The FEBS journal最新文献

Keratins assemble into mechanically resilient polymers that physically stabilize epithelial cells. When epithelial cells divide, keratin polymers must be severed to allow cell separation during cytokinesis. Phosphorylation has been implicated in this process, but how keratins are regulated during cell division is not understood. Aurora B kinase, which is part of the chromosome passenger complex (CPC), accumulates at the cell center during cytokinesis and has been implicated in regulating intermediate filaments. We mapped six Aurora B kinase sites in Keratin 8. Phosphorylation of Keratin 8 at S34 occurred specifically at the cleavage furrow and persisted at the midzone until the completion of cytokinesis. Inhibition of Aurora B or expression of a nonphosphorylatable Keratin 8 mutant impaired keratin disassembly at the cleavage furrow. We propose that Aurora B-mediated phosphorylation promotes localized keratin filament disassembly at the cleavage furrow, allowing spatially regulated disassembly during cytokinesis. Aurora B binds to keratin filaments, and its localization to midzones was reduced in Keratin 8 knockout cells, showing that Keratin 8 facilitates Aurora B targeting during cytokinesis. This suggests a positive feedback cycle whereby Keratin 8 promotes midzone localization of Aurora B and, in turn, is locally disassembled by its kinase activity. This cycle is required for successful furrow ingression and completion of cell division in cancer cells of epithelial origin and might provide a target for solid tumor treatment.

Glucocorticoid receptor (GR) is a critical regulator of erythroid progenitor proliferation, while Nemo-like kinase (NLK) is reported to be hyperactivated in Diamond-Blackfan anemia (DBA), suggesting a possible cross talk. Here, we demonstrate that NLK directly interacts with multiple domains of GR and promotes its ubiquitin-mediated proteasomal degradation. Co-immunoprecipitation assays confirmed endogenous NLK-GR interaction in K562 cells, enhanced by proteasomal inhibition. NLK overexpression downregulated levels of GR in a kinase-dependent manner across HEK293T, K562, and MEL cells, an effect reversed by MG132 or a ubiquitination-defective mutant. NLK directly phosphorylated GR at Ser226, as shown by in vitro kinase assays and site-specific immunoblotting. Conversely, NLK depletion reduced basal GR phosphorylation while increasing total GR. We identified OTS167 as a direct NLK inhibitor through cellular thermal shift and kinase assays. OTS167 suppressed NLK autophosphorylation and decreased Ser226 phosphorylation of GR, stabilizing GR protein. Functionally, among all the inhibitors tested, OTS167 maximally inhibited proliferation of K562 and MEL cells by 40-90%. OTS167 also induced erythroid differentiation in K562 and MEL by increasing CD71/TER119 expression and benzidine-positive cells by 60-80%, while NLK overexpression inhibited hemin-induced benzidine staining by 25%. In primary human CD34⁺ cells, NLK and GR exhibited inverse temporal expression during erythropoiesis. OTS167 or dexamethasone expanded CD71⁺ and CFU-E populations and enhanced proliferation (Ki67⁺) across BFU-E, CFU-E, and proerythroblast stages. Conversely, dexamethasone upregulated NLK, suppressing GR and suggesting a feedback loop. Thus, NLK-mediated GR downregulation constrains erythropoiesis, and its inhibition by OTS167 promotes erythroid expansion, revealing a targetable pathway in erythroid disorders.

Molecular chaperones ensure that proteins attain their mature state by assisting in proper folding, preventing aggregation, refolding misfolded proteins, and targeting irreparably misfolded proteins for degradation. This comprehensive role is vital for maintaining cellular homeostasis and responding to stress conditions. In this review, I focus on the multifaceted roles of chaperones in regulating protein production, spanning from ribosome biogenesis to controlling translation rate and translation fidelity through the folding of essential translation factors in eukaryotes. I discuss the function of ribosome- and nascent chain-bound molecular chaperones for the translation machinery and protein synthesis. Finally, I highlight findings on the interdependence of the two pillars of protein homeostasis when cells experience cellular stress and organisms face pathophysiological conditions.

