In this research letter, we report the development and validation of a new subset of fluorescence-based CRISPR interference (CRISPRi) tools for our scientific community. The pJL series is directly derived from the original pIRL CRISPRi vectors and conserves all the elements to perform inducible targeted gene repression. These vectors carry two distinct fluorescent markers under the constitutive promoter psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the glycopeptidolipid translocase mmpL4b and the essential genes rpoB and mmpL3. Finally, we describe an efficient single-step procedure to co-transform mycobacterial species with this integrative genetic tool alongside episomal vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research.

Serratia sp. ATCC 39006 has two tandemly positioned genes, ser4 and ser5, both annotated as sugar aminotransferases, in a putative secondary metabolite biosynthetic gene cluster. Ser5 possesses a complete fold-type I aminotransferase fold, while Ser4 lacks the N- and C-terminal regions and a catalytically important lysine residue of fold-type I aminotransferase. We herein revealed that Ser4 and Ser5 formed a heterotetrameric complex (SerTA) with aminotransferase activity and determined the crystal structures. MD simulations and activity assays with SerTA variants indicated that residues from helix α-8* of inactive Ser4 are important for activity, confirming the importance of heterocomplex formation for activity. Furthermore, the structures suggest that SerTA recognizes a substrate loaded on the carrier protein.

Understanding the intricate mechanisms underlying brain-related diseases hinges on unraveling the pivotal role of the blood-brain barrier (BBB), an essential dynamic interface crucial for maintaining brain equilibrium. This review offers a comprehensive analysis of BBB physiology, delving into its cellular and molecular components while exploring a wide range of in vivo and in vitro BBB models. Notably, recent advancements in 3D cell culture techniques are explicitly discussed, as they have significantly improved the fidelity of BBB modeling by enabling the replication of physiologically relevant environments under flow conditions. Special attention is given to the cellular aspects of in vitro BBB models, alongside discussions on advances in stem cell technologies, providing valuable insights into generating robust cellular systems for BBB modeling. The diverse array of cell types used in BBB modeling, depending on their sources, is meticulously examined in this comprehensive review, scrutinizing their respective derivation protocols and implications. By synthesizing diverse approaches, this review sheds light on the improvements of BBB models to capture physiological conditions, aiding in understanding BBB interactions in health and disease conditions to foster clinical developments.

d-Galactofuranose (Galf) is widely distributed in glycoconjugates of pathogenic microbes. β-d-Galactofuranosidase (Galf-ase) from Streptomyces sp. JHA19 (ORF1110) belongs to glycoside hydrolase (GH) family 2 and is the first identified Galf-specific degradation enzyme. Here, the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, d-iminogalactitol (K = 65 μm) was solved. ORF1110 binds to the C5-C6 hydroxy groups of d-iminogalactitol with an extensive and integral hydrogen bond network, a key interaction that discriminates the substrates. The active site structure of ORF1110 is largely different from those of β-glucuronidases and β-galactosidases in the same GH2 family. A C-terminal domain of ORF1110 is predicted to be a carbohydrate-binding module family 42 that may bind Galf. The structural insights into Galf-ase will contribute to the investigation of therapeutic tools against pathogens.

HUWE1, a HECT E3 ligase, is critical for processes like protein degradation and tumor development. Contrary to previous findings which suggested minimal non-covalent interactions between the HUWE1 HECT domain and ubiquitin, we identified a non-covalent interaction between the HUWE1 HECT N-lobe and ubiquitin using NMR spectroscopy, revealing a conserved ubiquitin-binding mode shared across HECT E3 ligases. Molecular dynamics simulations not only confirmed the stability of this interaction but also uncovered conformational changes in key residues, which likely influence binding affinity. Additionally, we highlighted the roles of both conserved and unique residues in ubiquitin binding. These findings advance our understanding of the interactions between the HUWE1 HECT domain and ubiquitin, and highlight potential targets for therapeutic intervention in the ubiquitin-proteasome pathway.

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.

Cordycepin (3' deoxyadenosine) has been widely researched as a potential cancer therapy, but many diverse mechanisms of action have been proposed. Here, we confirm that cordycepin triphosphate is likely to be the active metabolite of cordycepin and that it consistently represses growth factor-induced gene expression. Bioinformatic analysis, quantitative PCR and western blotting confirmed that cordycepin blocks the PI3K/AKT/mTOR and/or MEK/ERK pathways in six cell lines and that AMPK activation is not required. The effects of cordycepin on translation through mTOR pathway repression were detectable within 30 min, indicating a rapid process. These data therefore indicate that cordycepin has a universal mechanism of action, acting as cordycepin triphosphate on an as yet unknown target molecule involved in growth factor signalling.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by accumulation of α-synuclein cross-β amyloid filaments in the brain. Previous structural studies of these filaments by cryo-electron microscopy (cryo-EM) revealed three discrete folds distinct from α-synuclein filaments associated with other neurodegenerative diseases. Here, we use cryo-EM to identify a novel, low-populated MSA filament subtype (designated Type I) in addition to a predominant class comprising MSA Type II filaments. The 3.3-Å resolution structure of the Type I filament reveals a fold consisting of two asymmetric protofilaments, one of which adopts a novel structure that is chimeric between two previously reported protofilaments. These results further define MSA-specific folds of α-synuclein filaments and have implications for designing MSA diagnostics and therapeutics.

