The FEBS journal最新文献

Photodamage to the outer segments of photoreceptor cells and their impaired utilization by retinal pigment epithelium (RPE) cells contribute to the development of age-related macular degeneration (AMD) leading to blindness. Degeneration of photoreceptor cells and RPE cells is triggered by reactive oxygen species (ROS) produced by photochemical reactions involving bisretinoids, by-products of the visual cycle, which accumulate in photoreceptor discs and lipofuscin granules of RPE. Carotenoids, natural antioxidants with high potential efficacy against a wide range of ROS, may protect against the cytotoxic properties of lipofuscin. To solve the problem of high hydrophobicity of carotenoids and increase their bioaccessibility, specialized proteins can ensure their targeted delivery to the affected tissues. In this study, we present new capabilities of the recombinant water-soluble protein AstaP from Coelastrella astaxanthina Ki-4 (Scenedesmaceae) for protein-mediated carotenoid delivery and demonstrate how zeaxanthin delivery suppresses oxidative stress in a lipofuscin-enriched model of photoreceptor and pigment epithelium cells. AstaP in complex with zeaxanthin can effectively scavenge various ROS (singlet oxygen, free radical cations, hydrogen peroxide) previously reported to be generated in AMD. In addition, we explore the potential of optimizing the structure of AstaP to enhance its thermal stability and resistance to proteolytic activity in the ocular media. This optimization aims to maximize the prevention of retinal degenerative changes in AMD.

Atherosclerosis (AS) is a vascular disease associated with endothelial damage, plaque formation, and retinal neovascularization (RNV), leading to visual impairment. Research indicates that vascular endothelial dysfunction, lipid deposition, and inflammatory responses contribute to the formation of plaques and atherosclerotic lesions. Among the common complications, studies have shown that RNV and the molecular mechanisms of AS hold significant clinical importance. In this study, we identified the overexpression of the gene heat shock protein 90 (HSP90) through transcriptome sequencing. Subsequent protein expression analysis and inhibition experiments in corresponding animal models confirmed the crucial role of HSP90 in the modulation of this disease. Research findings revealed an increase in the expression of HSP90, HMGA2, Snail family transcriptional repressor 2 gene (SNAI2), CXC chemokine receptor 4 (CXCR4), and vascular endothelial growth factor (VEGF) in atherosclerotic mouse tissues. Inhibition of HSP90 expression reduced vascular neovascularization and downregulated the expression of HMGA2 and VEGF. Given that HSP90 can promote HMGA2 expression, which, in turn, facilitates angiogenesis, we conducted lentiviral infection experiments on primary retinal endothelial cells obtained from atherosclerotic mice, confirming the regulatory role of HSP90 in modulating HMGA2 expression through the SNAI2/CXCR4 signaling pathway and its involvement in retinal endothelial neovascularization. In conclusion, our study highlights the significant regulatory role of HSP90 in AS-induced RNV, providing a new target for disease treatment. Furthermore, this research extensively explores the mechanism of HSP90 in regulating RNV and associated signaling pathways, offering novel insights and laying a solid foundation for future studies in this disease domain.

There are many occurrences of enzymes catalysing the same reaction but having significantly different structures. Leveraging the comprehensive information on enzymes stored in the Mechanism and Catalytic Site Atlas (M-CSA), we present a collection of 34 cases for which there is sufficient evidence of functional convergence without an evolutionary link. For each case, we compare enzymes which have identical Enzyme Commission numbers (i.e. catalyse the same reaction), but different identifiers in the CATH data resource (i.e. different folds). We focus on similarities between their sequences, structures, active site geometries, cofactors and catalytic mechanisms. These features are then assessed to evaluate whether all the evidence for these structurally diverse proteins supports their independent evolution to catalyse the same chemical reaction. Our approach combines published literature information with knowledge-based computational resources from, amongst others, M-CSA, PDBe and PDBsum, supported by tailor-made software to explore active site structures and assess similarities in mechanism. We find that there are multiple types of convergent functional evolution observed to date, and it is necessary to investigate sequence, structure, active site geometry and enzyme mechanisms to describe such convergence accurately.

