The FEBS journal最新文献

Calcium influx via the L-type voltage-gated Ca_v1.2 calcium channel in smooth muscle cells regulates vascular contraction. Calcium channel blockers (CCBs) are widely used to treat hypertension by inhibiting Ca_v1.2 channels. Using the vascular smooth muscle cell line, A7r5 and primary culture of cerebral vascular smooth muscle cells, we found that the expression and function of Ca_v1.2 channels are downregulated during hypoxia. Furthermore, hypoxia induces structural changes in Ca_v1.2 channels via alternative splicing. The expression of exon 9* is upregulated, whereas exon 33 is downregulated. Such structural alterations of Ca_v1.2 channels are caused by the decreased expression of RNA-binding proteins RNA-binding protein fox-1 homolog 1 and 2 (RbFox1 and RbFox2). Overexpression of RbFox1 and RbFox2 prevents hypoxia-induced exon 9* inclusion and exon 33 exclusion. Importantly, such structural alterations of the Ca_v1.2 channel partly contribute to the enhanced sensitivity of Ca_v1.2 to isradipine (a CCB) under hypoxia. Overexpression of RbFox1 and RbFox2 successfully reduces isradipine sensitivity in hypoxic smooth muscle cells. Our results suggest a new strategy to manage ischemic diseases such as stroke and myocardial infarction.

Actin is a highly conserved and fundamental protein in eukaryotes and participates in a broad spectrum of cellular functions. Cells maintain a conserved ratio of actin isoforms, with muscle and non-muscle actins representing the main actin isoforms in muscle and non-muscle cells, respectively. Actin isoforms have specific and redundant functional roles and display different biochemistries, cellular localization, and interactions with myosins and actin-binding proteins. Understanding the specific roles of actin isoforms from the structural and functional perspective is crucial for elucidating the intricacies of cytoskeletal dynamics and regulation and their implications in health and disease. Here, we review how the structure contributes to the functional mechanisms of actin isoforms with a special emphasis on the questions of how post-translational modifications and disease-linked mutations affect actin isoforms biochemistry, function, and interaction with actin-binding proteins and myosin motors.

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur-mediated mechanism independent of iron, which affects Fur DNA-binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur-regulated genes in vivo, is specific and does not require co-regulator metal under reducing conditions. Fur treatment with H₂O₂ produces dose-dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH-thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection.

Cultivated rice (Oryza sativa) produces a variety of diterpenoid-type phytoalexins. Diterpene synthase genes that are responsible for the biosynthesis of momilactones, phytocassanes, and oryzalexins have been identified in O. sativa cv. Nipponbare. OsKSL10 (Os12t0491800 in RAP and LOC_Os12g30824 in MSU) was previously identified as an enzyme catalyzing the conversion of ent-copalyl diphosphate to ent-sandaracopimaradiene for the production of oryzalexins A to F. Our previous study on Oryza rufipogon, a wild progenitor of Asian cultivated rice, showed that both OrKSL10 and OrKSL10ind from O. rufipogon accessions W1943 and W0106, respectively, closely related to the japonica and indica subspecies, converted ent-copalyl diphosphate to ent-miltiradiene. Thus, the functional conversion of ent-miltiradiene synthase into ent-sandaracopimaradiene synthase is implied to have occurred through natural amino acid mutations, the details of which have not been elucidated. In this study, we show that introduction of A654G substitution into OrKSL10 significantly alters its function into more closely resembling that of OsKSL10. Moreover, double substitution V546I/A654G almost completely converts the function of OrKSL10 into that of OsKSL10. On the other hand, the reversed substitution I546V/G654A was insufficient to convert the function of OsKSL10 into OrKSL10, indicating the introduction of additional substitution S522I is required for the functionality of OsKSL10. Lastly, point mutations at the ⁶⁵⁴A residue in OrKSL10 suggest that hydrophobic side chains at this position have a negative influence on the production of ent-sandaracopimaradiene.

Cancer cells exhibit a unique metabolic preference for the glycolytic pathway over oxidative phosphorylation for maintaining the tumor microenvironment. Lactate dehydrogenase A (LDHA) is a key enzyme that facilitates glycolysis by converting pyruvate to lactate and has been shown to be upregulated in multiple cancers due to the hypoxic tumor microenvironment. Diclofenac (DCF), a nonsteroidal anti-inflammatory drug, has been shown to exhibit anticancer effects by interfering with the glucose metabolism pathway. However, the specific targets of this drug remain unknown. Using in silico, biochemical, and biophysical studies, we show that DCF binds to LDHA adjacent to the substrate binding site and inhibits its activity in a dose-dependent and allosteric manner in HeLa cells. Thus, DCF inhibits the hypoxic microenvironment and induces apoptosis-mediated cell death. DCF failed to induce cytotoxicity in HeLa cells when LDHA was knocked down, confirming that DCF exerts its antimitotic effects via LDHA inhibition. DCF-induced LDHA inhibition alters pyruvate, lactate, NAD⁺, and ATP production in cells, and this could be a possible mechanism through which DCF inhibits glucose uptake in cancer cells. DCF-induced ATP deprivation leads to mitochondria-mediated oxidative stress, which results in DNA damage, lipid peroxidation, and apoptosis-mediated cell death. Reduction in intracellular ATP levels additionally activates the sensor kinase, adenosine monophosphate-activated protein kinase (AMPK), which further downregulates phosphorylated ribosomal S6 kinase (p-S6K), leading to apoptosis-mediated cell death. We find that in LDHA knocked down cells, intracellular ATP levels were depleted, resulting in the inhibition of p-S6K, suggesting the involvement of DCF-induced LDHA inhibition in the activation of the AMPK/S6K signaling pathway.

