Photosynthesis uses the energy of sunlight to convert water and atmospheric CO2 into sugars, providing food and oxygen for life. The fixation of atmospheric CO2 in this crucial biological process is mediated by the enzyme Rubisco. The inefficiencies of Rubisco have inspired researchers for decades to explore ways to improve its function with the goal of increasing crop yields [1-4], and more recently to combat global warming [5]. In this graphical review we highlight the challenges involved in engineering plant Rubisco, with a focus on the extensive chaperone requirement for its biogenesis. We discuss strategies for engineering the catalytic properties of Rubisco and for sequestering the enzyme in membraneless compartments to increase CO2 fixation.

Cancer cells hijack metabolic pathways in order to provide themselves with building blocks to support their proliferation and survival. Upregulation and addiction to de novo serine/glycine synthesis is an example of metabolic rewiring in cancer cells whereby serine and glycine are synthesised via a side branch of glycolysis. In this review, we focus on upregulation of endogenous serine/glycine production in acute leukemia, namely T-cell acute leukemia (T-ALL) and acute myeloid leukemia (AML). Several genetic lesions directly driving the serine/glycine addiction in acute leukemia have been established. Additionally, indirect regulation of de novo serine/glycine synthesis is observed in acute leukemia.

Fluctuations in nutrient and biomass availability, often as a result of disease, impart metabolic challenges that must be overcome in order to sustain cell survival and promote proliferation. Cells adapt to these environmental changes and stresses by adjusting their metabolic networks through a series of regulatory mechanisms. Our understanding of these rewiring events has largely been focused on those genetic transformations that alter protein expression and the biochemical mechanisms that change protein behavior, such as post-translational modifications and metabolite-based allosteric modulators. Mounting evidence suggests that a class of proteome surveillance proteins called molecular chaperones also can influence metabolic processes. Here, we summarize several ways the Hsp90 and Hsp70 chaperone families act on human metabolic enzymes and their supramolecular assemblies to change enzymatic activities and metabolite flux. We further highlight how these chaperones can assist in the translocation and degradation of metabolic enzymes. Collectively, these studies provide a new view for how metabolic processes are regulated to meet cellular demand and inspire new avenues for therapeutic intervention.

DNA methylation (5mC) is an essential epigenetic mark associated with transcriptional silencing. The role of 5mC in transcriptional repression is well established for a few hundred genes through methylation of their promoters. Yet, whether 5mC contributes more broadly to gene expression is an important open question. 5mC removal has recently been associated with the activation of enhancers, opening the possibility that 5mC may globally contribute to the expression of genes defining cell identities. Here, we will review the evidence and molecular mechanisms that link 5mC with the activity of enhancers. We will discuss the spread and amplitude of the potential gene expression changes controlled by 5mC at enhancers, and how these may contribute to the determination of cell identities during development.

Mitochondria are organelles indispensable for the correct functioning of eukaryotic cells. Their significance for cellular homeostasis is manifested by the existence of complex quality control pathways that monitor organellar fitness. Mitochondrial biogenesis relies on the efficient import of mitochondrial precursor proteins, a large majority of which are encoded by nuclear DNA and synthesized in the cytosol. This creates a demand for highly specialized import routes that comprise cytosolic factors and organellar translocases. The passage of newly encoded mitochondrial precursor proteins through the cytosol to the translocase of the outer mitochondrial membrane (TOM) is under tight surveillance. As a result of mitochondrial import defects, mitochondrial precursor proteins accumulate in the cytosol or clog the TOM complex, which in turn stimulates cellular stress responses to minimize the consequences of these challenges. These responses are critical for maintaining protein homeostasis under conditions of mitochondrial stress. The present review summarizes recent advances in the field of mitochondrial protein import quality control and discusses the role of this quality control within the network of cellular mechanisms that maintain the cellular homeostasis of proteins.

RNA-binding proteins (RBPs) play vital roles in organisms through binding with RNAs to regulate their functions. Small molecules affecting the function of RBPs have been developed, providing new avenues for drug discovery. Herein, we describe the perspectives on developing small molecule regulators of RBPs. The following types of small molecule modulators are of great interest in drug discovery: small molecules binding to RBPs to affect interactions with RNA molecules, bifunctional molecules binding to RNA or RBP to influence their interactions, and other types of molecules that affect the stability of RNA or RBPs. Moreover, we emphasize that the bifunctional molecules may play important roles in small molecule development to overcome the challenges encountered in the process of drug discovery.

The nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide mononucleotide (NMN) is a proposed therapy for age-related disease, whereby it is assumed that NMN is incorporated into NAD+ through the canonical recycling pathway. During oral delivery, NMN is exposed to the gut microbiome, which could modify the NAD+ metabolome through enzyme activities not present in the mammalian host. We show that orally delivered NMN can undergo deamidation and incorporation in mammalian tissue via the de novo pathway, which is reduced in animals treated with antibiotics to ablate the gut microbiome. Antibiotics increased the availability of NAD+ metabolites, suggesting the microbiome could be in competition with the host for dietary NAD+ precursors. These findings highlight new interactions between NMN and the gut microbiome.

Mitochondria contain 902 (yeast) to 1.136 (mouse, humans) verified proteins. Except for a very small number of mitochondrially encoded core components of the respiratory chain, mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol. Different import pathways direct proteins to their respective mitochondrial subcompartment (outer membrane, intermembrane space (IMS), inner membrane and matrix). Specific targeting signals in their sequence direct proteins to their target destination and allow the proteins to embark on their respective import pathway. The main import pathways are shown here on the poster and are introduced in the following, using the mitochondrial import system of the baker's yeast Saccharomyces cerevisiae as example. However, the mitochondrial import system of mammalian cells is highly similar and deviates only in minor aspects. Even the mitochondrial import machineries of less closely related eukaryotes, such as plants and trypanosomes, are very similar and adhere to the same general principles.

The nuclear envelope (NE) encloses the genetic material and functions in chromatin organization and stability. In Saccharomyces cerevisiae, the NE is bound to the ribosomal DNA (rDNA), highly repeated and transcribed, thus prone to genetic instability. While tethering limits instability, it simultaneously triggers notable NE remodeling. We posit here that NE remodeling may contribute to genome integrity maintenance. The NE importance in genome expression, structure, and integrity is well recognized, yet studies mostly focus on peripheral proteins and nuclear pores, not on the membrane itself. We recently characterized a NE invagination drastically obliterating the rDNA, which we propose here as a model to probe if and how membranes play an active role in genome stability preservation.

