The FEBS journal最新文献

The stress-inducible protein Sestrin2 (SESN2) has recently emerged as an orchestrator of mitochondrial signaling. The regulation of mitochondria-related pathways, such as aerobic respiration, is thought to be mediated by SESN2, but the underlying mechanisms are not fully understood. Here, we characterized mitochondria in Sesn2-knockdown myoblasts under physiological conditions using oxygen consumption rate measurements, fluorescence microscopy, and protein content analysis. We discovered that SESN2 is essential for sustaining oxidative phosphorylation and maintaining the mitochondrial network organization. SESN2 loss diminished ATP production, decreased the levels of nuclear- and mitochondrial-encoded complex IV subunits, and increased superoxide generation. Moreover, the assessment of mitochondrial distribution in Sesn2-knockdown cells revealed a more fragmented network. This was associated with an increased ratio of short to long optic atrophy 1 (OPA1) forms. Remarkably, disruption of mitochondrial signaling suppressed cellular proliferation and altered both cell and nuclear morphology. In summary, our findings suggest that SESN2 plays an important role in maintaining cellular homeostasis, partly through its impact on mitochondrial function.

This study analyzed B-cell heterogeneity in lacrimal gland tissues from 4 patients with IgG4-related ophthalmic disease (IgG4-ROD) and 2 patients with IgG4-positive MALT lymphoma using single-cell transcriptome sequencing (scRNA-seq). The results revealed that while the B-cell differentiation trajectories have similarities between the two diseases, critical differences were evident. IgG4-ROD was predominantly composed of naïve B cells, with memory B cells mainly being the central memory type. These cells were enriched in hormone/innate immunity pathways, and the plasma cells exhibited features of functional exhaustion. Conversely, MALT lymphoma was dominated by memory B cells, particularly enriched in terminally differentiated subtypes, with aberrant activation of oncogenic pathways (BCR/NF-κB). In MALT lymphoma, naïve B cells showed upregulated expression of immunoglobulin genes (IGHG3/IGHG4) and abnormal activation of EBV/BCR/T-cell differentiation pathways, despite having suppressed basal metabolism. Germinal center-like B cells in MALT lymphoma revealed upregulated gene expression enriched in T-cell activation and PD-1/PD-L1 pathways. Plasma cells in MALT lymphoma displayed monoclonal expansion (high expression of IGHG2/IGHG3/IGHG4) and enhanced antibody secretion. Therefore, MALT lymphoma is characterized by abnormal activation and malignant transformation of B cells, whereas IgG4-ROD manifests as functional exhaustion and metabolic suppression. The differentiation state of memory B cells may represent a critical juncture for malignant transformation.

The accumulation of misfolded and unfolded proteins within the endoplasmic reticulum (ER) lumen induces ER stress, which in turn triggers various consequences, such as the unfolded protein response (UPR). AMP-activated protein kinase (AMPK) is also a cellular stress sensor. However, the interplay between AMPK and ER stress remains poorly understood. In this study, we report that in the fission yeast Schizosaccharomyces pombe, the deletion of erd2, a central component for the retrieval of ER-resident proteins, leads to the accumulation of the canonical ER luminal chaperone Bip1 in the cytosol. Moreover, we demonstrate that erd2 deletion increases the levels of the AMPK upstream kinase Ssp1 in a Bip1-dependent manner, thereby promoting AMPK phosphorylation. Intriguingly, although these phenotypes are not dependent on UPR, they can also be caused by ER stress. We further identify multiple E3 ubiquitin ligases that are responsible for the regulation of Ssp1 stability, and Bip1 physically interacts with and stabilises Ssp1 by inhibiting ubiquitination of Ssp1. Additionally, we elucidate that AMPK activation, mediated by the stabilised Ssp1, is required to sustain cell viability, particularly in cells lacking Erd2. Collectively, our findings demonstrate the important role of Erd2 in the maintenance of cellular homeostasis and establish a link between ER stress and AMPK signalling.