Type 1 diabetes (T1D) is characterized by immune cell infiltration in the islets of Langerhans, leading to the destruction of insulin-producing beta cells. This destruction is driven by secreted cytokines and cytotoxic T cells inducing apoptosis in beta cells. Butyrate, a metabolite produced by the gut microbiota, has been shown to have various health benefits, including anti-inflammatory and anti-diabetic effects. In this study, we investigated the potential protective effects of butyrate on cytokine-induced apoptosis in beta cells and explored the underlying mechanisms. Insulin-secreting INS-1E cells and isolated mouse islets were treated with interleukin-1beta (IL-1β) or a combination of IL-1β and interferon-gamma (IFN-γ) in the presence or absence of butyrate. We analyzed apoptosis, nitric oxide (NO) levels, expression of stress-related genes, and immune cell migration. Our results demonstrated that butyrate significantly attenuated cytokine-induced apoptosis in both INS-1E cells and mouse islets, accompanied by a reduction in NO levels. Butyrate also decreased the expression of endoplasmic reticulum (ER) stress markers such as Chop, phosphorylated eIF2α and Atf4, as well as some pro-apoptotic genes including Dp5 and Puma. Butyrate reduced the cytokine-induced expression of the chemokine genes Cxcl1 and Cxcl10 in mouse islets, as well as the chemotactic activity of THP-1 monocytes toward conditioned media from IL-1β-exposed islets. In conclusion, these findings indicate that butyrate protects beta cells from cytokine-induced apoptosis and ER stress, suggesting its potential as a therapeutic agent to prevent beta cell destruction in T1D.

The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is widely used for the generation of a variety of gene products, including proteins, vaccines, and gene therapy vectors; however, it has some limitations, including a constrained host range and low protein yields. In a previous study, we established the RIRI-PA1 cell line, which was derived from Periplaneta americana. This cell line is susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection, which results in a higher yield production of recombinant protein within a short post-infection period of 24-48 h compared to the commonly used engineered cell line Sf21. To elucidate the basis for this phenomenon, we used RNA sequencing and transcriptome analysis of RIRI-PA1 and Sf21 cells infected with AcMNPV-GFP at 24, 72, and 168 h post-infection. Differentially expressed genes (DEGs) were identified in both cell lines. GO, eggNOG, and KEGG annotation analyses were used to identify DEGs and select candidate genes that could regulate recombinant protein expression. The results indicated a significant link between ribosomal pathway regulation and recombinant protein expression. After 24 h of AcMNPV-GFP infection, relatively high levels of protein were produced in RIRI-PA1 cells compared to Sf21 cells, which exhibited lesser enrichment of ribosomal protein-related DEGs (7 : 12). Moreover, a correlation was observed in the gene expression patterns between AcMNPV-GFP infection and recombinant protein synthesis, including genes associated with the ribosome, Toll and Imd signaling, and the cytochrome P450 pathway. Overall, our findings suggested that the ribosomal pathway might be more involved in regulation of protein expression during the early stages of RIRI-PA1 infection. The mechanisms underlying this process could have potential future applications in engineering cell modifications to reduce production time for recombinant proteins and to promote the use of IC-BEVS.

Tissue specificity can render mitochondrial uncouplers more promising as leading compounds for creating drugs against serious diseases. In search of tissue-specific uncouplers, we address anilinothiophenes as possible glutathione-S-transferase substrates (GST). Earlier, 'cyclic' uncoupling activity was reported for 5-bromo-N-(4-chlorophenyl)-3,4-dinitro-2-thiophenamine (BDCT) in isolated rat liver mitochondria (RLM). The mechanism by which BDCT induced two-phase changes in mitochondrial respiration (stimulation followed by deceleration) was unknown. To clarify this issue, we synthesized BDCT and its two analogues. Among these, 5-bromo-3,4-dinitro-N-(4-nitrophenyl)-2-thiophenamine (BDNT) appeared to be the most effective as a mitochondrial uncoupler, decreasing membrane potential and stimulating respiration at submicromolar concentrations. Importantly, BDNT exerted two-phase changes in both mitochondrial membrane potential and respiration rate of RLM, which were enhanced by the addition of glutathione (GSH) but inhibited by the compounds capable of GSH depleting, such as 1-chloro-2,4-dinitrobenzene (CDNB). By contrast, the phase of recoupling was not observed in rat heart mitochondria (RHM). Remarkably, BDNT elicited mitochondrial depolarization in primary human fibroblasts but not in cultured human liver (HepG2) cells. By detecting proton-selective electrical current through planar bilayer lipid membranes, we demonstrated the ability of BDCT and BDNT to transfer protons across membranes. BDNT proved to be an anionic protonophore with a pK_a of 7.38. By using LC-MS and capillary electrophoresis, we directly showed the formation of BDNT conjugates with GSH upon incubation with RLM but not RHM. Therefore, we hypothesize that GST is involved in the disappearance of the anilinothiophene uncoupling activity in RLM, ensuring the tissue-specific behavior of the uncoupler.