Conserved catabolic pathways operate to remove aberrant polypeptides from the endoplasmic reticulum (ER), the major biosynthetic organelle of eukaryotic cells. The best known are the ER-associated degradation (ERAD) pathways that control the retrotranslocation of terminally misfolded proteins across the ER membrane for clearance by the cytoplasmic ubiquitin/proteasome system. In this review, we catalog folding-defective mammalian, yeast, and plant proteins that fail to engage ERAD machineries. We describe that they rather segregate in ER subdomains that eventually vesiculate. These ER-derived vesicles are captured by double membrane autophagosomes, engulfed by endolysosomes/vacuoles, or fused with degradative organelles to clear cells from their toxic cargo. These client-specific, mechanistically diverse ER-phagy pathways are grouped under the umbrella term of ER-to-lysosome-associated degradation for description in this essay.

The cAMP-dependent protein kinase A (PKA) is the archetypical eukaryotic kinase. The catalytic subunit (PKA-C) structure is highly conserved among the AGC-kinase family. PKA-C is a bilobal enzyme with a dynamic N-lobe, harbouring the Adenosine-5'-triphosphate (ATP) binding site and a more rigid helical C-lobe. The substrate-binding groove resides at the interface of the two lobes. A distinct feature of PKA-C is the positive binding cooperativity between nucleotide and substrate. Several PKA-C mutations lead to the development of adenocarcinomas, myxomas, and other rare forms of liver tumours. Nuclear magnetic resonance (NMR) spectroscopy shows that these mutations disrupt the allosteric communication between the two lobes, causing a drastic decrease in binding cooperativity. The loss of cooperativity correlates with changes in substrate fidelity and reduced kinase affinity for the endogenous protein kinase inhibitor (PKI). The similarity between PKI and the inhibitory sequence of the kinase regulatory subunits suggests that the overall mechanism of regulation of the kinase may be disrupted. We surmise that a reduced or obliterated cooperativity may constitute a common trait for both orthosteric and allosteric mutations of PKA-C that may lead to dysregulation and disease.

Despite great progress in recent years, gender equity in science is still missing. Women are underrepresented in senior/leadership positions and struggle to be funded and awarded. Social norms, gender bias, stereotypes in education, and lack of support for the family are just some of the issues that need to be addressed to revert this trend. Historically, many women have been overshadowed by their men colleagues. Although it is hard to give the deserved credit to all women who went unnoticed for centuries, it is time to properly recognize the growing number of them who succeded in science despite the challenges. These women can inspire many more who intend to make of science their future.

Tumor-elicited inflammation confers tumorigenic properties, including cell death resistance, proliferation, or immune evasion. To focus on inflammatory signaling in tumors, we investigated linear ubiquitination, which enhances the nuclear factor-κB signaling pathway and prevents extrinsic programmed cell death under inflammatory environments. Here, we showed that linear ubiquitination was augmented especially in tumor cells around a necrotic core. Linear ubiquitination allowed melanomas to tolerate the hostile tumor microenvironment and to extend a necrosis-containing morphology. Loss of linear ubiquitination resulted in few necrotic lesions and growth regression, further leading to repression of innate anti-PD-1 therapy resistance signatures in melanoma as well as activation of interferon responses and antigen presentation that promote immune-mediated tumor eradication. Collectively, linear ubiquitination promotes tumor-specific tissue remodeling and the ensuing immune evasion.

Mitochondria are the powerhouses of the cell as they produce the majority of ATP with their oxidative phosphorylation (OXPHOS) machinery. The OXPHOS system is composed of the F1 Fo ATP synthase and four mitochondrial respiratory chain complexes, the terminal enzyme of which is the cytochrome c oxidase (complex IV) that transfers electrons to oxygen, generating water. Complex IV comprises of 14 structural subunits of dual genetic origin: while the three core subunits are mitochondrial encoded, the remaining constituents are encoded by the nuclear genome. Hence, the assembly of complex IV requires the coordination of two spatially separated gene expression machinery. Recent efforts elucidated an increasing number of proteins involved in mitochondrial gene expression, which are linked to complex IV assembly. Additionally, several COX1 biogenesis factors have been intensively biochemically investigated and an increasing number of structural snapshots shed light on the organization of macromolecular complexes such as the mitoribosome or the cytochrome c oxidase. Here, we focus on COX1 translation regulation and highlight the advanced understanding of early steps during COX1 assembly and its link to mitochondrial translation regulation.

Chromophore-bearing proteins that are (reversibly) altered after light illumination are major functional components of nature. They gained considerable attention in the last decades since the dynamic interactions of the chromophore and protein matrix can be used to control downstream effects altering the functionality of proteins, cells, or complete organisms with light (optogenetics). Additionally, the photophysical effects can be employed to add capabilities to optical imaging. For example, light can be used to reversibly switch the signal on or off (e.g., fluorescence). In this article, we review chromophore and protein matrix interactions, focusing on photoswitching fluorescent proteins of the GFP family (RSFPs) and natively photoswitching bacteriophytochromes (BphPs). This review aims to provide an in-depth understanding of the dynamic interplay between photoswitching photophysics and the protein matrix and a thorough discussion on how this connection has been harnessed for the development of optogenetic and imaging tools.

Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.

Cytoplasmic capping returns a cap to specific mRNAs, thus protecting uncapped RNAs from decay. Prior to the identification of cytoplasmic capping, uncapped mRNAs were thought to be degraded. Here, we test whether long noncoding RNAs (lncRNAs) are substrates of the cytoplasmic capping enzyme (cCE). The subcellular localisation of 14 lncRNAs associated with sarcomas were examined in U2OS osteosarcoma cells. We used 5' rapid amplification of cDNA ends (RACE) to assay uncapped forms of these lncRNAs. Inhibiting cytoplasmic capping elevated uncapped forms of selected lncRNAs indicating a plausible role of cCE in targeting them. Analysis of published cap analysis of gene expression (CAGE) data shows increased prevalence of certain 5'-RACE cloned sequences, suggesting that these uncapped lncRNAs are targets of cytoplasmic capping.

The Beautiful Brain is a documentary web series that breaks down borders between science and art. Five episodes retrace in a simple, but visually effective, way five key steps of brain development, using awe-inspiring masterpieces of art as analogies. This unconventional series focuses on fundamental research in neuroscience, the communication of which is not always easy and straightforward. In this article, we share our experience of how we tried to overcome the difficulties of communicating fundamental science to the lay audience. Moreover, we give insights into the path we followed to create The Beautiful Brain, with the hope that our experience may be an inspiration to other basic scientists who wish to communicate their own research.

Human aldehyde oxidase (hAOX1) is a molybdoenzyme that oxidizes aldehydes and N-heterocyclic compounds, thereby generating hydrogen peroxide (H2 O2 ) and superoxide during turnover. hAOX1 has been shown previously to be inactivated under turnover conditions by H2 O2 . Here, we investigated the effect of exogenously added H2 O2 on the activity of hAOX1. We show that exogenously added H2 O2 did not affect the enzyme activity under aerobic conditions, but completely inactivated the enzyme under anaerobic conditions. We propose that this effect is based on the reducing power of H2 O2 and the susceptibility of the reduced molybdenum cofactor (Moco) to lose the sulfido ligand. When oxygen is present, the enzyme is rapidly reoxidized. We believe that our study is significant in understanding the detailed effect of reactive oxygen species on the inactivation of hAOX1 and other molybdoenzymes.

