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The acetylation of histones is a central component of reversible chromatin modification that governs genome regulation. Understanding the complex histone acetylation network requires knowledge about the contributions of individual acetyltransferases. These are not easily determined through perturbation studies in cells, due to indirect effects and limited selectivity of the antibodies that detect site-specific histone acetylation. The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histones H4 and the variant H2A.V, but the precise positions of substrate lysines and their relative acetylation rates were unknown. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V, and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns, and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzyme's mode of action and to evaluate its contribution to histone acetylation patterns in cells.

In the archaeal mevalonate pathway, the prototype of all existing mevalonate pathways, a unique intermediate, trans-anhydromevalonate phosphate, is decarboxylated to form isopentenyl phosphate. The key reaction is catalyzed by a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD) family decarboxylase, anhydromevalonate phosphate decarboxylase (EC:4.1.1.126). The yet-to-be-identified properties of the archaea-specific enzyme, such as the requirement for prenylated flavin mononucleotide (prFMN) as a coenzyme, were elucidated using an enzyme derived from the hyperthermophilic archaeon Aeropyrum pernix. The coenzyme can be supplied to the decarboxylase from coexisting prFMN synthase, which anaerobically catalyzes the prenylation of reduced flavin mononucleotide and subsequent cyclization. Kinetic analysis of A. pernix anhydromevalonate phosphate decarboxylase supported its physiological role in catalyzing the decarboxylation step and progressing the archaeal mevalonate pathway, which is characterized by lower ATP consumption than other mevalonate pathways and is therefore considered promising for future metabolic engineering. However, nuclear magnetic resonance and liquid chromatography-mass spectrometry analyses showed that the enzyme could form non-negligible amounts of secondary products, probably because of the reactivity of the intermediate cycloaddition adduct between prFMN and the substrate. This study provides deeper insights into the reaction mechanism of UbiD family decarboxylases via 1,3-dipolar cycloaddition.

Nutrient depletion triggers a starvation-induced calcium (Ca²⁺) signal (SICS) that promotes Ca²⁺-dependent responses. However, the components and regulations of SICS are unclear. Here, we explored SICS components and their regulation by the Ca²⁺ sensor calmodulin (CaM). Overexpression of the stromal interaction molecule 1 (STIM1), a key switcher of store-operated Ca²⁺ entry (SOCE), enhances SICS by fourfold. This effect is abolished by the truncation of the Ca²⁺-binding loop within STIM1. Consistently, SOCE inhibition strongly suppresses SICS. Nutrient removal or stimulation of the transient receptor potential mucolipin-1 (TRPML1, encoded by the Mcoln1 gene) triggers intracellular Ca²⁺ release that is prevented by pre-emptying of endoplasmic reticulum (ER) Ca²⁺. In the presence of extracellular Ca²⁺, inhibition and silencing of Mcoln1 reduces SICS by 35-40%. We identified a CaM-binding site in the second cytoplasmic loop of TRPML1 that interacts with Ca²⁺-bound CaM. Mcoln1 overexpression enhances the upstroke and peak of SICS, effects that are absent with a mutated CaM-binding domain. Further, we generated a genetically encoded biosensor for TRPML1 (BS-ML1). BS-ML1 produces a robust signal upon nutrient deprivation, which is substantially reduced with the Mcoln1 mutant. The response of BS-ML1 to nutrient depletion is equally reduced by extracellular Ca²⁺ removal or SOCE inhibition. Reduced CaM availability significantly prolongs SICS, consistent with an ~40% reduction in cytoplasmic Ca²⁺ removal rate. Our data thus indicate that (1) SICS comprises multiple components, including linked Ca²⁺ release from the lysosome and ER and subsequent SOCE; and (2) CaM regulates the kinetics and magnitude of SICS by controlling cytoplasmic Ca²⁺ removal and a perilysosomal feedforward mechanism that promotes TRPML1 activity. The dynamics of this feedforward mechanism likely regulate subsequent tissue responses to nutrient starvation.

