Sleep and circadian disruptions are early symptoms of Parkinson's disease (PD), which is one of the most common neurodegenerative disorders. However, PD has an idiopathic origin, and the factors accelerating the progress...Sleep and circadian disruptions are early symptoms of Parkinson's disease (PD), which is one of the most common neurodegenerative disorders. However, PD has an idiopathic origin, and the factors accelerating the progression of symptoms are not fully understood. One genetic factor associated with PD is a mutation in the parkin gene, which impairs mitophagy and increases oxidative stress. In this research, we used Drosophila melanogaster as a model of PD, employing both a park mutant and cell-specific park silencing, and followed the progression of main clock disruption. Our data suggest that pacemaker neurons are sensitive to oxidative stress, and increased ROS levels disrupt daily changes in the morphology of their termini, affecting circadian network communication and sleep regulation.
Mitochondrial oxidative phosphorylation relies on cytochrome c transferring electrons between complexes III and IV. Earlier studies using detergent-purified complex III-IV supercomplexes from S. cerevisiae showed that th...Mitochondrial oxidative phosphorylation relies on cytochrome c transferring electrons between complexes III and IV. Earlier studies using detergent-purified complex III-IV supercomplexes from S. cerevisiae showed that this transfer is limited by two-dimensional cytochrome c diffusion. This study investigates this process in membrane-embedded mitoplasts. The results show that membrane embedment shifts the rate-limiting step from cytochrome c-mediated electron transfer to the catalytic activity of the supercomplex itself. Up to a cytochrome c : supercomplex ratio of unity, turnover increases sharply regardless of ionic strength. At higher ratios, the rate levels out at 15-20 s, indicating that the process is no longer limited by salinity-dependent electron transfer, but rather by the catalytic capacity of complex IV.
To sustain rapid proliferation, cancer cells increase protein synthesis, intensifying reliance on protein disulfide isomerase A1 (PDIA1). It is largely unknown whether disulfide bond formation of PDIA1 substrates is driv...To sustain rapid proliferation, cancer cells increase protein synthesis, intensifying reliance on protein disulfide isomerase A1 (PDIA1). It is largely unknown whether disulfide bond formation of PDIA1 substrates is driven by one or both CGHC motifs. Using active-site trapping mutants in prostate cancer cells combined with mass spectrometry, we identified 29 proteins uniquely bound to the CGHC domain and 20 proteins uniquely bound to the CGHC domain. Hyaluronan-mediated motility receptor (HMMR) was validated as a PDIA1 CGHC-specific substrate, with PDIA1 catalysing disulfide bond formation between Cys242 and Cys293. PDIA1 knockdown induced HMMR ubiquitination, blocked androgen receptor nuclear translocation, and suppressed prostate cancer cell growth, survival, and migration. These findings reveal a previously unknown role of PDIA1 in prostate cancer biology.
Depolarizing stimuli regulate acetylcholine (ACh) storage and release via distinct synaptic vesicle pools in cholinergic terminals, but the mechanisms remain unclear. Here, we examined the effects of preliminary depolari...Depolarizing stimuli regulate acetylcholine (ACh) storage and release via distinct synaptic vesicle pools in cholinergic terminals, but the mechanisms remain unclear. Here, we examined the effects of preliminary depolarization on choline uptake, ACh synthesis, and release in rat striatal segments using [H]choline incorporation and [H]ACh release assays. Preliminary KCl depolarization significantly increased functional high-affinity choline transporter-1 (CHT1) availability, enhancing subsequent [H]choline uptake, [H]ACh synthesis, and release. In contrast, preliminary electrical stimulation selectively increased evoked [H]ACh release without affecting choline uptake or tissue ACh content. These results suggest that electrical stimulation primarily mobilizes vesicles from the readily releasable pool (RRP) and recycling pool, whereas KCl depolarization additionally recruits vesicles from the reserve pool and may directly trigger ACh release.
Alphavirus is a genus of enveloped, single-stranded positive-sense RNA viruses with an icosahedral virion consisting of two T = 4 layers. These viruses are transmitted by invertebrates, are present in almost every ecosys...Alphavirus is a genus of enveloped, single-stranded positive-sense RNA viruses with an icosahedral virion consisting of two T = 4 layers. These viruses are transmitted by invertebrates, are present in almost every ecosystem, and infect a wide range of vertebrates. The assembly and budding of these viruses are highly coordinated processes that involve capsid protein-RNA, capsid protein-capsid protein, capsid protein-glycoprotein, and glycoprotein-glycoprotein interactions. Assembly and budding have been studied for more than 40 years, and in the past decade, key, sometimes paradigm-shifting results have changed our understanding of their structure and assembly. However, there are fundamental aspects of RNA selective packaging, core and virion assembly, budding, and the interactions that control these processes that remain to be fully understood. Here, I provide an updated overview of these processes and highlight areas to further improve our understanding of alphaviruses.
