The Ton and Tol-Pal systems are molecular machines that are essential for survival of Gram-negative bacteria.Both use the energy derived from the proton gradient at the inner membrane to generate force on protein components at the outer membrane. Ton and Tol share extensive homology, but they fulfill different functions: Ton is involved in the active transport of essential nutrients from the extracellular media into the cell, while Tol maintains the outer membrane integrity and participates in the cell division process. Despite decades of biochemical and biophysical studies, the molecular mechanism coupling the proton gradient at the inner membrane with the propagation of force and movement to the outer membrane is not understood. In this review, we discuss the recent high-resolution structures obtained for both systems, and how these structures fit with existing mechanistic models.

WRKY transcription factors, a plant-specific family of transcriptional regulators, are classified into four groups (I–IV) and play pivotal roles in plant defense, development, and stress responses. These proteins are characterized by conserved WRKY domains that preferentially bind to the W-box cis-element C/TTGACC/T in target gene promoters. In Gossypium hirsutum (Gh; upland cotton), the group IId member GhWRKY17 regulates cotton fiber development by activating downstream target genes such as GhHOX3 through promoter W-box binding. However, the structural basis for its DNA recognition specificity remains elusive. Here, we present the 1.8 Å resolution crystal structure of the GhWRKY17 WRKY domain in complex with the GhHOX3 promoter DNA—the first structural characterization of a group IId WRKY protein. Structural analysis reveals that it consists of four antiparallel β-strands, with the β2-strand (harboring the conserved 249WRKYGQK255 motif) and β3-strand co-operatively engaging the DNA major groove. Key residues (R250, K251, Y252, Q254, K255, R264, Y266, Y267) form an intricate hydrogen-bonding network essential for recognizing the extended G/TTTGACC motif. Comparative structural analyses with group I/IIa/III WRKY–DNA complexes reveal that GhWRKY17’s dual-strand engagement and extensively hydrogen bond-mediated specific interaction represent novel mechanistic features distinguishing group IId members from other WRKY subgroups, emphasizing the necessity for subgroup-specific investigations. These findings not only establish a structural paradigm for group IId WRKY function but also provide molecular insights for engineering cotton fiber traits through transcriptional regulation.

Titin kinase (TK) is an enigmatic pseudokinase specific to the striated muscle of vertebrates. Embedded within the contractile sarcomere and flanked by extensible regulatory tails, TK is thought to act as a mechanoreceptor that senses mechanical signals arising from muscle function. Studies on TK so far have focused narrowly on the human representative, whose phosphotransfer activity remains questioned. To investigate whether the pseudokinase character is a hallmark of TK, we studied sequences of distantly evolved fish representatives and rationalized conservation patterns by solving the crystal structure of TK from medaka (isoform b). We find that sequence deviations in functional motifs involved in ATP and magnesium binding, respectively θxK (θ: bulky hydrophobic residue) and EFG, are evolutionarily conserved in TK. Beyond the kinase domain, N- and C-terminal flanking tails show remarkable structural similarity across orthologues, even though sequence conservation is limited to individual residues and short motifs: a YD-motif in the N-terminal tail; a [R/K]H[R/K]RYY sequence, a R-7x-R motif and position -2 of the latter in the C-terminal tail. Motifs in the C-terminal tail consistently covary with the divergent functional motifs of TK, being part of its pseudokinase signature. Contrary with these general features, the putatively inhibitory interaction of the catalytic aspartate with a tyrosine from loop P+1 is primarily confined to mammals. Finally, based on sequence clustering analysis, we identify TK subgroupings and propose a classification of titin genes from fish into a and b isoforms (ttna and ttnb) that can assist future studies. A curated genomic annotation is provided.

The authors of the article “The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl- co-transporters” (DOI: 10.1042/BJ20131478) wish to retract the article from the Biochemical Journal after being alerted by a reader to multiple errors across Figures 3–7 of the published work.These errors included misalignment of digitised Western blot data, incorrect figure labelling and instances of blot duplication due to miscommunication between laboratory members involved in preparing the figures following peer review. Unfortunately, these errors were not detected during the final pre-publication review.The authors have located and verified the original raw data and laboratory notebooks for all experiments. These data reaffirm the study’s central conclusion, that WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit K+-Cl- co-transporters. These findings have been independently replicated and supported by numerous peer-reviewed publications over the past decade, and the phospho-specific antibodies generated in this study have also been widely used to investigate KCC regulation (e.g. DOI: 10.1074/jbc.m117.817841; DOI: 10.1074/jbc.m705095200; DOI: 10.1073/pnas.1810134115; and DOI: 10.1038 /s41467-019-13851-6). The authors therefore stand by the conclusions of the paper.However, given the number of errors identified, and in the interest of maintaining scientific transparency and integrity of the published record, the authors collectively agree that retraction of this article is the most appropriate course of action.The Publisher reviewed original data provided by the authors and agrees with retraction of the paper.The authors sincerely apologise to the editors, reviewers and readers of the Biochemical Journal for the inconvenience caused by these unintentional errors. The authors intend to repeat the key experiments to verify the findings, and the confirmed results will be submitted for consideration of publication in due course.