CRISPR-Cas systems provide adaptive immunity to bacteria, although bacteriophages counter these defenses with anti-CRISPR (Acr) proteins. Acr expression is frequently regulated by anti-CRISPR associated (Aca) proteins, which repress transcription by binding inverted repeat (IR) sequences in operon promoters. Here, we report the first identification of an IR motif within the AcrIF11-Aca7 operon promoter from Halomonas caseinilytica and present the crystal structure of Aca7 bound to this IR DNA. Biochemical assays demonstrated that Aca7 specifically recognizes the IR element, and structural analysis revealed a symmetric Aca7 dimer engaging both major grooves via helix-turn-helix motifs while stabilizing DNA bending through minor groove contacts. Residue-level interactions, including those mediated by R38, Q42, K46, and K49, establish a detailed basis for sequence-specific recognition. Comparison with Aca2 highlights distinct dimer architectures and DNA deformation strategies among Aca proteins. Our findings uncover the molecular mechanism by which Aca7 represses AcrIF11 expression and broaden the understanding of Aca-mediated transcriptional regulation.

The labile iron pool in the cell is required for ferroptosis, a form of regulated cell death resulting from excessive lipid peroxidation and membrane damage. Glutathione (GSH) is critical for lipid-peroxide scavenging, and cysteine is the rate-limiting amino acid in GSH synthesis. Cysteine metabolism intricately intertwines with iron metabolism, either directly by participating in assembly of the iron-sulfur cluster or indirectly through the pantothenate pathway and coenzyme A (CoA) synthesis. However, the regulation of iron homeostasis in cystine (Cys₂)-deprivation-induced ferroptosis is poorly understood. Here, we show that Cys₂ deprivation promotes ferroptosis, at least in part, by activating the iron-starvation response (ISR), and CoA can mitigate ferroptosis by suppressing the ISR. Mechanistically, Cys₂ deprivation promotes the oxidation of cytosolic iron-sulfur clusters to activate the ISR; CoA and related small-molecule thiols in the pantothenate pathway suppress the ISR and ferroptosis by preventing the oxidation of iron-sulfur clusters in Cys₂-deprived cells. Our findings provide important insight into the regulation of the ISR in Cys₂-deprivation-induced ferroptosis, and show that CoA can protect cells from Cys₂-deprivation-induced ferroptosis by suppressing the ISR.

DNAJC7, a member of the J-domain protein (JDP/Hsp40) family, plays a key role in protein homeostasis by regulating Hsp70 activity and preventing protein aggregation. Mutations in DNAJC7 have been linked to amyotrophic lateral sclerosis (ALS); yet, the molecular mechanisms by which these variants impair chaperone function remain poorly understood. DNAJC7 is a conserved chaperone featuring both a canonical J-domain, essential for Hsp70 activation, and three TPR domains, which serve as protein-protein binding interfaces. Here, we investigate the structural and functional consequences of the ALS-associated E425K mutation located within the conserved J-domain. Using NMR spectroscopy, we show that although the E425K mutation does not alter the structure of the protein, it significantly disrupts the conserved J-domain-Hsp70 interaction. We further identify a second Hsp70-binding interface within the TPR domains, which interacts with the C-terminal EEVD motif of Hsp70. This TPR-EEVD interaction is preserved in the E425K mutant but cannot compensate for the loss of J-domain binding or restore DNAJC7-dependent Hsp70 activation. Functionally, we show that the TPR domains of DNAJC7 directly bind TDP-43 and prevent its aggregation and that this holdase activity is retained in the E425K mutant. However, the mutant fails to support client transfer to Hsp70 and the subsequent Hsp70-mediated substrate refolding. Together, these findings demonstrate that DNAJC7 requires coordinated action of both J-domain and TPRs to regulate Hsp70 function and that disruption of J-domain-mediated activation uncouples DNAJC7 from the Hsp70 cycle, providing a mechanistic basis for its dysfunction in ALS.

