Self-assembling protein filaments are at the heart of cell function. Among them, tubulin-like proteins are essential for cell division, DNA segregation and cytoskeletal functions across the domains of life. FtsZ and tubulin share their core structures, a characteristic nucleotide-binding pocket and similar protofilament architecture. GTP hydrolysis between consecutive subunits drives their assembly dynamics. Two recent studies provide previously missing, filament atomic structures of bacterial FtsZ and a recently discovered archaeal tubulin in their nucleotide triphosphate-bound states. Both filament structures reveal strikingly conserved interfacial GTPase active sites, with Mg2+ and K+ /Na+ cations and an NxDxxD/E triad of catalytic residues, probably inherited from the common ancestor of FtsZs and tubulins. Moreover, both proteins exhibit nucleotide-regulated subunit association mediated by interfacial water bridges, as well as polymerization-induced structural changes, likely enabling related dynamic assembly mechanisms.

Androgen receptor (AR) signalling is known to be dispensable for the biology of castration-resistant prostate cancer (CRPC), whereas the AR itself and the residual androgens after castration are crucial for the growth and progression of CRPC. Therefore, there is high demand for novel therapeutic candidates targeting AR itself or aberrant AR signalling to suppress the progression to or the growth of CPRC. Here, we report that ginsenoside compound K (GCK), the primary bioactive metabolite biotransformed from protopanaxadiol (PPD) ginsenoside, acts as a novel AR signalling inhibitor by transcriptionally suppressing AR expression and tumour growth in athymic nude mice. GCK inhibited cell growth in LNCaP, PC-3 and 22Rv1 prostate cancer cell lines and suppressed the expression levels of cell cycle regulators. GCK down-regulated epithelial-mesenchymal transition markers such as vimentin and matrix metalloproteinase 9 (MMP9), whereas E-cadherin was significantly increased in GCK-stimulated LNCaP and 22Rv1 cells. Moreover, GCK treatment markedly decreased both AR and AR-V7 protein levels in LNCaP and 22Rv1 cells, possibly by decreasing AR promoter activity. Experiments with AR promoter-deleted constructs revealed that the region between -412 and -227 is critical for GCK regulation. GCK treatment in athymic nude mice implanted with 22Rv1 CRPC cell lines significantly suppressed tumour growth and AR expression levels in tumour tissues. Collectively, our results suggest that GCK, as a novel AR inhibitor, could be a potential therapeutic agent against prostate cancer and an effective chemopreventive agent to delay the progression to CRPC.

Cytosine deaminases AID/APOBEC proteins act as potent nucleic acid editors, playing important roles in innate and adaptive immunity. However, the mutagenic effects of some of these proteins compromise genomic integrity and may promote tumorigenesis. Here, we demonstrate that human APOBEC3G (A3G), in addition to its role in innate immunity, promotes repair of double-strand breaks (DSBs) in vitro and in vivo. Transgenic mice expressing A3G successfully survived lethal irradiation, whereas wild-type controls quickly succumbed to radiation syndrome. Mass spectrometric analyses identified the differential upregulation of a plethora of proteins involved in DSB repair pathways in A3G-expressing cells early following irradiation to facilitate repair. Importantly, we find that A3G not only accelerates DSB repair but also promotes deamination-dependent error-free rejoining. These findings have two implications: (a) strategies aimed at inhibiting A3G may improve the efficacy of genotoxic therapies used to cure malignant tumours; and (b) enhancing A3G activity may reduce acute radiation syndrome in individuals exposed to ionizing radiation.

Fatty acid-binding protein 7 (FABP7), one of the fatty acid (FA) chaperones involved in the modulation of intracellular FA metabolism, is highly expressed in glioblastoma, and its expression is associated with decreased patients' prognosis. Previously, we demonstrated that FABP7 requires its binding partner to exert its function and that a mutation in the FA-binding site of FABP7 affects tumour biology. Here, we explored the role of FA ligand binding for FABP7 function in tumour proliferation and examined the mechanism of FABP7 and ligand interaction in tumour biology. We discovered that among several FA treatment, oleic acid (OA) boosted cell proliferation of FABP7-expressing cells. In turn, OA increased FABP7 nuclear localization, and the accumulation of FABP7-OA complex in the nucleus induced the formation of nuclear lipid droplet (nLD), as well as an increase in colocalization of nLD with promyelocytic leukaemia (PML) nuclear bodies. Furthermore, OA increased mRNA levels of proliferation-related genes in FABP7-expressing cells through histone acetylation. Interestingly, these OA-boosted functions were abrogated in FABP7-knockout cells and mutant FABP7-overexpressing cells. Thus, our findings suggest that FABP7-OA intracellular interaction may modulate nLD formation and the epigenetic status thereby enhancing transcription of proliferation-regulating genes, ultimately driving tumour cell proliferation.