Using a zebrafish ionocyte model, transcriptomics and genetic analyses were performed to identify pathways and genes involved in cell quiescence-proliferation regulation. Gene ontology and Kyoto encyclopedia of genes and genomes pathway analyses revealed that genes involved in transcription regulation, cell cycle, Foxo signalling and Wnt signalling pathway are enriched among the up-regulated genes while those involved in ion transport, cell adhesion and oxidation-reduction are enriched among the down-regulated genes. Among the top up-regulated genes is FK506-binding protein 5 (Fkbp5). Genetic deletion and pharmacological inhibition of Fkbp5 abolished ionocyte reactivation and impaired Akt signalling. Forced expression of a constitutively active form of Akt rescued the defects caused by Fkbp5 inhibition. These results uncover a key role of Fbkp5 in regulating the quiescence-proliferation decision via Akt signalling.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

Polysaccharide-degrading mono-copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H2 O2 co-substrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH-dependent manner. Between pH 6.0 and 8.0, reactions with ascorbic acid show little pH dependency, whereas reactions with gallic acid become much faster at increased pH. These dependencies correlate with the reductant ionization state, which affects its ability to react with molecular oxygen and generate H2 O2 . The correlation does not apply to l-cysteine because, as shown by stopped-flow kinetics, increased H2 O2 production at higher pH is counteracted by increased binding of l-cysteine to the copper active site. The findings highlight the importance of the choice of reductant and pH in LPMO reactions.

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split-luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split-mCitrine bimolecular fluorescence complementation and co-immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn-induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed.

Liquid-ordered (Lo) membrane domains have been proposed to play important roles in a wide variety of biological processes, such as protein sorting and cell signaling. However, the mechanisms by which they are formed and maintained remain poorly understood. Lo domains are formed in the vacuolar membrane of yeast in response to glucose starvation. Here, we show that the deletion of proteins that localize to vacuole membrane contact sites (MCSs) caused a marked decrease in the number of cells with Lo domains. In addition to Lo domain formation, autophagy is induced upon glucose starvation. However, the deletion of core autophagy proteins did not inhibit Lo domain formation. Thus, we propose that vacuolar Lo domain formation during glucose restriction is regulated by MCSs but not by autophagy.

Using a murine model of high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD), we found that the expression of the epidermal growth factor receptor (EGFR) significantly decreased in hepatocytes. In vitro, free fatty acid influx decreased EGFR in hepatocytes. In HFD-fed mice, ectopic expression of EGFR alleviated intrahepatic lipid accumulation and reduced serum triglyceride and cholesterol, whereas knockdown of EGFR aggravated hepatic steatosis. Notably, EGFR inhibited the induction of lipogenic genes, including Srebf1, Srebf2, Fasn, Acc1 and Ppara, both in vitro and in vivo. Mechanistically, EGFR potentiates TGF-β/Smad signalling and augments the inhibitory effects of TGF-β1 on lipogenic genes in hepatocytes. Our findings suggest a hitherto unknown paradigm in the pathogenesis of NAFLD, thereby providing a rational basis for future therapeutic considerations.

Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose-xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re-evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose-associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.

This is the first report on the molecular characterization of isoprene synthase (ISPS) from the moss Calohypnum plumiforme. After isoprene emission from C. plumiforme was confirmed, the cDNA encoding C. plumiforme ISPS (CpISPS) was narrowed down using a genome database associated with protein structure prediction, and a CpISPS gene was identified. The recombinant CpISPS, produced in Escherichia coli, converted dimethylallyl diphosphate to isoprene. Phylogenetic analysis indicated similarity between the amino acid sequences of CpISPS and moss diterpene cyclases (DTCs) but not ISPSs of higher plants, implying that CpISPS is derived from moss DTCs and is evolutionarily unrelated to canonical ISPSs of higher plants. CpISPS is a novel class I cyclase of the terpene synthase-c subfamily harboring αβ domains. This study will help further study of isoprene biosynthesis and the physiological functions of isoprene in mosses.

The telomeric repeat-containing RNA, TERRA, associates with both telomeric DNA and telomeric proteins, often forming RNA:DNA hybrids (R-loops). TERRA is most abundant in cancer cells utilizing the alternative lengthening of telomeres (ALT) pathway for telomere maintenance, suggesting that persistent TERRA R-loops may contribute to activation of the ALT mechanism. Therefore, we sought to identify the enzyme(s) that regulate TERRA metabolism in mammalian cells. Here, we identify that the 5'-3' exoribonuclease XRN2 regulates the stability of TERRA RNA. Moreover, while stabilization of TERRA alone was insufficient to drive ALT, depletion of XRN2 in ALT-positive cells led to a significant increase in TERRA R-loops and exacerbated ALT activity. Together, our findings highlight XRN2 as a key determinant of TERRA metabolism and telomere stability in cancer cells that rely on the ALT pathway.

GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into β-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.

Sortase-mediated pili are flexible rod proteins composed of major and minor/tip pilins, playing important roles in the initial adhesion of bacterial cells to host tissues. The pilus shaft is formed by covalent polymerization of major pilins, and the minor/tip pilin is covalently attached to the tip of the shaft involved in adhesion to the host cell. The Gram-positive bacterium Clostridium perfringens has a major pilin, and a minor/tip pilin (CppB) with the collagen-binding motif. Here, we report X-ray structures of CppB collagen-binding domains, collagen-binding assays and mutagenesis analysis, demonstrating that CppB collagen-binding domains adopt an L-shaped structure in open form, and that a small β-sheet unique to CppB provides a scaffold for a favourable binding site for collagen peptide.

Decreased NADH-induced and increased reduced FADH2 -induced respiration rates at high temperatures are associated with thermal tolerance in Drosophila. Here, we determined whether this change was associated with adjustments of adenosine triphosphate (ATP) production rate and coupling efficiency (ATP/O) in Drosophila melanogaster. We show that decreased pyruvate + malate oxidation at 35°C is associated with a collapse of ATP synthesis and a drop in ATP/O ratio. However, adding succinate triggered a full compensation of both oxygen consumption and ATP synthesis rates at this high temperature. Addition of glycerol-3-phosphate (G3P) led to a huge increase in respiration with no further advantage in terms of ATP production. We conclude that succinate is the only alternative substrate able to compensate both oxygen consumption and ATP production rates during oxidative phosphorylation at high temperature, which has important implications for thermal adaptation.

