FEBS Letters最新文献

Carbonyl sulfide hydrolase (COSase) is a unique enzyme that exhibits high activity towards carbonyl sulfide (COS) but low carbonic anhydrase (CA) activity, despite belonging to the CA family. COSase was initially identified in a sulfur-oxidizing bacterium and later discovered in the ascomycete Trichoderma harzianum strain THIF08. The COSase from T. harzianum has been recognized as a key enzyme in the assimilation of gaseous COS, and homologous genes are widely present not only in Ascomycota but also in Basidiomycota. Here, we characterized the COSases from the basidiomycete Gloeophyllum trabeum NBRC 6430 and T. harzianum to obtain detailed characteristics of fungal COSase. This study contributes to a better understanding of COS metabolism in fungi.

Soluble, circulating Klotho (sKlotho) is essential for normal health and renal function. sKlotho is shed from the renal distal convoluted tubule (DCT), its primary source, via enzymatic cleavage. However, the physiologic mechanisms that control sKlotho production, trafficking, and shedding are not fully defined. We previously found that the G protein-coupled calcium-sensing receptor (CaSR) co-localizes with membrane-bound αKlotho and the disintegrin/metalloprotease ADAM10 in the DCT and controls sKlotho in response to CaSR ligands and pHo by activating ADAM10. Here, we advance understanding of this process by showing that tetraspanin 5 (Tspan5), a scaffolding and chaperone protein, contributes to the cell surface expression and specificity of a protein complex that includes Tspan5, ADAM10, Klotho, and CaSR. These results support a model of multiprotein complexes that confer signaling specificity beyond CaSR on G protein-coupled processes. Impact statement Systemic circulating sKlotho is a determinant for normal physiology. Studies of knockout animals established its role as an anti-aging protein. The regulatory mechanisms for Klotho production and secretion are largely unknown. We report that Tspan 5 contributes to CaSR- and ADAM10-dependent Klotho shedding from the kidney, its primary source.

Protein-protein interactions involving 14-3-3 proteins regulate various cellular activities in normal and pathological conditions. These interactions have mostly been reported to be phosphorylation-dependent, but the 14-3-3 proteins also interact with unphosphorylated proteins. In this work, we investigated whether phosphorylation is required, or, alternatively, whether negative charges are sufficient for 14-3-3ε binding. We substituted the pThr residue of pT(502-510) peptide by residues with a varying number of negative charges and investigated the binding of the peptides to 14-3-3ε using MD simulations and biophysical methods. We demonstrated that at least one negative charge is required for the peptides to bind 14-3-3ε, although phosphorylation is not necessary, and that two negative charges are preferable for high affinity binding. This discovery opens up new approaches for designing peptide-based 14-3-3 protein inhibitors.

The mitochondrial outer membrane iron-sulphur ([Fe-S]) protein mitoNEET has been extensively studied as a target of the anti-inflammatory and type-2 diabetes drug pioglitazone and as a protein affecting mitochondrial respiratory rate. Despite these extensive past studies, its molecular function has yet to be discovered. Here, we applied an interdisciplinary approach and discovered an explicit nitric oxide (NO) access site to the mitoNEET [2Fe-2S] cluster. We found that O₂ and pioglitazone block NO access to the cluster, suggesting a molecular function for the mitoNEET [2Fe-2S] cluster in mitochondrial signal transduction. Our discovery hints at a new pathway via which mitochondria can sense hypoxia through O₂ protection of the mitoNEET [2Fe-2S] cluster, a new paradigm in understanding the importance of [Fe-S] clusters for gasotransmitter signal transduction in eukaryotes.

Lipid nanodiscs have become a widely used approach for studying membrane proteins thanks to several advantages they offer. They have been especially useful for studying ABC transporters, despite the growing concern about the possible restriction of the conformational changes of the transporters due to the small size of the discs. Here, we performed a systematic study to determine the effect of the nanodisc size on the ATPase activity of model ABC transporters from human, plant, and bacteria. Our data confirm that the activity of the transporters and their response to regulatory molecules is affected by the nanodisc size. Our findings suggest the use of larger membrane scaffold proteins (MSPs), such as MSP2N2 nanodiscs, to minimize alterations caused by the commonly used small MSP1D1.