Metrics play a vital part in the valuation and funding of research for scientists worldwide. We review the challenges that metrics pose in providing a fair and equitable system for research funding. We highlight the attempts with declarations, including the San Francisco Declaration on Research Assessment (SF-DORA), to improve the research environment and specific impacts that metric choice can have on the evaluation and progression of Early Career Lecturers (ECLs). While there is much evidence that metrics will never be entirely satisfactory, we conclude there are opportunities that would benefit ECLs and reason for optimism for researchers.

Inflammation is an essential immune response critical for responding to infection, injury and maintenance of tissue homeostasis. Upon injury, regenerative inflammation promotes tissue repair by a timed and coordinated infiltration of diverse cell types and the secretion of growth factors, cytokines and lipids mediators. Remarkably, throughout evolution as well as mammalian development, this type of physiological inflammation is highly associated with immunosuppression. For instance, regenerative inflammation is the consequence of an in situ macrophage polarization resulting in a transition from pro-inflammatory to anti-inflammatory/pro-regenerative response. Immune cells are the first responders upon injury, infiltrating the damaged tissue and initiating a pro-inflammatory response depleting cell debris and necrotic cells. After phagocytosis, macrophages undergo multiple coordinated metabolic and transcriptional changes allowing the transition and dictating the initiation of the regenerative phase. Differences between a highly efficient, complete ad integrum tissue repair, such as, acute skeletal muscle injury, and insufficient regenerative inflammation, as the one developing in Duchenne Muscular Dystrophy (DMD), highlight the importance of a coordinated response orchestrated by immune cells. During regenerative inflammation, these cells interact with others and alter the niche, affecting the character of inflammation itself and, therefore, the progression of tissue repair. Comparing acute muscle injury and chronic inflammation in DMD, we review how the same cells and molecules in different numbers, concentration and timing contribute to very different outcomes. Thus, it is important to understand and identify the distinct functions and secreted molecules of macrophages, and potentially other immune cells, during tissue repair, and the contributors to the macrophage switch leveraging this knowledge in treating diseases.

Reactive oxygen species (ROS) are considered a major cause of ageing and ageing-related diseases through protein carbonylation. Little is known about the molecular mechanisms that confer protection against ROS. Here, we observed that, compared with nondiapause-destined pupae, high protein carbonyl levels are present in the brains of diapause-destined pupae, which is a 'non-ageing' phase in the moth Helicoverpa armigera. Protein carbonyl levels respond to ROS and decrease metabolic activity to induce diapause in order to extend lifespan. However, protein carbonylation in the brains of diapause-destined pupae still occurs at a physiological level compared to young adult brains. We find that ROS activate Akt, and Akt then phosphorylates the transcription factor CREB to facilitate its nuclear import. CREB binds to the promoter of carbonyl reductase 1 (CBR1) and regulates its expression. High CBR1 levels reduce protein carbonyl levels to maintain physiological levels. This is the first report showing that the moth brain can naturally control protein carbonyl levels through a distinct ROS-Akt-CREB-CBR1 pathway to extend lifespan.

The yeast mitogen-activated protein kinase pathways serve as a model system for understanding how network interactions affect the way in which cells coordinate the response to multiple signals. We have quantitatively compared two yeast strain backgrounds YPH499 and Σ1278b (both of which have previously been used to study these pathways) and found several important differences in how they coordinate the interaction between the high osmolarity glycerol (HOG) and mating pathways. In the Σ1278b background, in response to simultaneous stimulus, mating pathway activation is dampened and delayed in a dose-dependent manner. In the YPH499 background, only dampening is dose-dependent. Furthermore, leakage from the HOG pathway into the mating pathway (crosstalk) occurs during osmostress alone in the Σ1278b background only. The mitogen-activated protein kinase Hog1p suppresses crosstalk late in an induction time course in both strains but does not affect the early crosstalk seen in the Σ1278b background. Finally, the kinase Rck2p plays a greater role suppressing late crosstalk in the Σ1278b background than in the YPH499 background. Our results demonstrate that comparisons between laboratory yeast strains provide an important resource for understanding how signalling network interactions are tuned by genetic variation without significant alteration to network structure.