Cyclic GMP-AMP synthase (cGAS) senses cytosolic self and microbial DNA to produce cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a secondary messenger that activates the endoplasmic reticulum-resident transmembrane protein, stimulator of interferon genes (STING). After binding to cGAMP, STING undergoes oligomerisation, exits the endoplasmic reticulum (ER), recruits tank-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) on Golgi membranes, resulting in the activation of type I interferons (IFNs). STING is found to be a preformed dimer in the ER; however, it is yet unknown whether protein-protein interactions maintain STING in its resting state. Optineurin (OPTN) functions as an adaptor or a scaffold to coordinate autophagy, type I IFN response, vesicle trafficking, and mitophagy. TBK1 commonly binds OPTN and STING to activate type I IFNs in response to extracellular and intracellular cues. However, it remains unclear whether OPTN participates in STING-mediated type I interferon (IFN) response. As STING initiates inflammatory signalling and OPTN functions as an adaptor protein, we asked if OPTN is necessary for STING to mediate type I IFN response. To answer this question, we examined STING-mediated type I IFN response in human and mouse cells depleted of OPTN and elucidated STING-OPTN binding. We found that modulating OPTN levels alters STING-mediated type I IFN response. Further, the N-terminal domain of STING binds to the C-terminal ubiquitin-binding domain of OPTN. In addition, we found that OPTN engages with STING and TBK1. Thus, we conclude that OPTN calibrates STING-mediated type I IFN response. Based on our observations, approaches that include developing tailored molecular glue-like compounds binding STING-OPTN, and determining STING activation might be valuable avenues for understanding and treating autoimmune diseases.

GATA1 is a crucial transcription factor involved in hematopoiesis and mutations in this gene are linked to severe hematological disorders, including anemia, thrombocytopenia, Down syndrome-related transient abnormal myelopoiesis (DS-TAM), and myeloid leukemia of Down syndrome (ML-DS). Despite significant clinical interest in the molecular level characterization of GATA1 mutations, a comprehensive understanding of their impact on DNA binding is limited. Efforts to conduct detailed studies on full-length recombinant GATA1 have faced significant technical challenges, while alternative approaches are limited by low throughput or qualitative nature. Here, we introduce a native holdup (nHU) assay designed to systematically quantify DNA-protein interactions and is suitable for studying the impact of transcription factor mutations on DNA binding affinity. First, using the erythroid-specific ATP2B4 promoter as a model, we demonstrate that nHU can capture sequence-specific interactions and detect even subtle differences in DNA binding affinities. Then, we quantitatively characterize the impact of pathological mutations on DNA binding affinities in the context of full-length human GATA1. Our findings reveal that the GATA1s isoform, lacking the N-terminal transactivation domain (N-TAD), binds to DNA with increased affinity, while the R307C mutation reduces binding to the ATP2B4 erythroid promoter. In harmony with these observations, GATA1s exhibits increased functional activity, while the R307C mutation results in decreased activity. This study demonstrates the power of the nHU assay for studying DNA interactions of transcription factor variants and providing insight into the molecular mechanism of related diseases.

Endonuclease G (ENDOG) is involved in DNA replication, mitochondrial DNA maintenance, and late apoptosis. Its role in genome maintenance and cell death necessitates strict regulation to prevent unwanted DNA damage. Although inhibitors of ENDOG have been identified in nonmammalian species, no such inhibitors have been discovered in mammals. Here, we identify KU80 as a novel negative regulator of ENDOG in human cells. The KU80 C-terminal domain (CTD) shares structural homology with Drosophila EndoG inhibitor (EGI), directly binds ENDOG and inhibits its nuclease activity in vitro. In silico modeling and immunoprecipitation reveal the KU80 core domain mediates ENDOG interaction. Depletion of KU80 increases nuclear ENDOG accumulation and DNA damage under normal conditions, evidenced by elevated phosphorylated histone H2AX (γH2AX) levels. Conversely, ENDOG-deficient cells show reduced DNA damage and increased chemotherapeutic resistance. We uncover a mechanism where KU80 maintains genomic stability by inhibiting ENDOG activity and nuclear translocation. This study establishes KU80 as a key DNA damage response regulator, offering insights into potential therapies for diseases linked to dysregulated ENDOG activity.

