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The hyperthermophile Thermotoga maritima does not possess a typical branched-chain amino acid aminotransferase or aromatic amino acid aminotransferase, leaving the biosynthetic pathways of these amino acids unclear. In this study, we identified and characterized a novel branched-chain and aromatic amino acid aminotransferase (TM1131). We also characterized a histidinol-phosphate aminotransferase (TM1040) with reported aminotransferase activity toward aromatic amino acids. TM1131 exhibited broad substrate specificity and the highest activity toward branched-chain and aromatic amino acids as an amino donor and toward corresponding 2-oxoacids as an amino acceptor. TM1040 also showed broad substrate specificity, with the highest activity toward l-lysine and l-arginine as an amino donor, and toward 2-oxoacids corresponding to l-methionine, l-leucine, and l-phenylalanine. Additionally, we investigated the multifunctionality of these two enzymes to explore other potential amino acid metabolic activities. Intriguingly, TM1131 displayed aspartate 4-decarboxylase activity, albeit with lower catalytic efficiency than measured for aminotransferase activity. TM1131 is involved in the final step of the biosynthetic pathways of branched-chain and aromatic amino acids, to which TM1040 also likely contributes.

Dimerization between the histone-fold domains (HFD) of two Plasmodium Oocyst Rupture Proteins (ORP1 and ORP2) is essential for oocyst rupture in the Anopheles mosquito vector host, representing a key event in parasite transmission to humans. Notably, ORPs are a rare example of HFD-containing proteins that operate outside the nucleus and that lack DNA-binding functions, typically associated with core histones and transcription factors hosting deviant histones. ORP HFD heterodimerization occurs at the outer capsule of the oocyst, immediately prior to rupture, thus providing a temporal window to administer dimerization blocking molecules. In this context, we present the first detailed structural analysis of the HFD ORP heterodimer, solved by X-ray crystallography at 3.1 Å resolution, and analyze the oligomerization interface as a possible druggable target. Targeting the mosquito phase of the parasite lifecycle remains an under-exploited avenue as present antimalarial therapies mainly target the human blood stages of infection. We employed a GAL4-based yeast two-hybrid (Y2H) combinatorial library of cyclic peptides (CPs) to identify six candidates that inhibit dimerization in vitro. Molecular docking simulations confirmed that all six CPs bind at the dimer interface, allowing us to rank them for further in vivo testing of their efficacy in blocking oocyst rupture.

Modulation of extracellular matrix (ECM) turnover is a critical prerequisite process underlying the onset of melanoma metastasis. ECM proteases are involved in the degradation of matrix components during ECM turnover, which is associated with melanoma cell growth, migration, invasion, extravasation, metastasis, and modulation of melanoma tumor immunogenicity. During these processes, fluctuations in ECM protease activities and concentrations occur in response to complex regulatory mechanisms acting at both the transcriptional and post-transcriptional levels of protease gene expression. In this review, we examine the major factors of epigenetic machinery, specifically protease-regulating microRNAs (miRNAs), with respect to their ability to directly target ECM protease transcripts and influence melanoma progression. Furthermore, given that dysregulation of the intestinal microbiota has been identified as an etiological factor in melanoma resistance to contemporary immunotherapies, this review examines evidence linking gut dysbiosis-induced changes in matrix metalloproteinase-targeting miRNA profiles to the progression of melanoma. In conclusion, we evaluate the therapeutic potential of approaches involving modifications of gut microbiota populations, alongside direct miRNA targeting of ECM proteases. The integration of these strategies may facilitate the development of innovative adjuvant therapies aimed at overcoming resistance to current inhibitor checkpoint immunotherapies.