Analysis of ovarian cancer tissue and normal ovarian tissue revealed 31 RNA modifications with significant differences observed in cancer tissue compared with normal tissue. Moreover, we found Im and chm5U as characteristic RNA modifications in advanced and platinum-resistant ovarian cancers, respectively. Considering that these differences in RNA modifications may be due to the intra-tumour microenvironment, we xenografted the ovarian cancer cell line RMG-1 to create RMG-1 tumours and compared them with original RMG-1 cells. As a result, 14 of the 31 RNA modifications showed marked variations during tumorigenesis. Eight RNA modifications (m2,2G, t6A, m7G, m5U, m1G, i6A, m6t6A and m1A), which were upregulated in ovarian cancer tissues and in RMG-1-xenografted tumour, were also upregulated under hypoxic conditions. RNAseq analysis, using the matched RNA samples analysed for RNA modifications, showed that 2137 genes were highly expressed in ovarian cancer tissues compared with those in normal ovarian tissues. Of these, 134 genes, which were enriched in a gene set belonging to the hypoxia signalling pathway, were positively correlated with the above eight RNA modifications. These results suggest that the tumour microenvironment, including hypoxia, is important for cancer characteristic RNA modifications.

In previous work, we have developed a DNA methylation-based epigenetic memory system that operates in Escherichia coli to detect environmental signals, trigger a phenotypic switch of the cells and store the information in DNA methylation. The system is based on the CcrM DNA methyltransferase and a synthetic zinc finger (ZnF4), which binds DNA in a CcrM methylation-dependent manner and functions as a repressor for a ccrM gene expressed together with an egfp reporter gene. Here, we developed a reversible reset for this memory system by adding an increased concentration of ZnSO4 to the bacterial cultivation medium and demonstrate that one bacterial culture could be reversibly switched ON and OFF in several cycles. We show that a previously developed differential equation model of the memory system can also describe the new data. Then, we studied the long-term stability of the ON-state of the system over approximately 100 cell divisions showing a gradual loss of ON-state signal starting after 4 days of cultivation that is caused by individual cells switching from an ON- into the OFF-state. Over time, the methylation of the ZnF4-binding sites is not fully maintained leading to an increased OFF switching probability of cells, because stronger binding of ZnF4 to partially demethylated operator sites leads to further reductions in the cellular concentrations of CcrM. These data will support future design to further stabilize the ON-state and enforce the binary switching behaviour of the system. Together with the development of a reversible OFF switch, our new findings strongly increase the capabilities of bacterial epigenetic biosensors.

Endosomes play crucial roles in the cell, serving as focal and 'triage' points for internalized lipids and receptors. As such, endosomes are a critical branching point that determines whether receptors are sorted for degradation or recycling. This Viewpoint aims to highlight recent advances in endosome research, including key endosomal functions such as sorting and fission. Moreover, the Viewpoint addresses key technical and conceptual challenges in studying endosomes.

The classification of Acinetobacter baumannii by WHO as 'priority 1' antibiotic-resistant pathogen underlines the urgent need for novel antimicrobial agents towards this pathogen. In this work, screening of the A. baumannii phage AbTZA1 genome allowed the identification of a putative endolysin (AbLys1, EC3.2.1.17) that belongs to the glycoside hydrolase family 24 (GH24). The sequence of AbLys1 was cloned, expressed in E. coli and purified. The lytic activity and specificity of AbLys1 were evaluated against a range of Gram-positive and Gram-negative human pathogens. AbLys1 was found to display a high selectivity towards A. baumannii. Kinetic analysis was carried out to characterize the dependence of its lytic activity on pH. The enzyme shows its maximal activity at pH values 7-8. The structure of AbLys1 was determined by X-ray crystallography to 1.82 Å resolution. The overall structure revealed two helical domains: a small, antenna-like, N-terminal domain and a larger C-terminal domain with six α-helices and a β-hairpin. Both the antenna-like and β-hairpin regions contain short sequences (AMseq1 and AMseq2) with predicted antimicrobial activity. Engineering studies revealed a key role of AMseq1 and AMseq2 on the enzyme's lytic activity towards A. baumannii cells but not towards purified peptidoglycan. This suggests that both sequences affect the destabilization of the outer membrane, thus providing access of the catalytic domain to the peptidoglycan. In addition, the deletion of AMseq1 enhanced the enzyme stability, whereas the deletion of AMseq2 diminished it. The results suggest that AbLys1 is a promising new enzybiotic with efficient lytic and antimicrobial activity.