Viruses represent a major threat to human health, while simultaneously exhibiting great potential in a wide range of applications, from virus-inspired devices to therapeutic delivery agents. Addressing virus-related ques...Viruses represent a major threat to human health, while simultaneously exhibiting great potential in a wide range of applications, from virus-inspired devices to therapeutic delivery agents. Addressing virus-related questions from an interdisciplinary standpoint promises to open new avenues, both in the fight against viral diseases and in the exploitation of viral structures to advance technology. This has stimulated the development of 'physical virology', a growing research field gathering researchers from various scientific disciplines with a common interest in viruses. The FEBS|EMBO Lecture course on Physical Virology brought together top researchers working with viruses to inspire and further educate a new generation of transdisciplinary virus-oriented scientists and to cement the growing physical virology community.
Phthalates are ubiquitous environmental contaminants and suspected endocrine disruptors, used as plasticizers and constituents of fragrances. Regulation of their use in consumer products has largely been guided by male r...Phthalates are ubiquitous environmental contaminants and suspected endocrine disruptors, used as plasticizers and constituents of fragrances. Regulation of their use in consumer products has largely been guided by male reproductive outcomes. However, as evidenced in rodent models, extended contact with phthalates also affects ovarian function. Data from human ovarian samples, ovary-derived cell lines, and epidemiological studies of infertile patient cohorts have demonstrated adverse associations between phthalate-related chemical burden and female fertility. Due to the sustained use of phthalate-containing products, women may experience earlier fertility decline. These outcomes can be linked to molecular disturbances in ovarian follicles, where phthalate metabolites are frequently detected. This review synthesizes current human evidence on phthalate impacts on the ovary.
Hyperglycaemia is an independent risk factor for pancreatic cancer (PC). Here, we show that both chronic and intermittent high glucose cause a robust upregulation of the novel lncRNA LINC00538 (YIYA) in pancreatic ductal...Hyperglycaemia is an independent risk factor for pancreatic cancer (PC). Here, we show that both chronic and intermittent high glucose cause a robust upregulation of the novel lncRNA LINC00538 (YIYA) in pancreatic ductal adenocarcinoma (PDAC) cells. YIYA enhances PDAC cell proliferation in both 2D and 3D culture systems, and its expression shows a positive correlation with poor patient survival. Importantly, knockdown of YIYA attenuates the proliferative effect. Mechanistically, YIYA drives the Warburg phenotype in a KRAS-dependent manner. YIYA physically interacts with KRAS and the glycolytic enzyme pyruvate kinase 2 (PKM2). Notably, YIYA stabilises KRAS by preventing its autophagy-mediated degradation, thereby sustaining a proliferative state. This study identifies YIYA as a glucose-responsive lncRNA that links KRAS signalling to metabolic reprogramming in PDAC.
Gland cells dynamically regulate their secretory granule content via balancing synthesis, maturation, secretion, and lysosomal degradation (crinophagy). However, the signal(s) leading to crinophagic breakdown of secretor...Gland cells dynamically regulate their secretory granule content via balancing synthesis, maturation, secretion, and lysosomal degradation (crinophagy). However, the signal(s) leading to crinophagic breakdown of secretory granules are unknown. Here, we show that ubiquitination of unreleased or low-grade glue-containing secretory granules marks these vesicles for crinophagy in larval salivary gland cells of Drosophila. We identify the ubiquitin ligase Cnot4 as a key mediator of glue granule ubiquitination. Loss of Cnot4 prevents ubiquitination and impairs granule fusion with lysosomes. Overexpression of Cnot4 induces premature crinophagy via ectopic ubiquitination of granules. Our work establishes that Cnot4-dependent ubiquitination of secretory granules is a key trigger of crinophagy in Drosophila, paving the way for further analysis of this barely characterized degradation route in metazoans.
Viruses infect all living organisms and have been responsible for major epidemics and pandemics. Their ongoing evolutionary battle with host defenses creates a constant need for improved tools to study viral behavior. Ad...Viruses infect all living organisms and have been responsible for major epidemics and pandemics. Their ongoing evolutionary battle with host defenses creates a constant need for improved tools to study viral behavior. Advancing methods to probe viral attachment, fusion, and genome release deepen our understanding of how infections begin and support the development of effective antiviral strategies. In this graphical review, we provide an overview of selected microscopy-based biophysical methods, most relying on extracellular experimental systems, that allow interrogation of the various steps leading to viral entry in both enveloped and non-enveloped viruses.