Exo-β-(1,3)-glucanases are promising enzymes for use in the biofuel industry as they hydrolyse sugars such as laminarin, a major constituent of the algal cell wall. This study reports structural and biochemical characterizations of Aspergillus oryzae exo-β-(1,3)-glucanase (AoBgl) belonging to the GH5 family. Purified AoBgl hydrolyses β-(1,3)-glycosidic linkages of the oligosaccharide laminaritriose and the polysaccharide laminarin effectively. We have determined three high-resolution structures of AoBgl: (a) the apo form at 1.75 Å, (b) the complexed form with bound cellobiose at 1.73 Å and (c) the glucose-bound form at 1.20 Å. The crystal structures, molecular dynamics simulation studies and site-directed mutagenesis reveal the mode of substrate binding and interactions at the active site. The results also indicate that AoBgl effectively hydrolyses trisaccharides and higher oligosaccharides. The findings from our structural and biochemical studies would aid in rational engineering efforts to generate superior AoBgl variants and similar GH5 enzymes for their industrial use.

Enhancers are non-coding cis-regulatory elements crucial for transcriptional regulation. Mutations in enhancers can disrupt gene regulation, leading to disease phenotypes. Identifying enhancers and their tissue-specific activity is challenging due to their lack of stereotyped sequences. This study presents a sequence-based computational model that uses combinatorial transcription factor (TF) genomic occupancy to predict tissue-specific enhancers. Trained on diverse datasets, including ENCODE and Vista enhancer browser data, the model predicted 25 000 forebrain-specific cis-regulatory modules (CRMs) in the human genome. Validation using biochemical features, disease-associated SNPs, and in vivo zebrafish analysis confirmed its effectiveness. This model aids in predicting enhancers lacking well-characterized chromatin features, complementing experimental approaches in tissue-specific enhancer discovery.

Zinc transporters (ZnTs) act as H⁺/Zn²⁺ antiporters, crucial for zinc homeostasis. Brain-specific ZnT3 expressed in synaptic vesicles transports Zn²⁺ from the cytosol into vesicles and is essential for neurotransmission, with ZnT3 dysfunction associated with neurological disorders. Ubiquitously expressed ZnT4 localized to lysosomes facilitates the Zn²⁺ efflux from the cytosol to lysosomes, mitigating the cell injury risk. Despite their importance, the structures and Zn²⁺ transport mechanisms remain unclear. We characterized the three-dimensional structures of human ZnT3 (inward-facing) and ZnT4 (outward-facing) using cryo-electron microscopy. By combining these structures, we assessed the conformational changes that could occur within the transmembrane domain during Zn²⁺ transport. Our results provide a structural basis for a more comprehensive understanding of the H⁺/Zn²⁺ exchange mechanisms exhibited by ZnTs.

RNA is modified by > 170 chemical modifications that affect its structure and function. Accordingly, RNA modifications have been implicated in regulation of gene expression and cellular outcomes in a variety of species spanning the phylogenetic tree. The study of RNA modifications is accelerated by generation of high-throughput methods for detecting RNA modifications at single base resolution. Here, we review recent advancement in next generation sequencing based approaches for detection of 14 distinct RNA modifications present in rRNA, tRNA and mRNA. We further outline the molecular and computational principles underlying currently available methods.

Autophagy, a highly conserved form of cellular recycling, is essential for cellular homeostasis. Its dysregulation has been linked to neurodegenerative diseases and various cancers, including breast cancer. We set out to determine if the RNA-binding protein (RBP) YBX3 regulates autophagy mRNAs, as previous findings suggest YBX3 depletion reduces distinct autophagy transcripts. We found that YBX3 interacts with and stabilizes the mRNA of the autophagy initiation factor ATG13 in HeLa, which in turn increases ATG13 protein expression. We have shown that this requires the 3' untranslated region (UTR) of ATG13 and occurs in other human cell lines, including HEK293, HepG2, and HCT116. Together, our data suggest a novel regulatory role for YBX3 of autophagy initiation through posttranscriptional control of ATG13 mRNA stability.