O-sulfated N-acetyl-d-galactosamine (GalNAc) residues in chondroitin sulfate (CS) play a crucial role in chondroitinase ABC I (cABC-I) activity. CSA containing mainly 4-O-monosulfated GalNAc was a good substrate for the enzyme, but not CSE containing mainly 4,6-O-disulfated GalNAc [GalNAc(4S,6S)]. Each CS isomer exhibits structural heterogeneity; CSE has di-sulfated disaccharide units and mono-sulfated disaccharide units. Disaccharide composition analysis of digested products revealed that mono-sulfated disaccharide units in CSE contributed to the enzyme reactivity. Although enough substrate (CSA) was present in mixtures of CSA and CSE for reaction, the reactivity was reduced depending on the amount of CSE in the mixture. These results suggested that CSE is not only resistant to enzyme digestion but also attenuates enzyme activity. To understand the mechanism of action, crystallography of cABC-I in complex with unsaturated CSE-disaccharide, ΔDi-(4,6)S, was performed. Both 4-O- and 6-O-sulfate groups in ΔDi-(4,6)S interact with Arg500, suggesting that there was a greater interaction between ΔDi-(4,6)S and Arg500 than between mono-sulfated disaccharides and Arg500. Besides, this interaction attenuated enzyme activity by interfering with a function of Arg500, which is the charge neutralization of the carboxy group of D-glucuronic acid (GlcA) residues in CS. When interacting with the CSE-disaccharide unit [GlcAβ1-3GalNAc(4S,6S)] in CS, cABC-I cannot interact with other CS-disaccharide units until it has digested the CSE-disaccharide unit. The low reactivity of cABC-I with CSE is attributable to two suggested factors: (a) resistance of E-units in CSE molecules to digestion by cABC-I, and (b) tendency of E-units in CSE molecules to attenuate cABC-I activity.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.

The transcription factor STAT3 is a potent activator of transcription, but evidence exists that STAT3 can also repress gene expression. However, little is known about the molecular mechanisms involved in STAT3-dependent gene repression. Notably, STAT3 reduces the expression of the stress-induced mTOR inhibitor REDD1 by reducing REDD1 mRNA transcription. Here, we determined the functional domains of STAT3 responsible for the reduction of REDD1 mRNA and protein expression. Within STAT3, the N-terminal domain and tyrosine 705 are crucial for STAT3-dependent reduction of REDD1 expression. Interestingly, binding of STAT3 to canonical STAT-binding sides within the REDD1 promoter is not necessary for STAT3-mediated reduction of REDD1 expression. Still, STAT3 is recruited to the REDD1 promoter upon stimulation with IL-6, and reduces REDD1 promoter activity. The reduction of REDD1 expression is specific for STAT3, as neither expression nor activation of STAT1 reduces REDD1 mRNA and protein expression. In summary, we present a novel, non-canonical STAT3-dependent mechanism for reducing gene expression. This transcriptional repression increases the functions of STAT3 proteins beyond classical transcriptional activation of cytokine-regulated target genes to a more complex function in modulating gene expression in immunity and cellular stress.

Autophagy is a conserved cytoprotective process, aberrations in which lead to numerous degenerative disorders. While the cytoplasmic components of autophagy have been extensively studied, the epigenetic regulation of autophagy genes, especially in stem cells, is less understood. Deciphering the epigenetic regulation of autophagy genes becomes increasingly relevant given the therapeutic benefits of small-molecule epigenetic inhibitors in novel treatment modalities. We observe that, during retinoic acid-mediated differentiation of mouse embryonic stem cells (mESCs), autophagy is induced, and identify the Polycomb group histone methyl transferase EZH2 as a regulator of this process. In mESCs, EZH2 represses several autophagy genes, including the autophagy regulator DNA damage-regulated autophagy modulator protein 1 (Dram1). EZH2 facilitates the formation of a bivalent chromatin domain at the Dram1 promoter, allowing gene expression and autophagy induction during differentiation while retaining the repressive H3K27me3 mark. EZH2 inhibition leads to loss of the bivalent domain, with consequent 'hyper-expression' of Dram1, accompanied by extensive cell death. This study shows that Polycomb group proteins help maintain a balance between autophagy and cell death during stem cell differentiation, in part, by regulating the expression of the Dram1 gene.