The transition from enzyme to pseudoenzyme is thought to begin with moonlighting enzymes that have gained nonenzymatic functions. Subsequent gene duplication events allow for the separation of enzyme and pseudoenzyme function. We explored this enzyme-to-pseudoenzyme transition in the family of fungal metallocarboxypeptidases through bioinformatics approaches, molecular modeling, and biochemical analyses. Over 3000 predicted fungal metallocarboxypeptidases were first classified by phylogeny and active site signature into 14 clusters. Prediction of isoelectric point revealed potential subcellular location, while predictions of solvent accessible surface area and AlphaFold modeling of representative structures suggested a tendency for clusters rich in pseudoenzymes to have extensive surface loops and polar distribution of surface electrostatic potential, possible requirements for the addition of nonenzymatic function. Five basidiomycete carboxypeptidases were selected for experimental analysis by RNA-seq, western blotting following expression in Sf9 and HEK293T systems, and enzymatic activity. No activity was detected from predicted pseudoenzymes, either purified or unpurified. Both predicted-active enzymes were secreted from Sf9 cells, although only one could be purified, with expected carboxypeptidase activity and specificity toward large hydrophobic C-terminal amino acids. Altogether, our study suggests that the addition of surface loops may be a key feature in the acquisition of pseudoenzyme function, and that both enzymes and pseudoenzymes are likely to play important and unique roles in these fungal systems.

Lin Y., Stevens C., Hrstka R., Harrison B., Fourtouna A., Pathuri S., Vojtesek B., Hupp T. (2008) An alternative transcript from the death-associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing. FEBS J, 275: 2574–2584. https://doi.org/10.1111/j.1742-4658.2008.06404.x.

During the preparation of Figure 5 in the manuscript by Lin et al. [1], the authors did not denote where irrelevant lanes were excised from the immunoblot images. The authors and editors concluded that this omission does not affect the work performed or its conclusions. This Corrigendum replaces the original Figure 5 and its legend with a version that includes this mark-up. The authors have checked the entire document and assert that they found no further errors.

The corrected Figure 5 and its legend are shown below.

Thyroxine (T4) plays a crucial role in regulating various physiological functions in the human body. As a key biomolecule for molecular recognition, single-chain variable fragments (scFvs) offer advantages, such as small molecular size, high specificity, and strong affinity, making them ideal alternatives to traditional antibodies for diagnostic applications. This study aimed to achieve in vitro affinity maturation of T4-specific scFvs through computational design, identify variants with enhanced affinity, and elucidate their interaction mechanisms with T4. A T4-specific scFv (T4-scFv) was constructed based on the previously reported anti-T4 Fab fragment (PDB ID: 5MHE) using homology modeling, followed by molecular dynamics optimization. Molecular docking and virtual mutagenesis of eight key residues (Ser91, Ser92, Pro94, Tyr180, Tyr181, Pro182, Pro186, and Ser226) were performed, yielding three affinity-enhanced variants. These variants were expressed in E. coli and evaluated experimentally using indirect competitive ELISA (IC-ELISA). The results showed that the IC₅₀ values decreased from 34.59 ng·mL^-1 (T4-scFv) to 18.30 ng·mL^-1 (scFv-M92), 15.26 ng·mL^-1 (scFv-MS), and 11.84 ng·mL^-1 (scFv-M226), demonstrating a 2.9-fold improvement in affinity for the best mutant. The optimized scFv-M226 also exhibited high specificity, with cross-reactivity to T3 and rT3 of less than 2.0%. A quantitative T4 detection method based on scFv-M226 was developed, which showed a wide linear range (1.0-200 ng·mL^-1) and a detection limit (IC₁₀) of 0.93 ng·mL^-1. These findings confirm the accuracy of the computationally guided affinity maturation strategy and highlight the potential of scFv-M226 for clinical detection and diagnostic monitoring of T4 in serum.

The FEBS Journal publishes primary papers and reviews relating to the molecules and mechanisms underpinning biological processes. Editor-in-Chief Seamus Martin discusses some of the challenges posed by the increasing use of generative AI tools in scientific publishing and some of the highlights of the past year at the journal.