Naïve B cells become activated and differentiate into antibody-secreting plasma cells (PCs) when encountering antigens. Here, we reveal that the WW domain-containing adapter protein with coiled-coil (Wac), which is important for histone H2B ubiquitination (ubH2B), is essential for PC differentiation. We demonstrate that B cell-specific Wac knockout mice have severely compromised T cell-dependent and -independent antibody responses. PC differentiation is drastically compromised despite undisturbed germinal center B cell response in the mutant mice. We also observe a significant reduction in global ubH2B in Wac-deficient B cells, which is correlated with downregulated expression of some genes critical for cell metabolism. Thus, our findings demonstrate an essential role of Wac-mediated ubH2B in PC differentiation and shed light on the epigenetic mechanisms underlying this process.

Sa15-21, a monoclonal antibody against mouse Toll-like receptor (TLR) 4, can protect mice from lipopolysaccharide (LPS)/D-galactosamine-induced acute lethal hepatitis. Herein, we investigated the molecular mechanisms underlying Sa15-21-mediated regulation of TLR4 signaling in macrophages. Results showed that Sa15-21 enhanced the production of proinflammatory cytokines and attenuated the production of anti-inflammatory cytokines in LPS-stimulated macrophages. Western blotting analysis revealed that Sa15-21 pretreatment had no effect on NF-κB and MAPK signaling in LPS-stimulated macrophages; however, Sa15-21 treatment alone led to a weak and delayed activation of NF-κB and MAPK signaling without any effect on proinflammatory cytokine production. By contrast, Sa15-21 failed to induce the activation of interferon regulatory factor 3. Taken together, our results indicate that Sa15-21 sensitizes macrophages to facilitate the inflammatory response via TLR signaling.

An essential challenge in diagnosing states of nonalcoholic fatty liver disease (NAFLD) is the early prediction of progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) before the disease progresses. Histological diagnoses of NAFLD rely on the appearance of anomalous tissue morphologies, and it is difficult to segment the biomolecular environment of the tissue through a conventional histopathological approach. Here, we show that hyperspectral Raman imaging provides diagnostic information on NAFLD in rats, as spectral changes among disease states can be detected before histological characteristics emerge. Our results demonstrate that Raman imaging of NAFLD can be a useful tool for histopathologists, offering biomolecular distinctions among tissue states that cannot be observed through standard histopathological means.

The systematic identification of tumour vulnerabilities through perturbational experiments on cancer models, including genome editing and drug screens, is playing a crucial role in combating cancer. This collective effort is known as the Cancer Dependency Map (DepMap). The 1st European Cancer Dependency Map Symposium (EuroDepMap), held in Milan last May, featured talks, a roundtable discussion, and a poster session, showcasing the latest discoveries and future challenges related to the DepMap. The symposium aimed to facilitate interactions among participants across Europe, encourage idea exchange with leading experts, and present their work and future projects. Importantly, it sparked discussions on future endeavours, such as screening more complex cancer models and accounting for tumour evolution.

Bone morphogenetic protein 1 (BMP1), a member of the astacin family of zinc-metalloproteases, proteolytically cleaves the low-density lipoprotein receptor (LDLR) within its ligand-binding domain, reducing the binding and cellular uptake of LDL-cholesterol. Here, we aimed to determine whether astacin proteases other than BMP1 may also cleave LDLR. Although human hepatocytes express all six astacin proteases, including the meprins and mammalian tolloid, we found through pharmacological inhibition and genetic knockdown that only BMP1 contributed to the cleavage of LDLR in its ligand-binding domain. We also found that the minimum amino acid change required to render mouse LDLR susceptible to cleavage by BMP1 is mutation at the P1' and P2 positions of the cleavage site. When expressed in cells, the resulting humanised-mouse LDLR internalised LDL-cholesterol. This work provides insight into the biological mechanisms regulating LDLR function.

The size and shape of organelles can influence the rate of biochemical reactions in cells. Previous studies have suggested that organelle morphology changes due to intra- and extracellular environmental responses, affecting the metabolic efficiency of and signal transduction emanating from neighboring organelles. In this study, we tested the possibility that intracellularly distributed organelles exhibit a heterogeneous response to intra- and extracellular environments. We detected a high correlation between peroxisome morphology and distance to the nucleus in light-exposed cells. Moreover, the proximity area between chloroplasts and peroxisomes varied with distance to the nucleus. These results indicate that peroxisome morphology varies with proximity to the nucleus, suggesting the presence of a nucleus-peroxisome signal transduction cascade mediated by chloroplasts.

Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosis (TB), an ancient disease which causes 1.5 million deaths worldwide. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the MTB de novo pyrimidine biosynthesis pathway, and it is essential for MTB growth in vitro, hence representing a promising drug target. We present: (i) the biochemical characterization of the full-length MTB DHODH, including the analysis of the kinetic parameters, and (ii) the previously unreleased crystal structure of the protein that allowed us to rationally screen our in-house chemical library and identify the first selective inhibitor of mycobacterial DHODH. The inhibitor has fluorescence properties, potentially instrumental to in cellulo imaging studies, and exhibits an IC50 value of 43 μm, paving the way to hit-to-lead process.

P2K1 is a plant-specific purinoceptor that perceives extracellular ATP (eATP), a signaling molecular implicated in various physiological processes. Interestingly, P2K1 harbors a C-terminal intrinsically disordered region (IDR). When we overexpressed a truncated P2K1 (P2K1t ) lacking the IDR, primary root growth completely ceased in response to eATP. We investigated the functional roles of the IDR in P2K1 using a combination of molecular genetics, calcium imaging, gene expression analysis, and histochemical approaches. We found that the P2K1t variant gave rise to an amplified response to eATP, through accumulation of superoxide, altered cell wall integrity, and ultimate cell death in the primary root tip. Together, these observations underscore the significant involvement of the C-terminal tail of P2K1 in root growth regulation.

A number of multidrug-resistant bacterial pathogens code for S1-P1 nucleases with a poorly understood role. We have characterized a recombinant form of S1-P1 nuclease from Stenotrophomonas maltophilia, an opportunistic pathogen. S. maltophilia nuclease 1 (SmNuc1) acts predominantly as an RNase and is active in a wide range of temperatures and pH. It retains a notable level of activity towards RNA and ssDNA at pH 5 and 9 and about 10% of activity towards RNA at 10 °C. SmNuc1 with very high catalytic rates outperforms S1 nuclease from Aspergillus oryzae and other similar nucleases on all types of substrates. SmNuc1 degrades second messenger c-di-GMP, which has potential implications for its role in the pathogenicity of S. maltophilia.