High-mobility group box 1 (HMGB1) is critical for inflammatory homeostasis and successful pregnancy, and there is a strong association among elevated levels of HMGB1, polycystic ovary syndrome (PCOS), chronic inflammation and pregnancy loss. However, the mechanisms responsible for PCOS-driven regulation of uterine HMGB1 and its candidate receptors [toll-like receptor (TLR) 2 and 4] and inflammatory responses during pregnancy remain unclear. In this study, we found a gestational stage-dependent decrease in uterine HMGB1 and TLR4 protein abundance in rats during normal pregnancy. We demonstrated that increased expression of HMGB1, TLR2 and TLR4 proteins was associated with activation of inflammation-related signalling pathways in the gravid uterus exposed to 5α-dihydrotestosterone and insulin, mimicking the clinical features (hyperandrogenism and insulin resistance) of PCOS and this elevation was completely inhibited by treatment with the androgen receptor (AR) antagonist flutamide. Interestingly, acute exposure to lipopolysaccharide suppressed HMGB1, TLR4 and inflammation-related protein abundance but did not affect androgen levels or AR expression in the gravid uterus with viable fetuses. Furthermore, immunohistochemical analysis revealed that, in addition to being localized predominately in the nuclear compartment, HMGB1 immunoreactivity was also detected in the cytoplasm in the PCOS-like rat uterus, PCOS endometrium and pregnant rat uterus with haemorrhagic and resorbed fetuses, possibly via activation of nuclear factor κB signalling. These results suggest that both AR-dependent and AR-independent mechanisms contribute to the modulation of HMGB1/TLR2/TLR4-mediated uterine inflammation. We propose that the elevation of HMGB1 and its receptors and disruption of the pro-/anti-inflammatory balance in the gravid uterus may participate in the pathophysiology of PCOS-associated pregnancy loss.

Radiotherapy, as an important primary treatment, has effectively improved the survival of patients with cervical cancer (CC). Some patients, however, do not benefit optimally from radiotherapy because of radio-resistance. Therefore, identifying radio-resistance biomarkers and unravelling the underlying mechanisms is of critical importance for these patients. In the present study, we found significant upregulation of hepatocyte nuclear factor 1-alpha (HNF1α) expression in radio-resistant cervical cancer tissues and cell lines. Depletion of HNF1α reduced and overexpression of HNF1α promoted the resistance of CC cells to irradiation in vitro and in vivo. HNF1α positively regulated DNA repair protein RAD51 homologue 4 (RAD51D) at the protein level but not at the mRNA level. Mechanistically, upregulation of HNF1α enhanced YTH domain-containing family protein 3 (YTHDF3) transcription, which in turn promoted RAD51D mRNA N6 -methyladenosine (m6A) modification. YTHDF3 mediates HNF1α regulation of cervical cancer radio-resistance by promoting RAD51D translation in an m6A-dependent manner. The HFN1α/YTHDF3/RAD51D regulatory axis was found to play a critical role in conferring radio-resistance of CC cells. In conclusion, dysregulation of the HFN1α/YTHDF3/RAD51D axis may promote the radio-resistance of CC cells. Blocking this pathway may provide therapeutic benefits against CC radio-resistance.

Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype-phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype-phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome.

Plakophilin (PKP1) 1 is a member of the arm-repeat family of catenins and acts as a structural component of desmosomes, which are important stabilizers of cell-cell adhesion. Besides this, PKP1 also occurs in a non-junctional, cytoplasmic form contributing to post-transcriptional regulation of gene expression. Moreover, PKP1 is expressed in the prostate epithelium but its expression is frequently downregulated in prostate cancers with a more aggressive phenotype. This observation may imply a tumour-suppressive role of PKP1. We found that, in prostatic adenocarcinomas with PKP1 deficiency, the occurrence of T-cells, B-cells, macrophages and neutrophils were significantly increased. In a PKP1-deficient prostatic cancer cell line expressing IL8, these levels were statistically meaningfully reduced upon PKP1 re-expression. When analysing prostatic PKP1 knockdown cell lines, the mRNA and protein levels of additional cytokines, namely CXCL1 and IL6, were upregulated. The effect was rescued upon re-expression of a PKP1 RNAi-resistant form. The corresponding mRNAs were co-precipitated with cytoplasmic PKP1, indicating that they are components of PKP1-containing mRNA ribonucleoprotein particles. Moreover, the mRNA half-lives of CXCL1, IL8 and IL6 were significantly increased in PKP1-deficient cells, showing that these mRNAs were stabilized by PKP1. In an in vitro migration assay, the higher cytokine concentrations led to higher migration rates of THP1 and PBMC cells. This finding implies that PKP1 loss of expression in vivo correlates with the recruitment of immune cells into the tumour area to set up a tumour-specific environment. One may speculate that this newly established tumour environment has tumour-suppressive characteristics and thereby accelerates tumour progression and metastasis.