The destabilization of transthyretin (TTR) tetramers underlies the pathogenesis of ATTR amyloidosis, a disease manifesting in form of toxic amyloid deposits. Here we investigate two clinically relevant TTR variants, H88R and I107V, found among ATTR patients in the Carpathian Basin. While the I107V mutation exerts only a mild effect on tetramer assembly and monomer stability, it decreases the denaturation midpoint and enhances aggregation propensity at physiological pH, against both wild-type and monomeric TTR (MTTR) backgrounds. Structural analysis revealed that the absence of the methyl group of residue 107 perturbs the hydrophobic trapping of F87 across the primary tetramerization interface. We also found that the far more disruptive H88R mutation that fully abolishes tetramer formation and yields a highly amyloidogenic monomer also acts through the disarray of the F87-centered inter-chain contact. Its equilibrium ensemble includes unfolded components and displays reduced denaturation entropy, traits suggestive of a primed aggregation-prone state. Simulations reveal that the H88R mutation leads to the partial disordering of the EF helix/loop, which in wild-type TTR participates in a Trp-cage-like architecture (centered on W79) shielding the hydrophobic core of the monomeric form. Our results suggest that the H88R variant may serve as a more physiologically relevant model of aggregation-prone TTR than the widely used MTTR double mutant, which does not show amyloidogenic propensity at physiological pH. Based on their physicochemical properties and position with respect to the determined interaction loci, we offer explanation for the phenotypic presentation of D18G, A25T, and Y114H and H88R monomerizing mutations.

G protein-coupled receptors (GPCRs) initiate G protein-mediated signaling pathways upon ligand binding and are desensitized via β-arrestin-dependent internalization. However, emerging evidence indicates that β-arrestin also mediates G protein-independent signaling pathways that lead to distinct cellular responses. This signaling complexity has led to the development of GPCR-biased ligands that selectively modulate either G protein- or β-arrestin-dependent pathways for drug discovery, offering precise control of cellular processes with minimal side effects. We have developed biased ligands for the 5-hydroxytryptamine receptor subtype 7 (5-HT₇R), which is implicated in autism spectrum disorder (ASD). Notably, we observed that a G protein-biased ligand (2b), but not a β-arrestin-biased ligand (1 g), induced a characteristic ASD behavior in mice, suggesting differential signaling pathways between G protein- or β-arrestin-mediated pathways. We thus further explored the mechanisms of β-arrestin-biased signaling pathways of 5-HT₇R. Our study revealed that 1 g induced a slow but sustained activation of proto-oncogene tyrosine-protein kinase Src (Src), demonstrating temporal specificity of β-arrestin-biased signaling. Src was recruited to 5-HT₇R via β-arrestin-2, followed by the internalization and accumulation of this receptor complex at endosomes. The sustained colocalization of Src at the internalized receptor complex suggests a mechanism for the slow and sustained Src activation. These results highlight spatiotemporal regulation of Src activation in GPCR-biased signaling pathways, providing new insights into 5-HT₇R-targeted therapeutics.

Human ribonuclease 7 (RNase 7), originally isolated from skin, is a member of the RNase A Superfamily and primarily known to be among the endogenous proteins with the most pronounced antimicrobial properties. Nevertheless, to date, studies on this enzyme are mostly limited to its antimicrobial effects on epithelial tissue, which is surprising considering the numerous districts of the human body potentially susceptible to infections. This inference inspired the present work, which is mainly dedicated to uncovering new roles of the RNase 7 in human cells that have not yet been explored in relation to this antimicrobial agent: neuronal cells. In this context, we decided to address possible host defense properties of RNase 7 in the nervous system, and to do this, we have selected both neuroblastoma SH-SY5Y and glioblastoma U-87 MG cells as experimental models. As a result, we found that, in addition to endogenously expressing RNase 7 under altered growth conditions, both cell lines are also responsive to its administration. More specifically, we highlighted for the first time that recombinant RNase 7 reduces the expression levels of pro-inflammatory cytokines, the release of nitric oxide, and the production of reactive oxygen species in LPS-stimulated SH-SY5Y and U-87 MG cells, contributing to their innate immune response. Moreover, here we highlighted that internalized recombinant RNase 7 can contribute to bacterial clearance in both SH-SY5Y and U-87 MG. These findings extend the functional role of RNase 7 beyond epithelia, indicating its potential involvement in neuroimmune regulation and suggesting novel therapeutic implications.

Ubiquitination is a post-translational modification that plays a key role in the maintenance of protein homeostasis. Ubiquitin is covalently attached to the target proteins through a three-step enzymatic cascade in which substrate specificity is conferred by the E3 ligases. However, to match more than 600 E3s with their specific substrates is one of the major challenges in the field. The dynamic and reversible nature of ubiquitination requires the development of techniques to systematically address this question. Here we provide a comprehensive overview of the current methodologies used to reveal targets of E3 ligases, discussing their strengths and limitations. This is particularly relevant in light of emerging pharmacological strategies for targeted protein degradation.