The nucleocapsid (N) protein of SARS-CoV-2 is central to viral assembly and replication. It binds the viral RNA to form a helical nucleocapsid and enables genome packaging and its release into host cells. Human heterogen...The nucleocapsid (N) protein of SARS-CoV-2 is central to viral assembly and replication. It binds the viral RNA to form a helical nucleocapsid and enables genome packaging and its release into host cells. Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), one of the most abundant RNA-binding proteins in eukaryotes, regulates key aspects of RNA metabolism, including splicing, transcription, localisation, and transport. Here, we report a direct physical interaction between the SARS-CoV-2 N protein and hnRNPA1, primarily mediated through their intrinsically disordered regions. Furthermore, we found that these proteins co-phase separate in vitro and colocalise within stress granules in cells. In vivo studies reveal that hnRNPA1 suppresses viral replication, suggesting that the N protein-hnRNPA1 interaction plays an important role in modulating the viral life cycle.
The molecular mechanisms by which artificial electron mediators, such as ethyl viologen (EV), interact with tungsten-containing formate dehydrogenases (FDHs) during reversible CO reduction remain poorly understood. Here,...The molecular mechanisms by which artificial electron mediators, such as ethyl viologen (EV), interact with tungsten-containing formate dehydrogenases (FDHs) during reversible CO reduction remain poorly understood. Here, we reveal an unexpected alternative mediator interaction site in FDH1 of Methylorubrum extorquens AM1. Removing the native flavin mononucleotide cofactor abolishes NAD-dependent activity but preserves EV-driven catalysis. Through mutagenesis, kinetic analysis, and molecular docking, we identified a cooperative aromatic network-comprising residues F232, F471, and Y329-that stabilizes EV via stacking interactions near the proximal B1 iron-sulfur cluster. Disrupting these residues impairs EV-mediated electron transfer without destabilizing global structure. These findings reveal a dual interaction strategy for artificial mediators in tungsten FDHs, offering a structural framework to rationally engineer biocatalysts for CO conversion.
Maintaining a functional proteome is essential for cellular health and organismal longevity. Disruption of proteostasis is a hallmark of aging and a central driver of diverse pathologies, including neurodegeneration, can...Maintaining a functional proteome is essential for cellular health and organismal longevity. Disruption of proteostasis is a hallmark of aging and a central driver of diverse pathologies, including neurodegeneration, cancer, and metabolic disease. The ubiquitin-proteasome system (UPS) and autophagy represent the two principal degradative pathways safeguarding proteome integrity, particularly under conditions of stress. While historically viewed as mechanistically distinct, it is now clear that UPS and autophagy operate as an interconnected and adaptive network. This Perspective discusses three core principles that govern their coordination: (1) a shared molecular language of ubiquitin signals and shuttle proteins that determines cargo routing; (2) spatial compartmentalization through organelle-specific quality control modules and phase-separated degradation hubs; and (3) temporal regulation by stress-responsive signaling pathways that reprogram proteolytic output. Understanding this dynamic partnership not only reveals fundamental organizing principles of cellular homeostasis but also identifies new therapeutic nodes for diseases driven by proteostasis collapse.
The EVH1 domain of the Homer1 scaffold protein interacts with the proline-rich region of Shank3, forming a key network within the postsynaptic density. While two mutations (M65I and S97L) in the EVH1 domain have been sug...The EVH1 domain of the Homer1 scaffold protein interacts with the proline-rich region of Shank3, forming a key network within the postsynaptic density. While two mutations (M65I and S97L) in the EVH1 domain have been suggested to be associated with autism spectrum disorder, our results show that neither mutation has a substantial effect on the overall structure or the partner binding properties of Homer1. Compared to the S97L variant, the M65I mutant exhibits larger chemical shift perturbations both upon the mutation itself and during partner binding, while also showing signs of thermal destabilization. Finally, integration of computational and NMR data suggests that both mutations perturb the μs-ms timescale internal motions of the EVH1 domain.
Human papillomavirus (HPV) utilizes multiple host proteins to infect epithelial cells. Here we identified Rab14, a small GTPase belonging to the Rab family, as a critical host factor in HPV infection. Rab14 knockout (KO)...Human papillomavirus (HPV) utilizes multiple host proteins to infect epithelial cells. Here we identified Rab14, a small GTPase belonging to the Rab family, as a critical host factor in HPV infection. Rab14 knockout (KO) cells exhibited resistance to HPV pseudovirion (PsV) infection. Confocal microscopy revealed that PsV transport to the trans-Golgi network (TGN) was significantly reduced in Rab14 KO cells. Reintroduction of wild-type Rab14 restored infection levels, but neither GDP-bound (S25N) nor GTP-bound (Q70L) mutants did. S25N preferentially localized to the TGN, while Q70L exhibited similar localization in the early endosome and the TGN. PsV showed reduced colocalization with S25N, whereas the opposite was observed for Q70L. These results indicate that Rab14 GTPase cycling controls HPV transport to the TGN.