TDP-43 mislocalization and aggregation are common features of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the mechanisms underlying the transition of nuclear TDP-43 to cytoplasmic aggregates, and their contribution to disease pathogenesis, remain poorly understood. To address this gap, we present a methodology to chemically control the assembly and disassembly of cytoplasmic TDP-43 condensates. By fusing TDP-43 to a phase separation-prone protein scaffold, we can induce the formation of cytoplasmic TDP-43 condensates or, conversely, promote nuclear localization upon addition of a disassembly molecule. TDP-43 accumulates into various assemblies, ranging from submicrometric puncta to larger aggregate-like structures that display hallmarks of proteinopathy in a concentration-dependent manner. Furthermore, oxidative stress drives the maturation of TDP-43 assemblies from puncta into aggregates through interactions with stress granule components. Finally, we show that cytoplasmic TDP-43 aggregates deplete nuclear endogenous TDP-43 and induce cytotoxicity. Collectively, these findings highlight the local cytoplasmic concentration of TDP-43 and stress exposure as key determinants in the onset of TDP-43 proteinopathy, providing a relevant model to study pathological TDP-43 aggregation.

Spinocerebellar ataxia type 3 is a debilitating neurodegenerative disorder driven by the pathological aggregation of ataxin-3 (Atx3), a deubiquitinating enzyme with a cysteine-rich catalytic domain and an expandable polyglutamine (polyQ) tract. While the role of polyQ expansion in Atx3 aggregation is well documented, the influence of redox conditions on its self-assembly remains underexplored. Here, we demonstrate that reducing agents and a redox environment critically modulate Atx3 aggregation by regulating disulfide bond formation within the Josephin domain. We demonstrate that dithiothreitol (DTT), through progressive oxidation, promotes the formation of non-native and disulfide-linked conformers, which may serve as nucleation centers for fibril formation. In contrast, tris(2-carboxyethyl)phosphine preserves cysteine residues of Atx3 in the reduced state and inhibits aggregation, but concomitantly promotes cleavage of full-length Atx3. Furthermore, we identify a previously underappreciated role for 4,5-dihydroxy-1,2-dithiane, the DTT oxidation product, in directly triggering Atx3 aggregation. We also demonstrate that running aggregation assays at 50 °C circumvents the redox dependency of Atx3 aggregation, thereby streamlining the aggregation process and enabling the use of a simplified, robust platform for medium- to high-throughput screening of aggregation modulators. These findings provide new insights into the redox-dependent modulation of Atx3 aggregation and highlight critical considerations for in vitro aggregation assays of cysteine-rich proteins, with broad implications for therapeutic strategies targeting cysteine-rich, aggregation-prone proteins in neurodegenerative diseases. Although our study focuses on in vitro investigation, it suggests that redox dysregulation in cells could promote pathogenic aggregation of Atx3, reinforcing the link between cellular redox balance and polyglutamine disease progression.

Gene replacement therapies for muscular dystrophies show promise in preclinical models but often fail in clinical settings. A major difference between animal models and human pathology is the extent of fibrosis observed. Progressive and irreversible fibrosis needs to be targeted before or alongside genetic strategies. Fibrosis limits muscle function through a collagen-rich extracellular matrix (ECM) that forms a stiff barrier impeding penetration of gene therapy vectors, such as adeno-associated viruses (AAVs). It disrupts the satellite cell niche, compromising activation, proliferation, and differentiation. Even with successful gene delivery, regeneration in fibrotic muscle is severely impaired. Recent reports of acute liver toxicity leading to deaths in gene therapy trials using the AAVrh74 vector underscore the risks associated with high systemic AAV doses. If fibrosis can be alleviated, effective transduction might be achieved with lower vector quantities in a single therapeutic dose, reducing the systemic risks. Anti-fibrotic agents are being explored to counteract disease progression. Modulators of ECM maturation offer novel therapeutic targets. However, pleiotropic and context-dependent roles of these mediators complicate translation. Therapies must target pathological ECM remodeling without disrupting essential physiology elsewhere. In this Review, we examine therapeutic efforts targeting skeletal muscle dystrophies and emphasize fibrosis as a major barrier to gene and regenerative therapies. We highlight the need for a deeper investigation into fibrotic pathways, modulators, and extracellular maturation processes and propose that these underexplored areas may yield novel therapeutic targets for muscular dystrophies. A fibrosis-aware therapeutic framework that integrates insights across systems and pathologies is critical for improving treatment outcomes in skeletal muscle disorders.