Autophagy is a genetically regulated, eukaryotic catabolic pathway that responds to internal and external cellular signals. In plants, it plays crucial roles in development, and responses to abiotic and biotic stresses. Due to its role in limiting the hypersensitive response, research on the molecular mechanisms of autophagic signalling pathways in plant-microbe interactions has primarily focused on plant-pathogen responses. Although there is substantially less information on the role of autophagy signalling in symbiotic plant-microbe interactions, there is accumulating evidence that it is also a key regulator of mutualistic plant-microbe interactions. Here, we review recent progress on the roles of autophagy in symbiotic plant interactions and discuss potential future research directions. Once understood, the central role that autophagy plays within pathogenic and symbiotic plant-microbe interactions has significant potential application for crop improvement. Manipulating autophagy in legume crops could help support crop growth with reduced levels of fertiliser application while maintaining yields with increased protein content in the harvest.

DNA replication and RNA transcription processes compete for the same DNA template and, thus, frequently collide. These transcription-replication collisions are thought to lead to genomic instability, which places a selective pressure on organisms to avoid them. Here, we review the predisposing causes, molecular mechanisms, and downstream consequences of transcription-replication collisions (TRCs) with a strong emphasis on prokaryotic model systems, before contrasting prokaryotic findings with cases in eukaryotic systems. Current research points to genomic structure as the primary determinant of steady-state TRC levels and RNA polymerase regulation as the primary inducer of excess TRCs. We review the proposed mechanisms of TRC-induced DNA damage, attempting to clarify their mechanistic requirements. Finally, we discuss what drives genomes to select against TRCs.

Nanobodies (NB) are powerful tools for biotechnological and therapeutic applications. They strongly bind to their targets and are very stable. Early studies showed that NB unfolding is reversible and can be analyzed by equilibrium thermodynamics, whereas more recent studies focused on their kinetic stability in very harsh conditions that are far from storage or physiological temperatures (4-37 °C). Here, we show that the thermodynamic view of NB stability holds in a wide range of temperatures (18-100 °C). The thermodynamic stability of three different NBs did not correlate with binding affinity for their target. Alpha-Fold 2 analyses of these NBs showed structural differences in the binding site and hydrogen bond networks. We expect that our approach will be helpful to improve our capacity to enhance structure-function-stability relationships of NB.

Nanodiscs, consisting of a lipid bilayer surrounded by membrane scaffold proteins (MSPs), are extensively used to study membrane proteins (MPs) because they provide a stable lipid environment. However, the precise mechanism governing MP behavior within the nanodisc remains elusive. Here, we examined the cryo-EM structures of various MPs reconstituted in nanodiscs from EMPIAR. By analyzing the heterogeneity and interactions in the nanodiscs, we discovered that MPs display a distinct spatial preference toward the edges of the nanodisc shells. Furthermore, MPs can establish direct, amphipathic interactions with the MSPs, causing a reduction in local protein dynamics. These interactions may rearrange MSP-MSP interactions into MP-MSP interactions. Collectively, we provide structural insights into how nanodiscs contribute to MP structural behavior and dynamics. Impact statement Nanodiscs are used to study membrane proteins (MPs), but the mechanisms governing the behavior of MPs within nanodiscs remain elusive. Here, we provide structural insights into how nanodiscs contribute to the behavior of MPs, which will aid the interpretation of cryo-EM studies performed using nanodiscs.

The Federation of European Biochemical Societies (FEBS) was founded in 1964 to bring together the scientific societies of Europe and neighbouring regions and to provide a platform for scientific exchange. Today, FEBS is an organisation of more than 30 000 members across 39 biochemistry and molecular biology societies that promotes excellence in the life sciences in Europe and beyond. To mark the 60 anniversary of FEBS, FEBS Letters celebrated with a writing contest focussed on participants' visions of scientific societies in the year 2084. Here, we present the winning essay, in which Yussuf Ali (Jagiellonian University, Poland) considers a future in which FEBS transcends not only geographical boundaries, but also interdisciplinary, technological and financial boundaries.

