selectively IFNAR signaling in particular types of cell. We show that this antagonist can be used to target IFNAR at the surface of tumor cells that lead to the inhibition of IFNAR signaling and ISG expression in these cells rendering them more permissive to VSV replication. Beside antitumor virotherapy, these novel antagonist could be useful to study role of IFN-I in normal or pathological context.

Efficient transport of membrane proteins through the endosomal network is essential for brain development and function, with perturbation implicated in disease. Deficiencies in Retromer, a key regulator of endosomal transport, have been linked to aging-related neurodegenerative disorders including Alzheimers and Parkinsons disease. To better define the neuroprotective role of Retromer, we have applied cell surface restricted proteomics to identify those integral membrane proteins whose recycling to the plasma membrane is mediated by Retromer and associated cargo adaptors, sorting nexin 3 (SNX3), its paralogue sorting nexin 12 (SNX12), and sorting nexin 27 (SNX27) (data available via ProteomeXchange: PXD078277). By comparing primary rat cortical neurons and astrocytes we have identified several cargoes that require either SNX3/SNX12- or SNX27-Retromer complexes for endosomal recycling, including proteins involved in synapse organisation, synaptic signalling and Alzheimers disease pathology. We highlight that perturbed Retromer function leads to endosomal enlargement, and we establish a key role of SNX27-Retromer in modulating transport of glutamate across both neuronal and astrocytic membranes via recycling of glutamate transporters EAAT3 (SLC1A1) and EAAT1 (SLC1A3) respectively. Our study provides further mechanistic insight into the consequences of Retromer deficiency for neuronal and astrocytic function, offering new avenues of research in the treatment of neurodegenerative disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/724903v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@98277forg.highwire.dtl.DTLVardef@1490534org.highwire.dtl.DTLVardef@f4a9feorg.highwire.dtl.DTLVardef@c48402_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Suppression of Retromer and the sorting nexins (SNX27, SNX3/SNX12) leads to a significant change in the surface proteome of rat cortical neurons and astrocytes. Focusing on the glutamate transporters, SLC1A1 and SLC1A3, we have validated that SNX27-Retromer is required for their trafficking, with SNX27-Retromer suppression in astrocytes leading to a loss of glutamate uptake. C_FIG

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

NRF1 is a key mediator of the proteasome recovery pathway, yet its regulation by ER-resident factors is not fully elucidated. Here, we demonstrate that selenoproteins SELS and SELK are critical regulators for NRF1 protein dynamics. SELS stabilizes NRF1, while SELK induces its insolubilization. Their deficiency leads to a hyper-accumulation and increased nuclear localization of NRF1 under proteasome inhibition condition. This results in an augmented transcriptional response of proteasome subunits. These results indicate that SELS and SELK cooperatively gate NRF1 activity by controlling its retrotranslocation and solubility, highlighting a novel layer of selenoprotein-mediated quality control in the proteostasis network.

T cell activation is associated with, and dependent upon, the upregulation of amino acid uptake from the extracellular environment. Uptake of the non-essential amino acid asparagine (Asn) is mediated via amino transporters such as Slc1a5 whilst Asn can be synthesized within cells that express asparagine synthetase (ASNS). Previous work demonstrated that initial activation of CD8+ T cells is perturbed in the absence of Asn, whereas effector cytotoxic T cells cells upregulate ASNS and lose their dependence on Asn uptake. By contrast, less is known of the role of Asn uptake and ASNS in CD4+ T cell responses. Here we demonstrate that CD4+ T cells are more reliant than CD8+ T cells on Asn uptake for initial activation, differentiation, metabolic reprogramming and regulation of autophagy. These phenotypes are associated with enhanced expression of ASNS in CD8+ as compared to CD4+ effector T cells.