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3′ specificities of PPEP-1 and PPEP-2, while identifying the endogenous substrate of PPEP-2 as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.

White and brown adipocytes are central mediators of lipid metabolism and thermogenesis, respectively. Their function is tightly regulated by all three β-adrenergic receptor (β-AR) subtypes which are coupled to the production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). While known for decades in other cell types, compartmentation of adipocyte β-AR/cAMP signaling by spatial organization of the pathway and by cAMP degrading phosphodiesterases (PDEs) as well as its role in the regulation of lipolysis is only beginning to emerge. Here, we provide a short overview of recent findings which shed light on compartmentalized signaling using live cell imaging of cAMP in adipocytes and discuss possible future directions of research which could open up new avenues for the treatment of metabolic disorders.

Phosphatidylinositol phosphates are powerful signaling molecules that orchestrate signaling and direct membrane trafficking in the cytosol. Interestingly, phosphatidylinositol phosphates also localize within the membrane-less compartments of the cell nucleus, where they participate in the regulation of gene expression. Nevertheless, current models of gene expression, which include condensates of proteins and nucleic acids, do not include nuclear phosphatidylinositol phosphates. This gap is partly a result of the missing detailed analysis of the subnuclear distribution of phosphatidylinositol phosphates and their relationships with gene expression. Here, we used quantitative dual-color direct stochastic optical reconstruction microscopy to analyze the nanoscale co-patterning between RNA polymerase II transcription initiation and elongation markers with respect to phosphatidylinositol 4,5- or 3,4-bisphosphate in the nucleoplasm and nuclear speckles and compared it with randomized data and cells with inhibited transcription. We found specific co-patterning of the transcription initiation marker P-S5 with phosphatidylinositol 4,5-bisphosphate in the nucleoplasm and with phosphatidylinositol 3,4-bisphosphate at the periphery of nuclear speckles. We showed the specific accumulation of the transcription elongation marker PS-2 and of nascent RNA in the proximity of phosphatidylinositol 3,4-bisphosphate associated with nuclear speckles. Taken together, this shows that the distinct spatial associations between the consecutive stages of RNA polymerase II transcription and nuclear phosphatidylinositol phosphates exhibit specificity within the gene expression compartments. Thus, in analogy to the cellular membranes, where phospholipid composition orchestrates signaling pathways and directs membrane trafficking, we propose a model in which the phospholipid identity of gene expression compartments orchestrates RNA polymerase II transcription.

Docetaxel (Doc) currently serves as the primary first-line treatment for patients with castrate-resistant prostate cancer (CRPC). Erastin, a small molecule compound, can trigger inhibition of the cystine–glutamate reverse transport system and other pathways, leading to iron-dependent cell death (ferroptosis). Beyond its role in inducing cancer cell death, erastin demonstrates potential when combined with chemotherapy drugs to heighten cancer cell drug susceptibility. However, the augmentation by erastin of the effects of Doc treatment on prostate cancer, and the underlying mechanisms involved, remain unclear. In the present study, we determined the role and the underlying molecular mechanism of erastin against CRPC. The results showed that CRPC cell lines were resistant to Doc, and the expression of ferroptosis-related factors in drug-resistant cell lines was downregulated. Erastin, in synergy with Doc, exerts a pro-apoptotic effect. Erastin significantly inhibited the activity of ATP-binding cassette subfamily B member 1 (ABCB1) but did not change its protein expression and localization. Finally, in mice, erastin treatment dramatically reduced tumor growth in vivo. Taken together, our findings demonstrate that erastin enhances Doc-induced apoptosis to a certain extent and reverses Doc resistance in prostate cancer by inhibiting the activity of multidrug-resistant protein ABCB1.

Tauopathies exhibit a characteristic accumulation of misfolded tau aggregates in the brain. Tau pathology shows disease-specific spatiotemporal propagation through intercellular transmission, which is closely correlated with the progression of clinical manifestations. Therefore, identifying molecular mechanisms that prevent tau propagation is critical for developing therapeutic strategies for tauopathies. The various innate immune receptors, such as complement receptor 3 (CR3) and complement receptor 4 (CR4), have been reported to play a critical role in the clearance of various extracellular toxic molecules by microglia. However, their role in tau clearance has not been studied yet. In the present study, we investigated the role of CR3 and CR4 in regulating extracellular tau clearance. We found that CR4 selectively binds to tau fibrils but not to tau monomers, whereas CR3 does not bind to either of them. Inhibiting CR4, but not CR3, significantly reduces the uptake of tau fibrils by BV2 cells and primary microglia. By contrast, inhibiting CR4 has no effect on the uptake of tau monomers by BV2 cells. Furthermore, inhibiting CR4 suppresses the clearance of extracellular tau fibrils, leading to more seed-competent tau fibrils remaining in the extracellular space relative to control samples. We also provide evidence that the expression of CR4 is upregulated in the brains of human Alzheimer's disease patients and the PS19 mouse model of tauopathy. Taken together, our data strongly support that CR4 is a previously undescribed receptor for the clearance of tau fibrils in microglia and may represent a novel therapeutic target for tauopathy.