G protein-coupled receptors (GPCRs) transmit signals from drugs across cell membranes, leading to associated physiological effects. To study the structural basis of the transmembrane signalling, in-membrane chemical modification (IMCM) has previously been introduced for 19 F-labelling of GPCRs expressed in Spodoptera frugiperda (Sf9) insect cells. Here, IMCM is used with the A2A adenosine receptor (A2A AR) expressed in Pichia pastoris; 19 F-NMR revealed nearly complete solvent protection of the A2A AR transmembrane domain in the membrane and in 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG)/cholesteryl hemisuccinate (CHS) micelles, and extensive solvent accessibility for A2A AR in n-dodecyl β-D-maltoside (DDM)/CHS micelles. No Cys residue dominated non-specific labelling with 2,2,2-trifluoroethanethiol. These observations yield an improved protocol for IMCM 19 F-labelling of GPCRs and new insights into variable solvent accessibility for function-related characterization of GPCRs.

An acidic environment in bone is essential for bone metabolism and the production of decarboxylated osteocalcin, which functions as a regulatory hormone of glucose metabolism. Here, we describe the high-resolution X-ray crystal structure of decarboxylated osteocalcin under acidic conditions. Decarboxylated osteocalcin at pH 2.0 retains the α-helix structure of native osteocalcin with three γ-carboxyglutamic acid residues at neutral pH. This implies that decarboxylated osteocalcin is stable under an acidic environment in bone. In addition, site-directed mutagenesis revealed that Glu17 and Glu21 are important for the adiponectin-inducing activity of decarboxylated osteocalcin. These findings suggest that the receptor of decarboxylated osteocalcin responds to the negative charge in helix 1 of osteocalcin.

GPRC5C is an orphan G protein-coupled receptor (GPCR) that belongs to the class C GPCR family. Although GPRC5C is expressed in various organs, its function and ligand are still undetermined. We found that GPRC5C is expressed in mouse taste cells, enterocytes, and pancreatic α-cells. In functional imaging assays, HEK293 cells heterologously expressing GPRC5C and the chimeric G protein α subunit Gα16-gust44 showed robust intracellular Ca2+ increases in response to monosaccharides, disaccharides, and a sugar alcohol, but not an artificial sweetener or sweet-tasting amino acid. Notably, Ca2+ increases occurred after washout, not during stimulation. Our findings suggest that GPRC5C has receptor properties which lead to novel 'off' responses to saccharide detachment and may work as an internal or external chemosensor specifically tuned to natural sugars.

Human FoxP proteins share a highly conserved DNA-binding domain that dimerizes via three-dimensional domain swapping, although showing varying oligomerization propensities among its members. Here, we present an experimental and computational characterization of all human FoxP proteins to unravel how their amino acid substitutions impact their folding and dimerization mechanism. We solved the crystal structure of the forkhead domain of FoxP4 to then perform a comparison across all members, finding that their sequence changes impact not only the structural heterogeneity of their forkhead domains but also the protein-protein association energy barrier. Lastly, we demonstrate that the accumulation of a monomeric intermediate is an oligomerization-dependent feature rather than a common aspect of monomers and dimers in this protein subfamily.

The co-option of regulatory genes has the potential to play an important role in the evolutionary gain of new traits. However, the changes at the sequence level that underlie such a co-option event are still elusive. We identified the changes in the cis-regulatory sequence of wingless that caused co-option of wingless and led to its expression in new places in Drosophila guttifera, which has unique pigmentation patterns on its wings. The newly gained function of gene expression activation was acquired evolutionarily via a combination of pre-existing sequences containing a putative binding site for SMAD transcription factors that exhibit an ancestral function in driving expression at crossveins, and a sequence that is specific to the lineage leading to D. guttifera.

Macrophages play an important role in the response to infection and/or repair of injury in tissues. To examine the NF-κB pathway in response to an inflammatory stimulus, we used wild-type bone-marrow-derived macrophages (BMDMs) or BMDMs with knockout (KO) of myeloid differentiation primary response 88 (MyD88) and/or Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF) via CRISPR/Cas9. Following treatment of BMDMs with lipopolysaccharide (LPS) to induce an inflammatory response, translational signalling of NF-κB was quantified via immunoblot and cytokines were measured. Our findings reveal that MyD88 KO, but not TRIF KO, decreased LPS-induced NF-κB signalling, and 10% expression of basal MyD88 expression was sufficient to partially rescue the abolished inflammatory cytokine secretion observed upon MyD88 KO.

Tripartite efflux systems of the ABC-type family transport a variety of substrates and contribute to the antimicrobial resistance of Gram-negative bacteria. PvdRT-OpmQ, a member of this family, is thought to be involved in the secretion of the newly synthesized and recycled siderophore pyoverdine in Pseudomonas species. Here, we purified and characterized the inner membrane component PvdT and the periplasmic adapter protein PvdR of the plant growth-promoting soil bacterium Pseudomonas putida KT2440. We show that PvdT possesses an ATPase activity that is stimulated by the addition of PvdR. In addition, we provide the first biochemical evidence for direct interactions between pyoverdine and PvdRT.

Cyclin/cyclin-dependent kinase (CDK) heterodimers have multiple phosphorylation targets and may alter the activity of these targets. Proteins from different metabolic processes are among the phosphorylation targets, that is, enzymes of central carbon metabolism. This work explores the interaction of Cyc/CDK complex members with the glycolytic enzymes hexokinase 7 (HXK7) and glyceraldehyde-3-phosphate dehydrogenase (GAP). Both enzymes interacted steadily with CycD2;2, CycB2;1 and CDKA;1 but not with CDKB1;1. However, Cyc/CDKB1;1 complexes phosphorylated both enzymes, decreasing their activities. Treatment with a CDK-specific inhibitor (RO-3306) or with lambda phosphatase after kinase assay restored total HXK7 activity, but not GAP activity. In enzymatic assays, increasing concentrations of CDKB1;1, but not of CycD2;2, CycB2;1 or CycD2;2/CDKB1;1 complex, decreased GAP activity. Cell cycle regulators may modulate carbon channeling in glycolysis by two different mechanisms: Cyc/CDK-mediated phosphorylation of targets (e.g., HXK7; canonical mechanism) or by direct and transient interaction of the metabolic enzyme (e.g., GAP) with CDKB1;1 without a Cyc partner (alternative mechanism).

Aggregation of the 43 kDa TAR DNA-binding protein (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA binding and TDP-43 N-terminal domain dimerisation has been suggested to ameliorate TDP-43 aggregation. However, the relationship between these factors and the solubility of TDP-43 is largely unknown. Therefore, we developed new oligonucleotides that can recruit two TDP-43 molecules and interfere with their intermolecular interactions via spatial separation. Using these oligonucleotides and TDP-43-preferable UG-repeats, we uncovered two distinct mechanisms for modulating TDP-43 solubility by RNA binding: One is N-terminal domain dimerisation, and the other is the spatial separation of two TDP-43 molecules. This study provides new molecular insights into the regulation of TDP-43 solubility.