The epidermal growth factor receptor (EGFR) expression is often dysregulated in head and neck squamous cell carcinoma (HNSCC), driving cancer cell proliferation, invasion, and metastasis through diverse pathways, thereby contributing to aggressive chemo- and radio-therapy resistance. A GPI-anchored protein, CD109 is upregulated in multiple cancers, including HNSCC. While membrane-anchored CD109 (mCD109) is pro-tumorigenic in SCC via EGFR/STAT3 activation, the role of protease-cleaved soluble CD109 (sCD109) is poorly understood. Our groundbreaking findings demonstrate that sCD109 antagonizes EGFR signaling by directly binding to the EGFR extracellular domain, preventing mCD109-EGFR stabilizing interactions on the cell surface, followed by inhibition of EGFR phosphorylation at Y1068 and downstream signaling cascades (AKT, MAPK, and STAT3) consequently suppressing cancer cell migration, invasion, 3D tumor spheroid formation and angiogenic tube formation. In addition, we found that sCD109 regulates EGFR fates by inhibiting nuclear localization of phosphorylated EGFR and promoting EGFR degradation. Additionally, sCD109 significantly reduces EGF-induced expression of cancer stem cell markers (CD44 and CD133) and embryonic stem cell markers (Nanog and Sox2), suggesting a suppressive role in cancer stemness. Taken together, these results underscore the opposing roles of mCD109 and sCD109: with sCD109 acting as an antagonist by inhibiting mCD109/EGFR-driven oncogenic signaling and phenotypes. Our current findings reveal a complex interplay among mCD109, sCD109, and EGFR, identifying a mechanism for targeting EGFRs degradation in HNSCC, and lay the groundwork for future research on investigating sCD109s modulatory role in preclinical models of HNSCC.

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting {beta}-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and {beta}-arrestin interaction. However, the respective roles of {beta}-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)-based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with {beta}-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in {beta}-arrestin-deficient ({Delta}{beta}arr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. {beta}-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired {beta}-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in {Delta}GRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G{beta}{gamma} was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of {beta}-arrestins but requires GRKs. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

Enteroviruses are arguably the most numerous group of viruses infecting humans. While most enterovirus infections are benign and self-resolving, their sheer number inevitably increases the chances of multiple complications. The diversity of enteroviruses means that the development of vaccines is only economically feasible against a select few, and no direct-acting or host-targeted anti-virals are approved to treat enteroviral infections, largely due to the rapid development of resistance against all experimental drugs. Here, we explored a universal property of enterovirus infection - a massive upregulation of phospholipid synthesis as a target for anti-viral interventions. The increased phospholipid synthesis consumes endogenously- and exogenously-derived long-chain fatty acids (LCFA). We demonstrate that polyunsaturated LCFAs can have a broad anti-enteroviral effect, affecting multiple steps of the virus life cycle. The anti-viral activity of LCFAs did not strictly depend on the degree of unsaturation or their capacity to induce lipid peroxidation but significantly correlated with their conformation. This suggests that their incorporation into the phospholipid molecules makes the replication organelle membranes incapable of properly accommodating viral replication machinery. Accordingly, the inhibition of neutral lipid synthesis promoted LCFAs retargeting to the membranes in infected cells and increased their anti-viral potency. We show that this approach is effective against diverse enteroviruses in different cell types, including differentiated primary cells, and that attempts to establish viruses resistant to such treatment were unsuccessful.

Actin-related protein T2 (ACTRT2) localizes to the perinuclear theca (PT) of male germ cells, yet its functional significance remains unclear. ACTRT2 is evolutionarily conserved and exhibits significant sequence similarity to other testis-specific actin-related proteins, with the highest conservation observed within the canonical actin core domain. We generated Actrt2-deficient mice which displayed male subfertility with pronounced acrosomal malformations originating during the Cap phase of acrosome biogenesis. Actrt2-deficient male mice showed reduced fertilization rate and poor blastocysts quality. Co-immunoprecipitation identified ACTRT2 interactions with PT proteins ACTRT1, ACTRT3, ACTL7A, ACTL9, PFN3, SPEM2 and CCIN while the interaction with CYLC1 was not detected. ACTRT2 overexpression in HEK293T cells altered cell morphology and F-actin distribution. Further, cytoskeletal regulator CFL1 was enriched in testis from Actrt2-deficient mice. We propose that ACTRT2 is a structural component of the PT stabilizing the acroplaxome during spermiogenesis and acrosome biogenesis by modulating actin dynamics. Finally, the high degree of sequence conservation and similarity with ACTRT1 and ACTRT3 together with their similar phenotypes when deleted, indicate that ACTRT2 shares a partial functional redundancy and compensatory capacity with other Arp proteins in testis. Taken together, these findings establish ACTRT2 as a structural regulator of sperm head architecture and male fertility in mice.

