The FEBS journal最新文献

Small open reading frame (sORF)-encoded proteins, with less than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in classical methodologies. For the mesophilic archaeal model system Methanosarcina mazei, a high number of previously unannotated sORFs have recently been identified. However, the physiological role of most of the respective small proteins remains unknown. Here, we report on the functional characterization of the small ORF16-encoded small protein MtrR (49 amino acids). We demonstrate that MtrR forms oligomers localized at the cytoplasmic membrane. There, it interacts with the tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex of energy metabolism, and impacts its activity. In vitro interaction and in vivo copurification assays showed MtrR interaction with the Mtr complex, which was further validated by microscale thermophoresis analysis demonstrating a specific interaction with the MtrA subunit. Analyzing growth under varying molecular hydrogen (H₂) availability demonstrated that the mtrR deletion mutant showed significantly impaired growth in the presence of H₂, independent of the carbon source. Further, we observed induction of mtrR transcription in the presence of H₂. Consequently, we propose that MtrR fine-tunes the activity of the Mtr complex in response to fluctuating H₂ availabilities, allowing adaptation of the energy metabolism to changing environmental H₂ conditions.

Elkenani M, Nyamsuren G, Toischer K, Adham IM & Mohamed BA (2021) Perturbed differentiation of murine embryonic stem cells upon Pelota deletion due to dysregulated FOXO1/β-catenin signaling. FEBS J, 288: 3317–3329. https://doi.org/10.1111/febs.15643

During preparation of the figures for the manuscript by Elkenani et al., the authors inadvertently inserted wrong images in some of the figures. The images of ESC cultures were duplicated between Figs 3E and 2B, and the wrong loading control was inserted in the western blot array in Fig. 5A. Upon carefully reviewing the figures in the article, and the corresponding raw data, the authors also realized that the wrong FOXO western blot image was inserted in Fig. 3A during the revision stage of manuscript preparation. Upon reviewing the raw data for these experiments, the editors concluded that the errors, while regrettable, do not affect the conclusions of the work. This corrigendum replaces the incorrect images in Figs 2, 3 and 5. The authors have checked the entire document and assert that they found no further errors.

We apologize for these errors.

Ding, J., Yu, C., Sui, Y., Wang, L., Yang, Y., Wang, F., Yao, H., Xing, F., Liu, H., Li, Y., Shah, J.A., Cai, Y. and Jin, J. (2018) The chromatin remodeling protein INO80 contributes to the removal of H2A.Z at the p53-binding site of the p21 gene in response to doxorubicin. FEBS J, 285: 3270–3285. https://doi.org/10.1111/febs.14615

During the preparation of Fig. 8 in the manuscript by Ding et al., the authors inadvertently duplicated the GAPDH loading control immunoblot in Fig. 8C. Upon careful review of the available data, the authors and editors concluded that this does not affect the work performed or its conclusions. This Corrigendum replaces the original Fig. 8 with a version that shows the correct loading controls that correspond to the experiments shown in Fig. 8C. The authors have checked the entire document and assert that they found no further errors.

We apologize for this error.

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.