Theileria parasites sequester host eIF5A to escape elimination by host-mediated autophagy.

Intracellular pathogens develop elaborate mechanisms to survive within the hostile environments of host cells. Theileria parasites infect bovine leukocytes and cause devastating diseases in cattle in developing countries. Theileria spp. have evolved sophisticated strategies to hijack host leukocytes, inducing proliferative and invasive phenotypes characteristic of cell transformation. Intracellular Theileria parasites secrete proteins into the host cell and recruit host proteins to induce oncogenic signaling for parasite survival. It is unknown how Theileria parasites evade host cell defense mechanisms, such as autophagy, to survive within host cells. Here, we show that Theileria annulata parasites sequester the host eIF5A protein to their surface to escape elimination by autophagic processes. We identified a small-molecule compound that reduces parasite load by inducing autophagic flux in host leukocytes, thereby uncoupling Theileria parasite survival from host cell survival. We took a chemical genetics approach to show that this compound induced host autophagy mechanisms and the formation of autophagic structures via AMPK activation and the release of the host protein eIF5A which is sequestered at the parasite surface. The sequestration of host eIF5A to the parasite surface offers a strategy to escape elimination by autophagic mechanisms. These results show how intracellular pathogens can avoid host defense mechanisms and identify a new anti-Theileria drug that induces autophagy to target parasite removal.

Qualitative rather than quantitative phosphoregulation shapes the end of meiosis I in budding yeast.

Exit from mitosis is brought about by dramatic changes in the phosphoproteome landscape. A drop in Cyclin-dependent kinase (Cdk) activity, the master regulatory kinase, and activation of counteracting phosphatases such as Cdc14 in budding yeast, results in ordered substrate dephosphorylation, allowing entry into a new cell cycle and replication licensing. In meiosis however, two cell divisions have to be executed without intermediate DNA replication, implying that global phosphorylation and dephosphorylation have to be adapted to the challenges of meiosis. Using a global time-resolved phosphoproteomics approach in budding yeast, we compared the phosphoproteome landscape between mitotic exit and the transition from meiosis I to meiosis II. We found that unlike exit from mitosis, Cdk phosphomotifs remain mostly stably phosphorylated at the end of meiosis I, whereas a majority of Cdk-unrelated motifs are reset by dephosphorylation. However, inducing an artificial drop of Cdk at metaphase of meiosis I leads to ordered substrate dephosphorylation, comparable to mitosis, indicating that phosphoregulation of substrates at the end of meiosis I is thus mainly qualitatively rather than quantitatively ordered.

Multiomic analysis in fibroblasts of patients with inborn errors of cobalamin metabolism reveals concordance with clinical and metabolic variability.

BACKGROUND: The high variability in clinical and metabolic presentations of inborn errors of cobalamin (cbl) metabolism (IECM), such as the cblC/epicblC types with combined deficits in methylmalonyl-coA mutase (MUT) and methionine synthase (MS), are not well understood. They could be explained by the impaired expression/activity of enzymes from other metabolic pathways. METHODS: We performed metabolomic, genomic, proteomic, and post-translational modification (PTM) analyses in fibroblasts from three cblC cases and one epi-cblC case compared with three cblG cases with specific MS deficits and control fibroblasts. FINDINGS: CblC patients had metabolic profilings consistent with altered urea cycle, glycine, and energy mitochondrial metabolism. Metabolomic analysis showed partial disruption and increased glutamate/ketoglutarate anaplerotic pathway of the tricarboxylic acid cycle (TCA), in patient fibroblasts. RNA-seq analysis showed decreased expression of MT-TT (mitochondrial tRNA threonine), MT-TP (mitochondrial tRNA proline), OXCT1 (succinyl CoA:3-oxoacid CoA transferase deficiency), and MT-CO1 (cytochrome C oxidase subunit 1). Proteomic changes were observed for key mitochondrial enzymes, including NADH:ubiquinone oxidoreductase subunit A8 (NDUFA8), carnitine palmitoyltransferase 2 (CPT2), and ubiquinol-cytochrome C reductase, complex III subunit X (UQCR10). Propionaldehyde addition in ornithine aminotransferase was the predominant PTM in cblC cells and could be related with the dramatic cellular increase in propionate and methylglyoxalate. It is consistent with the decreased concentration of ornithine reported in 3 cblC cases. Whether the changes detected after multi-omic analyses underlies clinical features in cblC and cblG types of IECM, such as peripheral and central neuropathy, cardiomyopathy, pulmonary hypertension, development delay, remains to be investigated. INTERPRETATION: The omics-related effects of IECM on other enzymes and metabolic pathways are consistent with the diversity and variability of their age-related metabolic and clinical manifestations. PTMs are expected to produce cumulative effects, which could explain the influence of age on neurological manifestations. FUNDING: French Agence Nationale de la Recherche (Projects PREDICTS and EpiGONE) and Inserm.