Calreticulin mutations in myeloproliferative neoplasms result in the replacement of the C-terminus acidic sequence with a positively charged tail that causes pathological activation of the thrombopoietin. The two canonical variants are Type-1 and Type-2. The remaining are mainly classified as Type-1 or Type-2 like based on the wild type sequence retained. Here, we performed in silico biophysicochemical analyses of 76 CALR exon 9 frameshift variants by their sequence and predicted biophysical properties, complemented by structural modeling of the mutant homodimers. Beyond confirming the Type-1 versus Type-2 distinction, we found that the Type 1-like variants form a continuum of charge architecture along which two reproducible subgroups can be identified, rather than sharply separated classes. This work refines the conventional mechanism-based classification into a charge-resolved framework and provides testable hypotheses linking novel-tail chemistry to receptor activation in CALR-mutant neoplasms and paves the way for improved targeted therapies based on individual mutants characteristics

Despite advances in knowledge and medicine, hepatitis C virus (HCV) infection remains a global challenge. The viral life cycle heavily depends on lipid metabolism; therefore, HCV infection is associated with profound changes in host lipid homeostasis. The transcription factor nuclear factor erythroid 2 related factor-1 (Nrf1) is one of the regulators maintaining this homeostasis. Nrf1 exists in multiple proteoforms that differ in their capacity to serve as cholesterol sensor, activator or inhibitor of gene expression. We have previously identified that the amount of full-length Nrf1 protein in HCV-replicating cells is significantly reduced. Here, we investigate whether HCV affects the formation of the different proteoforms and their functionality using Western blot, qPCR, CLSM and FRET acceptor-photobleaching methods. We report that HCV infection does not alter the onset of Nrf1 proteoforms generated through proteasomal cleavage of the protein. However, the amount of different Nrf1 proteoforms is significantly reduced in HCV-positive cells due to enhanced Nrf1 turnover. Furthermore, the Nrf1 proteoforms with transcriptional activator functions are prevented from translocation into the nucleus. Reduced Nrf1 activity contributes to elevated cholesterol levels and favors lipid droplets formation, which serve as a central platform for viral morphogenesis. Conversely, rescue of Nrf1 activity in HCV-replicating cells is associated with decreased intracellular cholesterol levels, reduced number of lipid droplets and impaired viral release, which is reflected by intracellular accumulation of the core protein and intact viral particles. Taken together, our results characterize the so far not investigated complex interplay between HCV and Nrf1. HCV-mediated inhibition of Nrf1 functionality leads to intracellular cholesterol accumulation, resulting in enhanced lipid droplet formation that supports the HCV life cycle and contributes to HCV-associated pathogenesis. Author SummaryThe lack of a vaccine and limited access to effective drugs (pan-genotypic direct-acting antivirals) for curing hepatitis C virus (HCV) infection means that HCV remains an ongoing and urgent challenge worldwide. In light of this, a deeper understanding of the virus-host interaction is required. In this study, we investigate the interplay between HCV and lipid metabolism, focusing on the uncharacterized role of the cholesterol sensor and transcription factor Nrf1 in this interaction. We observe the inhibition of Nrf1 activity in HCV-replicating cells, which leads to enhanced intracellular cholesterol accumulation and lipid droplet formation, resulting in microenvironment favorable for viral morphogenesis. We reveal the underlying mechanisms and describe their relevance to the viral life cycle and virus-associated pathogenesis.

Angiotensin-converting enzyme 2 (ACE2) functions as the receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus utilizes the cellular endocytic machinery for entry by binding to defined residues on ACE2 with its spike protein (S protein), whose activation requires a priming process by another transmembrane protease, the transmembrane protease serine 2 (TMPRSS2). In addition, ACE2 itself is cleaved by TMPRSS2, which has been shown to be critical for viral pathology. This study aimed to elucidate the relationship between ACE2 and TMPRSS2 and the mechanism of ACE2 processing under normal cellular conditions. It is shown that interaction of ACE2 with TMPRSS2 results in altered processing, modification and cellular localization. Glycosylation of ACE2 has a major impact on TMPRSS2 interaction, trafficking and shedding of the enzyme. Studies in newly generated TMPRSS2-knockout rats reveal increased ACE2 levels in tissues supporting an important role of TMPRSS2 in ACE2 shedding also in vivo.

