The FEBS journal最新文献

The binding of tetrameric E. coli single-stranded DNA-binding protein (SSB) to single-stranded DNA (ssDNA) was investigated using genetically encoded noncanonical amino acids (ncAA) as site-specific probes for detection by nuclear magnetic resonance (NMR) spectroscopy. Under the conditions used (300 mm NaCl, pH 7.2), the NMR spectra confirmed the equivalence of the monomeric subunits in the absence of ssDNA. Most of the probes responded to the binding of ssDNA by changes in chemical shifts and line width and distinguished between the presence of segments of cytidine versus thymidine. Although ssDNA-binding breaks the fourfold symmetry of the SSB tetramer, the probes sensed closely similar chemical environments in all four monomeric subunits. By comparing the performance of twelve different NMR-active ncAAs, this work identified N⁶-trifluoroacetyl-L-lysine (TFAK) as the ncAA sensing different ssDNAs with the best spectral resolution. In addition, we report aminoacyl-tRNA synthetases for the genetic encoding of 3,5-difluoro-L-tyrosine (3,5-diFTyr), 2,6-difluoro-L-tyrosine (2,6-diFTyr), and mCF₃-phenylalanine. The SSB construct was sensitive to precipitation under NMR conditions. The fluorinated ncAAs altered the rates of precipitation which varied even between fluorotryptophan isomers installed at a barely solvent-accessible site. Nonetheless, the NMR-active ncAAs proved suitable for probing a marginally stable protein system of ca. 100 kDa molecular weight without isotope labelling and at low concentration. The current data suggest that ¹⁹F spins attached to flexible solvent-exposed amino acid side chains guard better against protein precipitation than fluorinated aromatic amino acids despite the latter being more attractive for their close structural similarity to their canonical amino acid counterparts.

Extracellular matrix (ECM) proteins, known as the matrisome, have long been recognized for their structural roles and their control of cell phenotypes in health and disease. The broad range of biological activities mediated by the ECM has been further expanded with the discovery that fragments released upon ECM remodeling are also bioactive with functions that can differ from those of their parent proteins. This review provides an overview of the latest findings describing the roles of major bioactive fragments from collagens I, IV, VI, and XVIII in various physiological and pathological contexts affecting heart, lung, adipose, and even brain tissues. Angiogenesis, inflammation, fibrosis, and cancer are the most frequent processes regulated by collagen fragments. Perspectives on how these bioactive fragments could impact translational research as drug candidates, drug targets, and biomarkers are also discussed together with their biomedical applications.

Located at the crossroads between mitochondria and cytosol, VDAC1 (Voltage-Dependent Anion Selective Channel isoform 1) serves as the chief actor in the regulation of cell metabolism and apoptosis. The crucial role in cell fate determination has long made VDAC1 a promising target in cancer research. The recent discovery of a highly conserved and druggable NADH-like binding pocket has led to the development of specific VDAC antagonists (VA) with potential antitumor activity. Here, we performed electrophysiological analysis in artificial lipid membranes to examine in detail how these drugs affect VDAC1 gating. Upon addition of VA molecules to a planar bilayer containing recombinant human VDAC1, single channel recordings showed a reliable reduction in the voltage dependence of the pore. Experiments performed in asymmetric KCl solution revealed that VA binding renders the channel predominantly anion selective, potentially disrupting cation fluxes and simultaneously affecting the transport of negatively charged metabolites. Taken together, these data represent a step forward into the comprehension of VDAC modulation as a potential therapeutic approach in cancer management.

Polyphosphate (polyP), a ubiquitous and highly conserved biopolymer, has emerged as a potential modulator of bacterial amyloidogenesis. In bacteria, polyP contributes to the formation of dense intracellular regions associated with transcriptional silencing. Gene regulation and chromatin organization are primarily controlled by nucleoid-associated proteins (NAPs), including the highly conserved RNA chaperone Hfq. Recent studies suggest that polyP alters Hfq function, promoting genomic instability through increased mutagenesis and DNA damage. In vitro, Hfq interacts with polyP and nucleic acids to form phase-separated condensates, a process mediated by its intrinsically disordered C-terminal region (CTR). In this study, we investigated the impact of polyP on the amyloidogenic behavior of Hfq. Our results reveal that, contrary to expectations, polyP alone does not induce amyloid formation in the isolated CTR. However, in the presence of polyadenylated RNA, polyP significantly enhances Hfq amyloidogenesis. These findings suggest a previously unrecognized role for polyP in RNA-mediated phase separation using amyloid self-assembly and provide new insights into the molecular mechanisms underlying bacterial stress tolerance.

Coenzyme A (CoA) is a vital cofactor involved in 8-10% of all metabolic reactions in human cells. Different inherited enzyme deficiencies in which the oxidation of acyl-CoAs is hampered have been hypothesised to share a phenotype characterised by toxic accumulation of acyl-CoA and a concomitant decline in free CoA (CoASH) levels, whereby CoASH becomes limiting for other metabolic reactions. This is referred to as CoASH sequestration. There is, however, limited experimental evidence for this hypothesis. Using a combination of approaches, we test this hypothesis in medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common deficiency of mitochondrial fatty acid oxidation (mFAO), under energetic stress. Both in vitro MCAD-knockout (KO) HepG2 cells and a kinetic model of mFAO showed decreased CoASH, elevated medium-chain acyl-CoA, and decreased long-chain acyl-CoA levels. MCAD-KO mice exposed to fasting and cold as energetic stressors had a significantly increased total CoA pool and increased expression of CoA biosynthetic enzymes in the liver, indicative of an upregulated CoA biosynthesis. Expression of carnitine acyltransferases and acyl-CoA thioesterases, enzymes that liberate CoASH from acyl-CoAs, was also upregulated, suggesting an adaptive response of CoA metabolism to decreased CoASH. Finally, computational model simulations showed that a combination of elevated total CoA and thioesterase activity led to normalisation of both CoASH and medium-chain acyl-CoA levels. Together, the results provide the first evidence for the CoA sequestration hypothesis in MCADD. The observed adaptation of CoA metabolism under energetic stress may act as a compensatory response that counteracts CoASH depletion and accumulation of toxic medium-chain acyl-CoAs.