Pyrroline-5-carboxylate metabolism protein complex detected in Arabidopsis thaliana leaf mitochondria.

Proline dehydrogenase (ProDH) and pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyse the oxidation of proline into glutamate via the intermediates P5C and glutamate-semialdehyde (GSA), which spontaneously interconvert. P5C and GSA are also intermediates in the production of glutamate from ornithine and α-ketoglutarate catalysed by ornithine δ-aminotransferase (OAT). ProDH and P5CDH form a fused bifunctional PutA enzyme in Gram-negative bacteria and are associated in a bifunctional substrate-channelling complex in Thermus thermophilus; however, the physical proximity of ProDH and P5CDH in eukaryotes has not been described. Here, we report evidence of physical proximity and interactions between Arabidopsis ProDH, P5CDH, and OAT in the mitochondria of plants during dark-induced leaf senescence when all three enzymes are expressed. Pairwise interactions and localization of the three enzymes were investigated using bimolecular fluorescence complementation with confocal microscopy in tobacco and sub-mitochondrial fractionation in Arabidopsis. Evidence for a complex composed of ProDH, P5CDH, and OAT was revealed by co-migration of the proteins in native conditions upon gel electrophoresis. Co-immunoprecipitation coupled with mass spectrometry analysis confirmed the presence of the P5C metabolism complex in Arabidopsis. Pull-down assays further demonstrated a direct interaction between ProDH1 and P5CDH. P5C metabolism complexes might channel P5C among the constituent enzymes and directly provide electrons to the respiratory electron chain via ProDH.

Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import.

N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.

An unconventional TOG domain is required for CLASP localization.

Cytoplasmic linker-associated proteins (CLASPs) form a conserved family of microtubule-associated proteins (MAPs) that maintain microtubules in a growing state by promoting rescue while suppressing catastrophe.1 CLASP function involves an ordered array of tumor overexpressed gene (TOG) domains and binding to multiple protein partners via a conserved C-terminal domain (CTD).2,3 In migrating cells, CLASPs concentrate at the cortex near focal adhesions as part of cortical microtubule stabilization complexes (CMSCs), via binding of their CTD to the focal adhesion protein PHLDB2/LL5ß.4,5 Cortical CLASPs also stabilize a subset of microtubules, which stimulate focal adhesion turnover and generate a polarized microtubule network toward the leading edge of migrating cells. CLASPs are also recruited to the trans-Golgi network (TGN) via an interaction between their CTD and the Golgin protein GCC185.6 This allows microtubule growth toward the leading edge of migrating cells, which is required for Golgi organization, polarized intracellular transport, and cell motility.7 In dividing cells, CLASPs are essential at kinetochores for efficient chromosome segregation and anaphase spindle integrity.8,9 Both CENP-E and ASTRIN bind and target CLASPs to kinetochores,10,11 although the CLASP domain required for this interaction is not known. Despite its high evolutionary conservation, the CTD remains structurally uncharacterized. Here, we find that the CTD can be structurally modeled as a TOG domain. We identify a surface-exposed and conserved arginine residue essential for CLASP CTD interaction with partner proteins. Together, our results provide a structural mechanism by which the CLASP CTD directs diverse sub-cellular localizations throughout the cell cycle.

Mechanisms of nuclear pore complex disassembly by the mitotic Polo-like kinase 1 (PLK-1) in C. elegans embryos.

The nuclear envelope, which protects and organizes the genome, is dismantled during mitosis. In the Caenorhabditis elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the maternal and paternal genomes. Nuclear pore complex (NPC) disassembly is a decisive step of NEBD, essential for nuclear permeabilization. By combining live imaging, biochemistry, and phosphoproteomics, we show that NPC disassembly is a stepwise process that involves Polo-like kinase 1 (PLK-1)-dependent and -independent steps. PLK-1 targets multiple NPC subcomplexes, including the cytoplasmic filaments, central channel, and inner ring. PLK-1 is recruited to and phosphorylates intrinsically disordered regions (IDRs) of several multivalent linker nucleoporins. Notably, although the phosphosites are not conserved between human and C. elegans nucleoporins, they are located in IDRs in both species. Our results suggest that targeting IDRs of multivalent linker nucleoporins is an evolutionarily conserved driver of NPC disassembly during mitosis.