Atypical chemokine receptors (ACKRs) are non-signaling GPCRs that regulate ligand availability, with ACKR3 functioning as a dual scavenger of chemokines and opioid peptides. Here, we demonstrate that following ligand stimulation, besides the canonical internalization, ACKR3 is released on extracellular vesicles (EVs). ACKR3 was also found on EVs released under basal conditions, although to a lesser extent. These observations were confirmed across multiple cellular contexts, including endogenous systems. Mechanistically, basal and ligand-induced EV release are independent of GRKs and {beta}-arrestin but each relies on distinct trafficking routes and C-terminal determinants. Ligand-induced EV release is associated with plasma membrane localization and receptor recycling pathways. In contrast, basal EV release is governed by intracellular sorting processes and influenced by receptor ubiquitination and RAMP3. Functionally, EV-associated ACKR3 retains high-affinity ligand binding, enabling sequestration of CXCL12 and opioid peptides and thereby attenuating their signaling through CXCR4 and MOR. We also show that the release on EVs, in particular under basal conditions, is observed for other receptors such as KOR, CXCR4 and several ACKRs. Collectively, these findings establish EVs as regulators in chemokine and opioid systems and as a previously underappreciated dimension of ACKR3 and more broadly GPCR biology.

Leydig cells (LCs) are essential for male reproductive function due to their role in testosterone synthesis, a process critically dependent on mitochondrial cholesterol transport mediated by the Steroidogenic Acute Regulatory protein (StAR). Despite their importance, LCs are highly sensitive to metabolic and exogenous stressors. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a key link between cellular metabolism and cell fate; however, its role in LCs and steroidogenesis remains poorly understood. In this study, we investigated the induction of ferroptosis in LCs and its impact on their steroidogenic capacity. We evaluated cellular responses to canonical ferroptosis inducers (Erastin and RSL3) alongside the transcriptional regulation of key genes. Our results demonstrate that LCs are vulnerable to ferroptotic stress, which significantly downregulates Star expression. Notably, we uncovered a novel endocrine-metabolic crosstalk: hormonal stimulation via hCG effectively rescues LCs from Erastin-induced toxicity and fully sustains maximal steroidogenesis. However, this hormone-driven cytoprotection fails against direct GPX4 inhibition by RSL3, indicating an absolute reliance on functional GPX4. These mechanistic findings highlight the paradoxical dual role of ACSL4 in Leydig cell biology and are further supported by bioinformatic analysis of public transcriptomic profiles from infertile patients, which reveal a detrimental imbalance in the ACSL4/GPX4 axis. Together, our data position ferroptosis as a critical disruptor of male endocrine function and reveal a hormone-mediated metabolic adaptation that could inform novel therapeutic strategies against oxidative stress in the testis. Highlights-Leydig cells exhibit a strong vulnerability to ferroptotic cell death. -Ferroptosis disrupts StAR expression and halts Leydig cell steroidogenesis. -hCG signaling promotes metabolic adaptation against Erastin-induced ferroptosis.

HighlightsO_LILamin-A and lamin-B1 are essential for midgestational embryogenesis. C_LIO_LILamin-A/B1 are required for proper yolk sac endoderm (YSE) gene regulation. C_LIO_LILamin-A/B1 maintain LADs organization and chromatin interactions in YSE. C_LIO_LILamin-A/B1 and YSE transcription factors support proper YSE gene expression. C_LI Lamins are intermediate filament proteins functioning as ubiquitous structural components of the nuclear lamina that interact with and organize the Lamina-Associated chromatin Domains (LADs). LADs remodel during development and lamins maintain LADs and gene expression profile specific to a given cell type. How ubiquitous lamins achieve cell-type-specific functions during development remains unknown. We show lamin-A and -B1 are required for mouse midgestational embryogenesis and maintain LADs, 3D chromatin interactions, and gene expression in the yolk sac endoderm (YSE). Both lamin-regulated genes and remodeled LADs in YSE cells contain binding motifs of YSE-relevant transcription factors. By analyzing changes in chromatin interactions upon lamin-A and -B1 knockout, we reveal that chromatin neighborhoods maintained by these lamins can influence gene expression orchestrated by YSE-relevant transcription factors. Our findings explain how the ubiquitously expressed lamins can collaborate with lineage-relevant transcription factors to maintain LADs and gene expression programs in specific cell types.