The immunity-related GTPases (IRGs) belong to the interferon-inducible GTPase protein family, which mediates cell autonomous and innate immunity response to intracellular pathogens. Yet, the cellular and physiological function of IRGC, a member of the IRG subfamily, has not been elucidated. Here, we show that testis-specific IRGC is specifically and highly expressed in mature spermatozoa and is required for sperm motility. IRGC induction results in the clustering of lipid droplets and initiation of their physical contact with mitochondria. When examining clinical semen samples, IRGC expression is significantly lower in asthenozoospermia patients relative to healthy individuals. These unique effects of IRGC identify it as an important player in sperm motility, and show the potential of lipid metabolism-targeting therapeutic intervention aimed at controlling asthenozoospermia.

The coronavirus disease 2019 (COVID-19) pandemic has affected tens of millions of individuals and caused hundreds of thousands of deaths worldwide. Here, we present a comprehensive, multiscale network analysis of the transcriptional response to the virus. In particular, we focused on key regulators, cell receptors, and host processes that were hijacked by the virus for its advantage. ACE2-controlled processes involved CD300e (a TYROBP receptor) as a key regulator and the activation of IL-2 pro-inflammatory cytokine signaling. We further investigated the age dependency of such receptors in different tissues. In summary, this study provides novel insights into the gene regulatory organization during the SARS-CoV-2 infection and the tissue-specific, age-dependent expression of the cell receptors involved in COVID-19.

Spindles are intracellular crystals of the fusolin protein that enhances the oral virulence of insect poxviruses by disruption of the larval chitinous peritrophic matrix. The enigmatic fusolin protein is classified as a lytic polysaccharide monooxygenase (LPMO) by sequence and structure. Although circumstantial evidence points towards a role for fusolin in chitin degradation, no biochemical data exist to verify this claim. In the present study, we demonstrate that fusolin released from over 40-year-old spindles, stored for 10 years at 4 °C, are chitin-degrading LPMOs. Not only was fusolin active after long-term storage, but it also withstood high temperature and oxidative stress in its crystalline form, highlighting extreme stability that is beneficial to viral persistence and desirable for potential biotechnology applications.

It is unknown whether heteromerization between chemokine (C-X-C motif) receptor 4 (CXCR4), atypical chemokine receptor 3 (ACKR3) and α1b -adrenoceptor (α1b -AR) influences effects of the CXCR4/ACKR3 agonist chemokine (C-X-C motif) ligand 12 (CXCL12) and the noncognate CXCR4 agonist ubiquitin on agonist-promoted G protein activation. We provide biophysical evidence that both ligands stimulate CXCR4-mediated Gαi activation. Unlike CXCL12, ubiquitin fails to recruit β-arrestin. Both ligands differentially modulate the conformation of CXCR4:ACKR3 heterodimers and its propensity to hetero-trimerize with α1b -AR. CXCR4:ACKR3 heterodimerization reduces the potency of CXCL12, but not of ubiquitin, to activate Gαi. Ubiquitin enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from hetero-oligomers comprising CXCR4. CXCL12 enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from CXCR4:α1b -AR heterodimers and reduces phenylephrine-stimulated α1b -AR-promoted Gαq activation from ACKR3 comprising heterodimers and trimers. Our findings suggest heteromer and ligand-dependent functions of the receptor partners.

Me31B/DDX6 is a DEAD-box family RNA helicase playing roles in post-transcriptional RNA regulation in different cell types and species. Despite the known motifs/domains of Me31B, the in vivo functions of the motifs remain unclear. Here, we used the Drosophila germline as a model and used CRISPR to mutate the key Me31B motifs/domains: helicase domain, N-terminal domain, C-terminal domain and FDF-binding motif. Then, we performed screening characterization on the mutants and report the effects of the mutations on the Drosophila germline, on processes such as fertility, oogenesis, embryo patterning, germline mRNA regulation and Me31B protein expression. The study indicates that the Me31B motifs contribute different functions to the protein and are needed for proper germline development, providing insights into the in vivo working mechanism of the helicase.

Autoimmune polyendocrine syndrome type I (APS-1) is caused by mutations in the autoimmune regulator (AIRE) gene and characterised clinically by multiple autoimmune manifestations and serologically by autoantibodies against tissue proteins and cytokines. We here hypothesised that lack of AIRE expression in thymus affects blood immune cells and performed whole-blood microarray analysis (N = 16 APS-I patients vs 16 controls), qPCR verification, and bioinformatic deconvolution of cell subsets. We identified B cell responses as being downregulated in APS-1 patients, which was confirmed by qPCR; these results call for further studies on B cells in this disorder. The type I interferon (IFN-I) pathway was also downregulated in APS-1, and the presence of IFN antibodies is the likely reason for this mild overall downregulation of the IFN-I genes in most APS-1 patients.

Centromere-associated protein E (CENP-E) is a kinesin motor protein essential for mitosis and a new target for anticancer agents with less side effects. To rationally design anticancer drug candidates based on structure, it is important to determine the three-dimensional structure of the CENP-E motor domain bound to its inhibitor. Here, we report the first crystal structure of the CENP-E motor domain in complex with a non-hydrolysable ATP analogue, adenylyl-imidodiphosphate (AMPPNP). Furthermore, the structure is compared with the ADP-bound form of the CENP-E motor domain as well as the AMPPNP-bound forms of other kinesins. This study indicates that helix α4 of CENP-E participates in the slow binding of CENP-E to microtubules. These results will contribute to the development of anticancer drugs targeting CENP-E.

The control of pH in chloroplasts is important to regulate photosynthesis, although details of the precise regulatory mechanisms of H+ homeostasis in chloroplasts are not fully understood. We recently found that the cyanobacterial PxcA homolog DLDG1 is involved in plastidial pH control. PxcA and DLDG1 have been thought to control light-dependent H+ extrusion across the cyanobacterial cytoplasmic and chloroplast envelope membranes, respectively. To investigate DLDG1-dependent pH control in chloroplasts, we crossed the dldg1 mutant with various mutants lacking known non-photochemical quenching (NPQ)-related proteins, such as fluctuating-light acclimation protein 1 (FLAP1), PsbS/NPQ4, and proton gradient regulation 5 (PGR5). Phenotypes of these double mutants revealed that PsbS works upstream of DLDG1, PGR5 affects NPQ independently from DLDG1, and the ΔpH regulation by FLAP1 and DLDG1 are independent of each other.

Human MutT homolog 1 (MTH1), also known as Nudix-type motif 1 (NUDT1), hydrolyzes 8-oxo-dGTP and 2-oxo-dATP with broad substrate recognition and has attracted attention in anticancer therapeutics. Previous studies on MTH1 have proposed that the exchange of the protonation state between Asp119 and Asp120 is essential for the broad substrate recognition of MTH1. To understand the relationship between protonation states and substrate binding, we determined the crystal structures of MTH1 at pH 7.7-9.7. With increasing pH, MTH1 gradually loses its substrate-binding ability, indicating that Asp119 is deprotonated at pH 8.0-9.1 in 8-oxo-dGTP recognition and Asp120 is deprotonated at pH 8.6-9.7 in 2-oxo-dATP recognition. These results confirm that MTH1 recognizes 8-oxo-dGTP and 2-oxo-dATP by exchanging the protonation state between Asp119 and Asp120 with higher pKa .