The mitochondrial disulphide relay machinery is essential for the import and oxidative folding of many proteins in the mitochondrial intermembrane space. Its core component, the import receptor MIA40 (also CHCHD4), serves as an oxidoreductase but also as a chaperone holdase, which initially interacts with its substrates non-covalently before introducing disulphide bonds for folding and retaining proteins in the intermembrane space. Interactome studies have identified diverse substrates of MIA40, among them the intrinsically disordered HCLS1-associated protein X-1 (HAX1). Interestingly, this protein does not contain cysteines, raising the question of how and to what end HAX1 can interact with MIA40. Here, we demonstrate that MIA40 non-covalently interacts with HAX1 independent of its redox-active cysteines. While HAX1 import is driven by its weak mitochondrial targeting sequence, its subsequent transient interaction with MIA40 stabilizes the protein in the intermembrane space. HAX1 solely depends on the holdase activity of MIA40, and the absence of MIA40 results in the aggregation, degradation and loss of HAX1. Collectively, our study introduces HAX1 as the first endogenous MIA40 substrate without cysteines and demonstrates the diverse functions of this highly conserved oxidoreductase and import receptor.

N^ω-hydroxy-l-arginine amidinohydrolase (EC:3.5.3.25), an enzyme in the d-cycloserine (d-CS) biosynthetic pathway of Streptomyces lavendulae, catalyzes the hydrolysis of an arginase inhibitor, N^ω-hydroxy-l-arginine, to produce l-ornithine and hydroxyurea, despite being homologous to arginase. Like arginase, the enzyme (DcsB) possesses two manganese ions (Mn_A and Mn_B) essential for the enzymatic reaction at the bottom of the cavity formed within the molecule. However, one of the Mn_A ligands in DcsB is Cys86, whereas the corresponding residues in arginase are histidine. In this study, we determined the crystal structure of Mn-free DcsB to elucidate the installation mechanism of the manganese ions. The flipping of the His111 residue after the formation of the coordination bond to the second manganese ion may facilitate the installation of Mn_B and the closing of the cavity entrance to retain Mn_A and Mn_B at the active site. Copper ions, which are known to be a positive regulator of many secondary metabolites in Streptomyces species, were found to irreversibly inactivate the catalytic activity of DcsB. Mass spectrometric and crystallographic analyses of the Cu(II)-treated DcsB indicated that Cys86 is oxidized to sulfinic acid. The d-CS biosynthesis in the producing microorganism may be negatively regulated by the concentration of intracellular copper ions, which mediates the oxidative stress.

Membrane fusion is an essential biological process that merges two separate lipid bilayers into a whole one. Membrane fusion proteins facilitate this process by bringing lipid bilayers in close proximity to reduce the repulsive energy between membranes. Along with their interactions with membranes, the structures and dynamics of membrane fusion proteins are key to elucidating the mechanisms of membrane fusion. Solid-state NMR (SSNMR) spectroscopy has unique advantages in determining the structures and dynamics of membrane fusion proteins in their membrane-bound states. It has been extensively applied to reveal conformational changes in intermediate states of viral membrane fusion proteins and to characterize the critical lipid-membrane interactions that drive the fusion process. In this review, we summarize recent advancements in SSNMR techniques for studying membrane fusion proteins and their applications in elucidating the mechanisms of membrane fusion.

Atherosclerosis (AS) is a chronic inflammatory disease of arteries. Endothelial inflammation is key to the initiation and development of AS. The calcium-sensing receptor (CaSR) is expressed in endothelial cells (ECs) but its role in endothelial inflammation during AS remains unclear. This study focused on the involvement of CaSR in regulating endothelial inflammation and its underlying mechanisms, providing novel insights for AS therapy. Here, we observed that CaSR agonist NPS-R568 significantly reduced atherosclerotic lesions and aortic inflammation in high-fat diet (HFD)-fed ApoE^-/- mice, while enhancing the expression of CaSR in aortic tissues. In vitro, human umbilical vein endothelial cells (HUVECs) exposed to oxidized low-density lipoprotein (oxLDL) at 20 μg·mL^-1 triggered inflammation, as indicated by the upregulation of vascular cell adhesion molecule-1 (VCAM-1), interleukin (IL)-6, and IL-1β expression, along with increased adherence of THP-1 or U937 cells to the HUVECs. Additionally, treatment with 20 μg·mL^-1 oxLDL led to downregulation of CaSR expression in HUVECs. The administration of CaSR agonist NPS-R568 or overexpression of CaSR in HUVECs resulted in a significant reversal of inflammation induced by oxLDL. Mechanistically, CaSR was found to mitigate NLRP3 inflammasome activation by downregulating the protein level of integrin β1. In conclusion, our study elucidates the beneficial role of CaSR in reducing endothelial inflammation in AS through the regulation of integrin β1 and the subsequent NLRP3 inflammasome. CaSR emerges as a promising target for potential therapeutic interventions in AS.