Heterologous expression systems have been instrumental in furthering our understanding of plant RNA editing proteins. In this commentary, we discuss how the establishment of yeast as a model for studying plant RNA editing by Ramanathan et al. could advance the engineering of pentatricopeptide repeat proteins, and how in return pentatricopeptide repeat proteins might be used to advance yeast engineering.

Ectoine is a chemical chaperone and osmoprotectant discovered in halophilic bacteria where it is used to protect from osmotic stress in high-salt environments. Ectoine has become a valuable product in pharmaceutical, biotechnological, cosmetical, and other applications due to its protective and stabilizing properties. The rate-limiting step of ectoine biosynthesis is catalyzed by the l-2,4-diaminobutyric acid (DABA) transaminase enzyme EctB, which converts aspartate-β-semialdehyde (ASA) to DABA. We studied the structural and functional properties of EctB from a novel halophilic bacterium Marinobacter sp. CK1. Crystal structures of complexes with mEctB/pyridoxamine-5'-phosphate and an inactive mutant K264A/pyridoxal 5'-phosphate reveal that the enzyme forms tetramers. We also investigated the stability/activity relationship and validated residues important for activity and flexibility by characterizing mEctB and a series of 17 mutants for oligomerization, thermal stability, and catalytic activity. The wild-type enzyme retained > 50% activity over a pH range of 2.5 units, with an optimum at pH 8, all NaCl concentrations (0-1 m), and temperatures between 30 °C and 45 °C. Enzyme activity was highest using DABA and GABA as substrates. Mutations at the dimer-dimer interface (K150A, E194A, E194Q, and N134A) overall lowered the enzyme's thermal stability while two (E194Q and N134A) completely abolished activity. Mutation of Arg96 at the surface significantly lowered activity and melting temperature, but with little effect on oligomerization. The active site mutants K264, Y14 and R295 were shown to be vital for activity. Overall, this study provides new information regarding the structure, stability and function of EctB.

Hepatocellular carcinoma (HCC) remains one of the most lethal malignancies worldwide, largely due to late diagnosis and limited therapeutic options. Tumor hypoxia is a hallmark of HCC and drives aggressive disease behavior, in part through activation of hypoxia-inducible factors (HIFs). While HIF-1α is classically understood as a hypoxia-responsive transcription factor, its role under normoxic conditions in cells is less clear. The study presented by Gkotinakou and colleagues in this issue provides important insight into this question by revealing a previously underappreciated dependence of HIF-1 signaling in controlling cell cycle progression in HCC, even in the presence of normal oxygen levels.

Cis-(+)-12-oxophytodienoic acid (OPDA) is a bioactive oxylipin and phytohormone participating in regulation of plant stress responses, growth, and development. Due to its α, β-unsaturated carbonyl moiety, OPDA covalently binds to free thiol groups by Michael addition. This binding, termed OPDAylation, alters the activity of target proteins, such as cyclophilin 20-3 (EC:5.2.1.8) and thioredoxins, that are essential components of the cellular redox regulatory network. To function as a reversible redox regulatory mechanism, OPDAylation should be complemented by a process of de-OPDAylation allowing for fine-tuning of OPDA-dependent regulation. This study explored OPDAylation and de-OPDAylation in vitro with emphasis on the involvement of glutathione. OPDA can be transferred from protein to glutathione (GSH) and vice versa. In a competition experiment, OPDAylation of thioredoxins (TRX) occurred rapidly in the presence of GSH, while over extended incubation times, de-OPDAylation of TRX occurred due to the stoichiometric excess of GSH. These results support the hypothesis that the initial TRX-based OPDAylation is proceeding under kinetic control due to the higher reactivity of the more nucleophilic cysteine moiety in TRX compared to the one of GSH, while the OPDAylation of GSH observed at prolonged incubation time is then the result of a thermodynamically controlled process. De-OPDAylation depends on the protein's sensitivity towards OPDA, the pH and the concentration of excess thiol groups. This likely allows for precise modulation of OPDA amounts, as the rapid modification of protein activity enables subsequent induction of OPDA signaling, whereas de-OPDAylation, triggered by increasing glutathione, increasing cellular reduction or presumably enzymatically, reverses this effect.

To maintain proteome integrity within distinct subcellular compartments, cells rely on tightly regulated proteostasis mechanisms, including protein synthesis, folding, trafficking, and degradation. Disruption of these processes leads to the accumulation of damaged proteins and structural changes that progressively compromise organelle function, contributing to aging and age-associated disorders, such as neurodegeneration, cancer, and metabolic dysfunction. Here, we discuss recent insights into how proteostasis influences the integrity and function of specific organelles, including the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes, as well as membraneless organelles, such as stress granules, processing bodies, the nucleolus, and nuclear speckles. We further discuss how dysfunction in these systems contributes to different hallmarks of aging and disease progression, highlighting potential therapeutic strategies aimed at maintaining organelle homeostasis to promote healthy aging.