Arsenite causes proteotoxicity by targeting nascent proteins for misfolding and aggregation. Here, we assessed how selected yeast chaperones and ubiquitin ligases contribute to proteostasis during arsenite stress. Loss of the ribosome-associated chaperones Zuo1, Ssz1, and Ssb1/Ssb2 reduced global translation and protein aggregation, and increased arsenite resistance. Loss of cytosolic GimC/prefoldin function led to defective aggregate clearance and arsenite sensitivity. Arsenite did not induce ribosomal stalling or impair ribosome quality control, and ribosome-associated ubiquitin ligases contributed little to proteostasis. Instead, the cytosolic ubiquitin ligase Rsp5 was important for aggregate clearance and resistance. Our study suggests that damage prevention, by decreased aggregate formation, and damage elimination, by enhanced aggregate clearance, are important protective mechanisms that maintain proteostasis during arsenite stress.

Upon heat shock, activated heat shock transcription factor 1 (HSF1) binds to the heat shock response elements (HSEs) in the promoters of mammalian heat shock protein (HSP)-encoding genes and recruits the preinitiation complex and coactivators, including Mediator. These transcriptional regulators may be concentrated in phase-separated condensates around the promoters, but they are too minute to be characterized in detail. We herein established HSF1-/- mouse embryonic fibroblasts harbouring HSP72-derived multiple HSE arrays and visualized the condensates of fluorescent protein-tagged HSF1 with liquid-like properties upon heat shock. Using this experimental system, we demonstrate that endogenous MED12, a subunit of Mediator, is concentrated in artificial HSF1 condensates upon heat shock. Furthermore, the knockdown of MED12 markedly reduces the size of condensates, suggesting an important role for MED12 in HSF1 condensate formation.

MicroRNAs are among the key modulators of placental transcriptome dynamics. This study aimed at comparative profiling of urinary (sampling at 228-230 gestational days, g.days), serum (217-230 g.days), and placental (279-286 g.days) microRNAs in three healthy pregnant women using miRNome sequencing. Placenta showed significant enrichment of microRNAs compared with serum and urine (1174, 341, and 193, respectively; P < 10-5 ). All sample types shared 153 microRNAs, representing candidate biomarkers for placental health. Urine samples contained eight of 56 transcripts from the placenta-specific chromosome 19 microRNA cluster C19MC and one of 91 transcripts (miR-432-5p) from the chromosome 14 cluster C14MC. These data suggest an active filtering at the maternal-fetal interface in passing through only selected microRNAs. Urine represents a valid source to monitor the signature of placenta-expressed microRNAs that are differentially expressed in pregnancy complications.

Each tRNA is aminoacylated (charged) with a genetic codon-specific amino acid. It remains unclear what factors are associated with tRNA charging and how tRNA charging is maintained. By using the individual tRNA acylation PCR method, we found that the charging ratio of tRNAGln (CUG) reflects cellular glutamine level. When uncharged tRNAGln (CUG) increased under amino acid starvation, the kinase GCN2, which is a key stimulator of the integrated stress response, was activated. Activation of GCN2 led to the upregulation of ubiquitin C (UBC) expression. Upregulated UBC, in turn, suppressed the further reduction in tRNAGln (CUG) charging levels. Thus, tRNA charging is sensitive to intracellular nutrient status and is an important initiator of intracellular signaling.

Puf3p regulates the stability of nuclear-encoded mRNAs acting in mitochondrial biogenesis and function in Saccharomyces cerevisiae. This work identifies the phosphorylation of Pop2p, a component of the deadenylase complex, as being critical for adapting Puf3p-mediated mRNA decay upon carbon source alterations. We demonstrate that the Puf3p-Pop2p association diminishes in mitochondria-reliant conditions and establish Yak1p, a kinase that phosphorylates Pop2p at threonine 97, as a new player in Puf3p-mediated regulation of mRNA decay. Yak1p deletion alters the half-life of Puf3p target mRNAs. Our findings outline a metabolism-driven regulatory switch, whereby, in mitochondria-independent conditions, Puf3p recruits Pop2p and the decay machinery to bound mRNAs for rapid decay. Conversely, in mitochondria-reliant conditions, the association of Puf3p with Yak1p increases, placing Yak1p proximal to neighboring Pop2p. Subsequent Pop2p phosphorylation reduces the Puf3p-Pop2p interaction and stabilizes Puf3p target mRNAs.

Copper radical oxidases (CROs) are redox enzymes able to oxidize alcohols or aldehydes, while only requiring a single copper atom as cofactor. Studied CROs are found in one of two subfamilies within the Auxiliary Activities family 5 (AA5) in the carbohydrate-active enzymes database. We here characterize an AA5 enzyme outside the subfamily classification from the opportunistic bacterial pathogen Burkholderia pseudomallei, which curiously was fused to a carbohydrate esterase family 3 domain. The enzyme was shown to be a promiscuous primary alcohol oxidase, with an activity profile similar to enzymes from subfamily 2. The esterase domain was inactive on all tested substrates, and structural predictions revealed this being an effect of crippling substitutions in the expected active site residues.

Autoimmune coagulation factor XIII (FXIII) deficiency (AiF13D) is a bleeding disorder caused by anti-FXIII autoantibodies. Recently, we generated human monoclonal antibodies (mAbs) from the peripheral blood of an AiF13D patient and classified them into three groups: FXIII-dissociation inhibitor, FXIII-assembly inhibitor, and non-neutralizing/inhibitory mAbs. However, the epitope region and molecular inhibitory mechanism of each mAb remain unknown. Here, we localized the epitope regions of the representative inhibitory mAbs A69K (dissociation inhibitor) and A78L (assembly inhibitor) to the β-barrel-2 domain and boundary of β-barrel-1&2 domains, respectively, of the FXIII-A subunit, by combining a binding assay using its synthesized peptides and a protease-protection assay. Our findings suggest that A69K inhibits the activation-related conformational changes and dissociation of FXIII and that A78L competitively inhibits FXIII-assembly.

The architectural chromatin factor high-mobility group AT-hook 2 (HMGA2) is causally involved in several human malignancies and pathologies. HMGA2 is not expressed in most normal adult somatic cells, which renders the protein an attractive drug target. An established cell-based compound library screen identified the fibroblast growth factor receptor (FGFR) inhibitor PD173074 as an antagonist of HMGA2-mediated transcriptional reporter gene activation. We determined that PD173074 binds the C-terminus of HMGA2 and interferes with functional coordination of the three AT-hook DNA-binding domains mediated by the C-terminus. The HMGA2-antagonistic effect of PD173074 on transcriptional activation may therefore result from an induced altered DNA-binding mode of HMGA2. PD173074 as a novel HMGA2-specific antagonist could trigger the development of derivates with enhanced attributes and clinical potential.