Serine/threonine (Ser/Thr) kinases of the Hanks-type family are widespread in bacteria, playing key roles in signal transduction. The transmembrane Ser/Thr kinase PknS (XAC4127) from the phytopathogenic bacterium Xanthomonas citri is required for the expression of a type VI secretion system, which confers resistance to predation by the soil amoeba Dictyostelium discoideum. PknS exerts its function via activation of the cognate ECF-type alternative sigma factor EcfK, ultimately triggering the expression of type VI secretion system (T6SS) genes. In this study, we characterize PknS, demonstrating its ability to undergo autophosphorylation both in vitro and within X. citri cells. Structural analysis of the PknS kinase domain revealed the conservation of the canonical fold characteristic of Hanks-type kinases. PknS directly phosphorylates EcfK at five Ser/Thr residues located in two distinct regions of the sigma factor: the conserved σ₂ domain (residue T51) and a nonconserved linker connecting domains σ₂ and σ₄ (residues T104, T106, S108, and S110). The conserved residue T51, previously shown to be essential for sigma factor activity in an EcfK homolog, corresponds to a site that directly interacts with the RNA polymerase β' subunit. Site-directed mutagenesis analyses further revealed that the conserved residue T106 is also critical for EcfK function. Structural studies indicated that, in addition to T51, phosphorylation at T106 activates EcfK by promoting its interaction with a positively charged pocket within the RNA polymerase β' subunit. Collectively, our findings describe a previously unknown signal transduction pathway involving a Hanks-type kinase and a sigma factor, providing new insights into the mechanisms of sigma factor activation via phosphorylation in bacteria.

The pro-apoptotic BCL-2-interacting mediator of cell death [BIM; also known as Bcl-2-like protein 11 (BCL2L11)] is a crucial regulator of programmed cell death in immune cells, with roles in T-cell development, homeostasis, and immune response modulation. However, the precise molecular mechanisms that regulate BIM expression in these processes are not completely understood. One possible regulatory mechanism involves microRNAs, small noncoding RNAs that silence target messenger RNAs (mRNAs). The miR-17-92 cluster, which has been implicated in immune regulation, has nine predicted binding sites in the 3' untranslated region of the Bcl2l11 mRNA (thereafter referred to as Bim mRNA). To explore whether direct miR-17-92-mediated regulation of BIM controls apoptosis in T cells, a genetically modified mouse model with disrupted miR-17-92:Bim interactions specifically in T cells has been used. The results revealed that loss of miR-17-92:Bim binding, although leading to a modest increase of BIM protein in double-positive (DP) thymocytes and naïve CD8⁺ T cells, does not measurably affect early T-cell development or peripheral T-cell numbers. However, the absence of this interaction led to a moderate reduction in Th₁₇ CD4⁺ T cells at a steady state. Collectively, these findings suggest that miR-17-92-mediated regulation of BIM does not play major roles in T-cell apoptosis and homeostasis, highlighting the existence of alternative regulatory mechanisms controlling BIM pro-apoptotic activity.

We are delighted to present a selection of original articles, curated by FEBS Junior Section member, Dr Ignazio Restivo. These articles were published in The FEBS Journal in 2024 and 2025, and cover molecular aspects of developmental biology and cell biology, showcasing our advances in disease mechanisms and biotechnology. We invite you to join us in revisiting these gems, and we hope they will help you find scientific inspiration.