Inter-organellar cross-talk is an important component of cellular stress response enabling adaptation and survival. We have demonstrated the activation of RTG retrograde signaling to sustain the peroxisomesmitochondria– nucleus axis in a model of osmostressed Saccharomyces cerevisiae yeast cells. In this work, we aimed to gain insight into the molecular mechanisms regulating the communication between these organelles upon NaCl treatment. A metabolomic analysis revealed that the homeostasis of citrate is a pivotal factor in the osmoadaptive response. Gene expression analysis and citrate synthase activity showed that the synthesis of citrate mainly derives from peroxisomes, as indicated by the up-regulation of CIT2, and not CIT1 and CIT3, under the control of the RTG pathway. Furthermore, the involvement of the mitochondrial citrate transporter, encoded by YHM2, in the osmoadaptive response, as judged by gene and protein expression analysis together with growth assay, is demonstrated. In the absence of YHM2, alternative pathways relying on ODC2 and ACO1 are activated, indicating possible compensatory mechanisms for osmoadaptation. We propose a model in which peroxisome-derived citrate is converted to cytosolic 2-oxoglutarate to replenish TCA cycle and promote its rewiring. This work reveals a new layer of metabolic co-ordination among organelles and identifies citrate shuttling as a crucial adaptive mechanism to osmotic stress.

Histidine-rich glycoprotein (HRG) is a multidomain plasma protein involved in immune modulation, angiogenesis, coagulation and fibrinolysis. Despite its broad biological relevance, structural investigations into HRG have yielded only limited information, with no experimentally resolved three-dimensional structures of the intact protein to date. In this study, we integrate hydrogen-deuterium exchange mass spectrometry (HDX-MS) with predictive insights from AlphaFold to map the conformational landscape of HRG in solution under near-native conditions. The N1/N2 domains displayed low solvent exchange overall. However, specific regions of high-solvent exchange were also apparent, providing evidence for more dynamic stretches of secondary structure and the presence of flexible loops within these regions. Our findings also reveal extensive solvent accessibility and rapid exchange kinetics within the histidine-rich region, proline-rich regions and large segments of the C-terminal domain, strongly indicating intrinsic disorder across these domains. These findings support a model in which structural flexibility underlies HRG’s capacity to engage with a wide range of molecular partners. This integrative approach offers new insight into the conformational architecture of HRG and lays the groundwork for uncovering the molecular mechanisms governing its biological activity.

RAS proteins have been studied for more than 40 years, mainly in the context of cancer. Given that RAS signaling promotes cell cycle progression, it is commonly assumed that its main function is to drive cell proliferation. In this review, we will, however, address the roles of RAS during cell differentiation, which is intertwined with cell division during organismal development and tissue homeostasis in the adult. Our analysis suggests a far-reaching and profound impact of RAS signaling in associated processes. During vertebrate embryonal development, the FGF-RAS-ERK signaling axis is involved as early as germ layer induction and embryonal patterning. Current evidence suggests that RAS fundamentally controls the balance between stem cells and their differentiated progeny. RAS signaling needs to be downmodulated to sustain pluripotent stem cells. Inhibition of RAS activity is also required to preserve adult stem cell quiescence. At the other end of the differentiation spectrum, a different kind of RAS inactivation by the GTPase-activating protein (GAP) neurofibromin 1 (NF1) appears central to permit terminal differentiation, e.g., of muscle tissue. This latter process is disabled in muscle-borne cancer and likely also in other cancer types. In the RAS-associated developmental diseases, the RASopathies, cell differentiation appears to be broadly perturbed throughout development. We suggest that loss of RAS pathway activity mainly manifests in the stem/progenitor cell compartment, whereas inhibition of NF1 GAP-mediated RAS inactivation blocks terminal differentiation. Given that defects accumulate during development, it is plausible to assume that only progressive pathological phenotypes of RASopathies offer a realistic chance for treatment, notably by repurposing RAS-MAPK pathway oncology drugs. Thus, the impact of RAS on cell differentiation represents, in our view, the common mechanistic denominator of cancer and RASopathies. We conclude by giving a perspective on how improving our insight into the functioning of RAS during cell differentiation could lead to the development of misdifferentiation-correcting drugs.

Type III CRISPR systems typically generate cyclic oligoadenylate second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA. These activate ancillary effector proteins which elicit a diverse range of immune responses. The Calp (CRISPR associated Lon protease) system elicits a transcriptional response to infection when CalpL (Calp Lon protease) binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS (Calp Sigma factor) for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2 (second anti-CRISPR of type III systems), which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.

It has come to the attention of the authors of the article “Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase” (DOI: 10.1042/bj3490605) that there is a repetition of blots representing FLAG (EGF panel) in Figure 1C and FLAG (EGF panel) in Figure 3A. This was an unintentional error made during the assembly of the figure.The raw data for Figures 1C and 3A have been identified, and it is confirmed that the incorrect data were used for Figure 3A. A new Figure 3A (EGF panel), containing the correct data, is provided here.The raw data and requested correction have been assessed by, and agreed with, the Publisher. The authors apologise for the error and any inconvenience this may have caused. The data analysis and conclusions are not affected by this error.

Saccharomyces cerevisiae, a widely studied unicellular eukaryotic model, multiplies and divides through an asymmetric budding process, where a mother cell produces a smaller daughter cell. Although cells age across successive cell divisions, provided the mother is not too old, each daughter cell inherits a full lifespan potential. Extensive studies in budding yeast have established a framework for understanding how asymmetric cell division contributes to this lifespan resetting. One postulate of this framework is that the capacity of mother cells to bud daughters with full replicative potential is critically dependent on membraneassociated mechanisms that enable asymmetric inheritance of aging factors. Despite the identification of numerous asymmetrically distributed proteins, an integrated catalog detailing their roles in aging has not been compiled. This review provides a comprehensive resource of asymmetrically distributed membrane proteins in yeast that have a role in replicative aging. Existing knowledge governing the establishment and maintenance of asymmetry is synthesized, and gaps in our understanding of how membrane asymmetry contributes to cellular aging are identified.