Homozygous mutations in the autoimmune regulator (AIRE) gene that cripple thymic negative selection of autoreactive T cells result in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). However, how AIRE regulates the T-cell response against foreign pathogens is not well understood. Here, we observed comparable primary CD8+ T cells but a markedly reduced memory T-cell population and protective function in Aire-/- mice compared with wild-type after infection with a strain of recombinant Listeria monocytogenes. In adoptive transfer models, exogenous congenic CD8+ T cells transferred into Aire-/- mice also showed a reduction in the memory T-cell population, indicating an important role for extrathymic Aire-expressing cells in shaping or sustaining memory T cells. Moreover, using a bone marrow chimeric model, we found that Aire expressed in radioresistant cells plays an important role in maintaining the memory phenotype. These results provide important insights into the role of extrathymic Aire in the T-cell response to infection.

A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. We trigger conditioned regeneration in the Caenorhabditis elegans ASJ neuron by laser surgery or genetic disruption of sensory pathways. Conditioning upregulates thioredoxin-1 (trx-1) expression, as indicated by trx-1 promoter-driven expression of green fluorescent protein and fluorescence in situ hybridization (FISH), suggesting trx-1 levels and associated fluorescence indicate regenerative capacity. The redox activity of trx-1 functionally enhances conditioned regeneration, but both redox-dependent and -independent activity inhibit non-conditioned regeneration. Six strains isolated in a forward genetic screen for reduced fluorescence, which suggests diminished regenerative potential, also show reduced axon outgrowth. We demonstrate an association between trx-1 expression and the conditioned state that we leverage to rapidly assess regenerative capacity.

miRNAs are a class of noncoding RNAs with gene regulation properties, and they function as key factors in cell homeostasis. The interaction of miRNAs with their target mRNAs is largely considered to rely on sequence complementarity; however, some evidence indicates that mature miRNAs can adopt diverse conformations with implications for their function. Using the oncogenic miR-181 family as a study model, we suggest that a potential relationship between the primary sequence and secondary structure of miRNAs may have an impact on the number and spectrum of targeted cellular transcripts. We further emphasize that specific alterations in miR-181 primary sequences might impose certain constraints on target gene selection compared with the wild-type sequences, leading to the targeting of new transcripts with upregulated function in cancer.

The extreme N-terminal residues of the mitochondrial ribosomal bL27m proteins reside within the ribosomal peptidyl transferase center (PTC) and are conserved from their bacterial ancestors. Mutation or truncation of the N-terminal region of the yeast Mrp7/bL27m protein did not inhibit protein synthesis but significantly impacted the efficacy of the mitochondrial translational process with respect to yielding proteins competent to assemble into functional oxidative phosphorylation enzymes. The requirement for the N-terminal residues of Mrp7/bL27m to support normal mitotranslation was more apparent under respiratory growth. We demonstrate that the N-terminal region of Mrp7/bL27m impacts the environment of the PTC and speculate the bL27m proteins serve to fine-tune and optimize mitoribosomal activity with respect to the downstream fate of the nascent chain.

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.

Feruloyl esterases (FAEs) hydrolyze the ester bonds between hydroxycinnamic acids and arabinose residues of plant cell walls and exhibit considerable diversity in terms of substrate specificity. Here, we report the crystal structure of an FAE from Fusarium oxysporum (FoFaeC) at 1.7 Å resolution in complex with p-coumaric acid, which is the first ligand-bound structure of a tannase-like FAE. Our data reveal local conformational changes around the active site upon ligand binding, suggesting alternation between an active and a resting state of the enzyme. A swinging tyrosine residue appears to be gating the substrate binding pocket, while the lid domain of the protein exerts substrate specificity by means of a well-defined hydrophobic core that encases the phenyl moiety of the substrate.

Human phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is a 2-oxoglutarate (2OG)-dependent dioxygenase implicated in Alzheimer's disease, some cancers, and immune cell functions. The substrate, kinetic and inhibitory properties, function and subcellular localization of PHYHD1 are unknown. We used recombinant expression and enzymatic, biochemical, biophysical, cellular and microscopic assays for their determination. The apparent Km values of PHYHD1 for 2OG, Fe2+ and O2 were 27, 6 and > 200 μm, respectively. PHYHD1 activity was tested in the presence of 2OG analogues, and it was found to be inhibited by succinate and fumarate but not R-2-hydroxyglutarate, whereas citrate acted as an allosteric activator. PHYHD1 bound mRNA, but its catalytic activity was inhibited upon interaction. PHYHD1 was found both in the nucleus and cytoplasm. Interactome analyses linked PHYHD1 to cell division and RNA metabolism, while phenotype analyses linked it to carbohydrate metabolism. Thus, PHYHD1 is a potential novel oxygen sensor regulated by mRNA and citrate.

In Vibrio cholerae, the master regulator FlrA controls transcription of downstream flagellar genes in a σ54 -dependent manner. However, the molecular basis of regulation by VcFlrA, which contains a phosphorylation-deficient N-terminal FleQ domain, has remained elusive. Our studies on VcFlrA, four of its constructs, and a mutant showed that the AAA+ domain of VcFlrA, with or without the linker 'L', remains in ATPase-deficient monomeric states. By contrast, the FleQ domain plays a pivotal role in promoting higher-order functional oligomers, providing the required conformation to 'L' for ATP/cyclic di-GMP (c-di-GMP) binding. The crystal structure of VcFlrA-FleQ at 2.0 Å suggests that distinct structural features of VcFlrA-FleQ presumably assist in inter-domain packing. VcFlrA at a high concentration forms ATPase-efficient oligomers when the intracellular c-di-GMP level is low. Conversely, excess c-di-GMP locks VcFlrA in a non-functional lower oligomeric state, causing repression of flagellar biosynthesis.

Na+ ,K+ -ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell membrane by alternating between the E1 (showing high affinity for Na+ and low affinity for K+ ) and E2 (low affinity to Na+ and high affinity to K+ ) forms. Presented here are two crystal structures of NKA in E1·Mg2+ and E1·3Na+ states at 2.9 and 2.8 Å resolution, respectively. These two E1 structures fill a gap in our description of the NKA reaction cycle based on the atomic structures. We describe how NKA converts the K+ -bound E2·2K+ form to an E1 (E1·Mg2+ ) form, which allows high-affinity Na+ binding, eventually closing the cytoplasmic gate (in E1 ~ P·ADP·3Na+ ) after binding three Na+ , while keeping the extracellular ion pathway sealed. We now understand previously unknown functional roles for several parts of NKA and that NKA uses even the lipid bilayer for gating the ion pathway.