The iron-sulfur (Fe-S) cluster assembly homolog 1 (ISCA1) is a ubiquitous protein conserved in various organisms. Previous work has shown that a pigeon ISCA1 (clISCA1) forms columnar oligomers in the 2Fe-2S cluster-bound state, the length of which has been known to change in response to magnetic fields. However, whether this unique property is conserved in ISCA1 proteins of other species, particularly humans (hsISCA1), is unclear. Moreover, a recent study revealed that clISCA1 binds to not only Fe-S clusters but also mononuclear iron atoms, which may impart some magnetic properties to clISCA1. In this study, the electron spin resonance revealed that hsISCA1 also binds to mononuclear iron atoms. Moreover, the magnetic responses of Fe-S cluster-unbound ISCA1s (Fe-ISCA1s), which bind only mononuclear iron atoms, were inspected by small-angle X-ray scattering analyses for pigeon (Fe-clISCA1) and human (Fe-hsISCA1). The results indicated that Fe-hsISCA1 formed columnar oligomers under geomagnetic conditions, whereas Fe-clISCA1 formed dumbbell-like oligomers. When a magnetic field (180 mT) was applied, the Fe-hsISCA1 oligomer was shortened within 1 min and gradually elongated again after 10 min. This result indicates that mononuclear iron atoms contribute to the magnetically induced structural ordering of ISCA1, whereas the contribution of the Fe-S clusters to the columnarization of ISCA1 varies among species. Although the physiological role of the magnetic properties of ISCA1 is not yet elucidated, this study demonstrated that the magnetic field responsiveness of ISCA1 is conserved in humans. The magnetic field responsiveness may be a hidden fundamental property of ISCA1 that is maintained even if the Fe-S cluster is released.

Protein aggregation and transmission are hallmarks of neurodegenerative diseases. Praja1 E3 ubiquitin ligase has been shown to suppress the aggregation of causative proteins in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Parkinson's disease, Huntington's disease, and spinocerebellar degeneration, which include transactivation response DNA-binding protein of 43 kDa, fused in sarcoma, superoxide dismutase 1, α-synuclein, huntingtin, and ataxin-3. Aoki et al. demonstrated that Praja1 ubiquitinates and degrades tau, a key molecule in tauopathies such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, and corticobasal syndrome, furthering our understanding of the role of Praja1 in neurodegenerative diseases and potential therapeutic approaches.

Rickets, a disorder of bone formation, was originally known as nutritional rickets due to vitamin D deficiency. Advances in science have since identified various genetic forms, typically involving loss-of-function mutations in vitamin D activation or other mineral metabolism pathways. Recently, type 3 rickets was identified as a previously undescribed gain-of-function mutation in CYP3A4 (Ile301Thr). This mutant enzyme leverages the unique features of cytochrome P450 to produce an inactive vitamin D metabolite, 11α,25(OH)₂D₃, resulting in insufficient active vitamin D. The discovery of this unique gain-of-function aetiology and its associated metabolite opens a significant new direction in rickets research.

Long interspersed element-1 (LINE-1 or L1) is actively jumping in humans, notably in germ cells, neurons, and certain types of cancer. An active L1 is ~6.0 kb in length and encodes two proteins, designated ORF1p and ORF2p. L1 RNA binds with L1-encoded proteins and forms L1-ribonucleoprotein particles (L1-RNPs), the retrotransposition intermediate. Although cells that support L1 retrotransposition express both proteins, the detection of ORF2 protein (ORF2p) is extremely challenging due to its limited expression and unavailability of a suitable antibody. Here, we characterize an anti-ORF2p antibody and show the presence of endogenous L1-ORF2p in multiple cancer cell lines, among which the MCF-7 cell line showed notably high expression. Complexes purified by immunoprecipitation (IP) with anti-ORF2p or anti-ORF1p from MCF-7 or HEK293T cells contain ORF2p and ORF1p and show ORF2p-mediated reverse transcriptase (RT) activity on L1, Alu, and GAPDH RNA templates. The ORF2 IP complex was further purified by size exclusion chromatography (SEC), which showed three major peaks with molecular weights around 796, 427, and 239 kDa. All three peaks showed the presence of L1 proteins, RNA, and ORF2p-mediated RT activity. Although many proteins have been identified that interact with L1 proteins, it is unclear which of these belong to the core L1 RNP. Our novel anti-ORF2p will provide a valuable resource for future studies involving ORF2p IP followed by SEC to identify the protein components of core L1 RNPs. In summary, we report the detection of endogenous L1 ORF2 protein and partial purification of its complex by ORF2p antibody-coupled IP and SEC.