Devil Facial Tumour Diseases (DFTD), threatening Tasmanian devils, consist of two distinct transmissible cancers, DFT1 and DFT2, with differing origins and geographic spread. We investigated the metabolic differences between DFT1 and DFT2, examining cell viability, metabolic outputs, and bulk gene expression. Using both DFT1 and DFT2 cell lines and biopsies, we found that glycolysis, oxidative phosphorylation, glutamate metabolism, and fatty acid synthesis are all essential for the survival of both tumour types. However, DFT2 exhibited higher rates of glycolysis and lactate generation compared to DFT1. This coincided with elevated ATP production, cholesterol biosynthesis, and ROS generation, as well as an increased reliance on fatty acid metabolism. Furthermore, DFT2 is less metabolically adaptable than DFT1, being unable to switch to oxidative phosphorylation as DFT1 can when required. These metabolic changes in DFT2, in conjunction with its higher growth rate, suggest a more aggressive cancer phenotype than DFT1. Our findings highlight distinct metabolic adaptations in DFT2 that may contribute to its competitive advantage.

Congenital dyserythropoietic anemia type I (CDA-I) is a rare hereditary disease marked by ineffective erythropoiesis, a characteristic spongy heterochromatin structure in erythroblasts, and mutations in the genes CDAN1 and CDIN1, which encode the proteins Codanin1 and CDIN1. Codanin1 regulates histone shuttling via the chaperone ASF1, yet the role of CDIN1 in CDA-I pathology remains unclear. Notably, CDIN1 is known to interact directly with the C-terminus of Codanin1. Although mutations in both genes are critical to the disease phenotype, their molecular-level effects have not been fully elucidated. Here, we present a comprehensive structural and functional analysis of the CDIN1-Codanin1 C-terminus complex. Using complementary biophysical techniques, we show that CDIN1 and Codanin1 C-terminus form a high-affinity heterodimeric complex with equimolar stoichiometry. We further delineate the essential interacting regions of CDIN1 and Codanin1. We demonstrate that CDA-I-associated mutations in either protein disrupt the CDIN1-Codanin1 interaction, suggesting a potential molecular mechanism underlying the disease.

The Nedd4 subfamily of HECT E3 ligases is a ubiquitous group of 10 enzymes that share the same domain structure, consisting of a C2 domain, several WW domains and a catalytic HECT domain. Over the past decade, significant progress has been made in characterizing the molecular details of their activity and regulation. Studies have shown that, in the inactive state, the HECT domain is shielded by its N-terminal domains, thereby blocking access to the active site. The catalytic functions of Nedd4 enzymes include accepting ubiquitin molecules from ubiquitin-conjugating enzymes, transferring them to substrates, and generating diverse polyubiquitin chains. The modulation of Nedd4 enzyme activity involves mechanisms that facilitate enzymatic activation, relay binding to components of the enzymatic cascade, and enable (auto)ubiquitination. This minireview provides a comprehensive overview of the structural features distinguishing the inactive and active conformations in this group of E3 ligases, while underscoring the need for further research necessary to develop pharmaceutical solutions targeting pathological conditions rooted in Nedd4 dysfunction.

Proteostasis maintains the balance between protein synthesis, folding, and degradation within the endoplasmic reticulum (ER). This quality-control system ensures that proteins undergo proper post-translational modifications-such as PDI-ERO1-mediated oxidative folding and STT3-dependent N-glycosylation-so that only correctly folded proteins proceed through the secretory pathway. Impairment of protein load, folding capacity, or degradation via the ER-associated degradation (ERAD) pathway leads to the accumulation of unfolded proteins, triggering ER stress and activating the unfolded protein response (UPR), which, in the first instance, is an adaptive signaling network designed to restore homeostasis by adjusting protein synthesis, enhancing folding capacity, and promoting the clearance of misfolded proteins. During ER stress, the ER undergoes morphological and functional remodeling to manage the increased folding burden, including an increase of ER-mitochondria contact sites (ERMCs). These nanometric junctions (~10-100 nm) facilitate lipid and metabolite exchange and mediate calcium and reactive oxygen species signaling to support cellular metabolism. However, chronic ER stress can further tighten ERMCs, leading to calcium overload, mitochondrial dysfunction, and apoptosis. This review examines the core mechanisms underlying ER proteostasis in the context of ER stress and explores how ER stress first boosts mitochondrial activity and later impairs it through ERMCs, contributing to cell death and disease. Finally, emerging therapeutic strategies aimed at restoring proteostasis and modulating the dynamics of ERMCs are highlighted as promising interventions for conditions, such as cancer and congenital myopathies, where ER and mitochondrial dysfunction play central roles in pathogenesis.