The small-molecule pyridazinone derivative IMB5036 (IMB) exhibits significant cytotoxicity against multiple cancer cell lines, and KH-type splicing regulatory protein (KSRP) is confirmed as its direct binding partner. Ho...The small-molecule pyridazinone derivative IMB5036 (IMB) exhibits significant cytotoxicity against multiple cancer cell lines, and KH-type splicing regulatory protein (KSRP) is confirmed as its direct binding partner. However, KSRP's functional role in neuroblastoma (NB) and the mechanism mediating IMB's antitumor effects remain unclear. This study analyzed public tumor databases and found KSRP expression negatively correlates with NB patients' median survival. Experiments showed IMB induces NB cell pyroptosis, immunogenic cell death (ICD, with calreticulin exposure), and cGAS-STING activation (to boost antitumor immunity), while CRISPR-Cas9-mediated KSRP knockout notably attenuates these effects. Transcriptome sequencing further confirmed KSRP mediates IMB's regulation of HSPA6/RSAD2. This study clarifies KSRP-dependent ICD drives NB antitumor immunity, providing a basis for KSRP-targeted NB immunotherapies.
Septins are cytoskeletal GTP-binding proteins that organize microtubules and scaffold polarity complexes. Here, we uncover a polybasic (PB)-domain-dependent mechanism by which septin 9 controls apico-basal polarity in ep...Septins are cytoskeletal GTP-binding proteins that organize microtubules and scaffold polarity complexes. Here, we uncover a polybasic (PB)-domain-dependent mechanism by which septin 9 controls apico-basal polarity in epithelial cells. Septin 9 regulates centrosome positioning and the asymmetric distribution of acetylated microtubules, which are required for ciliogenesis and lumen formation. Knockdown of septin 9 or deletion of its PB domains disrupts centrosome asymmetry, induces a symmetric distribution of acetylated tubulin, and impairs cilia formation, leading to polarity inversion. These defects are associated with increased expression of the microtubule deacetylase HDAC6. Importantly, inhibition of TGF-β signaling or selective HDAC6 inhibition with tubacin restores microtubule acetylation and rescues apico-basal polarity. Together, our findings identify septin 9 as a cytoskeletal integrator that links centrosome positioning and microtubule acetylation to epithelial morphogenesis through the TGF-β/HDAC6 pathway.
The developing central nervous system is regionalized into different domains to generate functionally distinct brain structures. Although this regionalization is crucial for constructing a functional brain, its regulator...The developing central nervous system is regionalized into different domains to generate functionally distinct brain structures. Although this regionalization is crucial for constructing a functional brain, its regulatory mechanisms remain unclear. Here, we investigated regulatory mechanisms of the regionalization of the fly visual center. The medulla and lamina, two different ganglia, originate from the same precursor. We found that loss of anterior open (Aop), a negative regulator of epidermal growth factor receptor (EGFR) signaling, resulted in the ectopic formation of medulla neural stem cells in the developing lamina. In contrast, the lamina was expanded and intruded into the medulla after misexpression of Aop. Our findings suggest that Aop inactivates EGFR signaling in the prospective lamina to escape differentiation into the medulla for successful regionalization.
Atomic force microscopy-AFM enables label-free quantification of cellular and tissue mechanics with nanoscale resolution under near-physiological conditions, providing access to physical properties that are not captured...Atomic force microscopy-AFM enables label-free quantification of cellular and tissue mechanics with nanoscale resolution under near-physiological conditions, providing access to physical properties that are not captured by conventional approaches. In cancer, mechanical remodeling spans multiple scales, from cytoskeletal reorganization and increased cell deformability to extracellular matrix deposition and desmoplastic stiffening of solid tumors. These alterations generate reproducible nanomechanical fingerprints that distinguish cancer states at both the single-cell and tissue level. This review summarizes the principles underlying AFM, discusses their application in cancer diagnosis via tumor biopsy analysis, and highlights emerging efforts to extend mechanophenotyping toward circulating tumor-associated analytes in liquid biopsy. Finally, we illustrate the broader applicability of this framework using pulmonary fibrosis and discuss key challenges for clinical translation.