FoxO transcription factors (FoxO1, FoxO3a, FoxO4, FoxO6) are a highly evolutionary conserved subfamily of the 'forkhead' box proteins. They have traditionally been considered tumor suppressors, but FoxO1 also exhibits oncogenic properties. The complex nature of FoxO1 is illustrated by its various roles in B cell development and differentiation, immunoglobulin gene rearrangement and cell-surface B cell receptor (BCR) structure, DNA damage control, cell cycle regulation, and germinal center reaction. FoxO1 is tightly regulated at a transcriptional (STAT3, HEB, EBF, FoxOs) and post-transcriptional level (Akt, AMPK, CDK2, GSK3, IKKs, JNK, MAPK/Erk, SGK1, miRNA). In B cell malignancies, recurrent FoxO1 activating mutations (S22/T24) and aberrant nuclear export and activity have been described, underscoring the potential of its therapeutic inhibition. Here, we review FoxO1's roles across B cell and myeloid malignancies, namely acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and multiple myeloma (MM). We also discuss preclinical evidence for FoxO1 targeting by currently available inhibitors (AS1708727, AS1842856, cpd10).

There are only a few studies on the function of neuronal axon guidance molecules during low brain pH conditions. We previously reported that roundabout (ROBO) 2, a receptor for the axon guidance molecule SLIT, can bind to the neural epidermal growth factor-like-like (NELL) ligands in acidic conditions by conformational change of its ectodomain. Here, we show that the ROBO3 receptor also exhibits a pH-dependent increase in binding to the NELL2 ligand. We found that the Glu592 residue of ROBO3 at the binding interface between NELL2 and ROBO3 is a pH sensor and that the formation of a new hydrogen bonding network, due to protonation of the Glu592, leads to increased binding in acidic conditions. These results suggest that NELL2-ROBO3 signaling could be regulated by extracellular pH.

Acinetobacter baumannii, a top-priority WHO pathogen, causes life-threatening infections in immunocompromised patients, leading to prolonged hospitalisation and high mortality. Here, we used the Galleria mellonella model to investigate community strain C98 (Ab-C98) virulence via transcriptomic analysis. Ab-C98 showed greater killing and faster colonisation in larvae than the clinical reference strain (ATCC BAA1605). Genes in three iron clusters, acinetobactin, baumannoferrin and the Feo system, were significantly up-regulated. Targeted knockout of siderophore genes (basC, bfnD, and the gene encoding isochorismatase) significantly increased the survival of infected larvae by at least 35.16%, identifying these genes as potential targets for developing anti-virulence agents against A. baumannii.

The epidermis is a stratified epithelium that functions as the first line of defense against pathogenic invasion and acts as a barrier preventing water loss. In this study, we aimed to decipher the role of 14-3-3ε in the development of the epidermis. We report that loss of 14-3-3ε in the epidermis of juvenile and adult mice reduces cell division in the basal layer and increases the percentage of cells with multiple centrosomes, leading to a reduction in the thickness of the basal and stratified layers. We also demonstrate a decrease in the expression of differentiation markers, although no gross morphological defects in the skin or adverse effects on the survival of the mice were observed. These results suggest that loss of 14-3-3ε in the epidermis may lead to defects in proliferation and differentiation.

Iqsec1 (IQ motif and Sec7 domain-containing protein 1), also known as BRAG2 (Brefeldin A-resistant Arf-GEF 2), is a guanine nucleotide exchange factor that regulates membrane trafficking, cytoskeletal organization, and signal transduction by activating class II and III ADP-ribosylation factors. To investigate the physiological role of Iqsec1 at the whole animal level, we generated Iqsec1-deficient mice using CRISPR/Cas9-mediated gene editing. Nearly all Iqsec1 mice (99%) exhibited embryonic lethality with severe growth retardation. Electron microscopy revealed that Iqsec1 embryos at embryonic day 8.5 lacked large apical vacuoles in visceral endoderm cells of the yolk sac, compared with controls. These findings suggest that Iqsec1 plays a critical role in embryogenesis, likely through regulation of membrane trafficking in visceral endoderm cells.

The tumor suppressor p53 plays a central role in suppressing tumor formation. Mouse double minute 2 homolog (Mdm2) serves as the principal ubiquitin E3 ligase responsible for the ubiquitination and subsequent degradation of p53. However, the regulatory mechanisms governing the Mdm2-p53 pathway are not comprehensively understood. Here, we report that F-box only protein 46 (FBXO46) directly binds to Mdm2 and inhibits its self-ubiquitination and degradation, leading to Mdm2 stabilization and subsequent Mdm2-mediated ubiquitination and degradation of p53. Functionally, FBXO46 promotes cell proliferation, accelerates G1/S cell cycle progression, and increases anchorage-independent cell growth by inhibiting p53. Collectively, these findings reveal a critical role for FBXO46 in controlling Mdm2 stability and establish FBXO46 as an important regulator of the Mdm2-p53 pathway.

Zinc transporters (ZnTs) act as H/Zn antiporters, crucial for zinc homeostasis. Brain-specific ZnT3 expressed in synaptic vesicles transports Zn 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 Zn efflux from the cytosol to lysosomes, mitigating the cell injury risk. Despite their importance, the structures and Zn 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 Zn transport. Our results provide a structural basis for a more comprehensive understanding of the H/Zn exchange mechanisms exhibited by ZnTs.