Congenital mirror movements are associated with defective polymerisation of RAD51.

BACKGROUND: Mirror movements are involuntary movements of one hand that mirror intentional movements of the other hand. Congenital mirror movements (CMM) is a rare genetic disorder with autosomal dominant inheritance, in which mirror movements are the main neurological manifestation. CMM is associated with an abnormal decussation of the corticospinal tract, a major motor tract for voluntary movements. RAD51 is known to play a key role in homologous recombination with a critical function in DNA repair. While RAD51 haploinsufficiency was first proposed to explain CMM, other mechanisms could be involved. METHODS: We performed Sanger sequencing of RAD51 in five newly identified CMM families to identify new pathogenic variants. We further investigated the expression of wild-type and mutant RAD51 in the patients' lymphoblasts at mRNA and protein levels. We then characterised the functions of RAD51 altered by non-truncating variants using biochemical approaches. RESULTS: The level of wild-type RAD51 protein was lower in the cells of all patients with CMM compared with their non-carrier relatives. The reduction was less pronounced in asymptomatic carriers. In vitro, mutant RAD51 proteins showed loss-of-function for polymerisation, DNA binding and strand exchange activity. CONCLUSION: Our study demonstrates that RAD51 haploinsufficiency, including loss-of-function of non-truncating variants, results in CMM. The incomplete penetrance likely results from post-transcriptional compensation. Changes in RAD51 levels and/or polymerisation properties could influence guidance of the corticospinal axons during development. Our findings open up new perspectives to understand the role of RAD51 in neurodevelopment.

Mechanisms of Nuclear Pore Complex disassembly by the mitotic Polo-Like Kinase 1 (PLK-1) in C. elegans embryos.

The nuclear envelope, which protects and organizes the interphase genome, is dismantled during mitosis. In the C. elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the parental genomes. During NEBD, Nuclear Pore Complex (NPC) disassembly is critical for rupturing the nuclear permeability barrier and removing the NPCs from the membranes near the centrosomes and between the juxtaposed pronuclei. By combining live imaging, biochemistry, and phosphoproteomics, we characterized NPC disassembly and unveiled the exact role of the mitotic kinase PLK-1 in this process. We show that PLK-1 disassembles the NPC by targeting multiple NPC sub-complexes, including the cytoplasmic filaments, the central channel, and the inner ring. Notably, PLK-1 is recruited to and phosphorylates intrinsically disordered regions of several multivalent linker nucleoporins, a mechanism that appears to be an evolutionarily conserved driver of NPC disassembly during mitosis. (149/150 words). One-Sentence Summary: PLK-1 targets intrinsically disordered regions of multiple multivalent nucleoporins to dismantle the nuclear pore complexes in the C. elegans zygote.

Extending the Range of SLIM-Labeling Applications: From Human Cell Lines in Culture to Caenorhabditis elegans Whole-Organism Labeling.

The simple light isotope metabolic-labeling technique relies on the in vivo biosynthesis of amino acids from U-[12C]-labeled molecules provided as the sole carbon source. The incorporation of the resulting U-[12C]-amino acids into proteins presents several key advantages for mass-spectrometry-based proteomics analysis, as it results in more intense monoisotopic ions, with a better signal-to-noise ratio in bottom-up analysis. In our initial studies, we developed the simple light isotope metabolic (SLIM)-labeling strategy using prototrophic eukaryotic microorganisms, the yeasts Candida albicans and Saccharomyces cerevisiae, as well as strains with genetic markers that lead to amino-acid auxotrophy. To extend the range of SLIM-labeling applications, we evaluated (i) the incorporation of U-[12C]-glucose into proteins of human cells grown in a complex RPMI-based medium containing the labeled molecule, considering that human cell lines require a large number of essential amino-acids to support their growth, and (ii) an indirect labeling strategy in which the nematode Caenorhabditis elegans grown on plates was fed U-[12C]-labeled bacteria (Escherichia coli) and the worm proteome analyzed for 12C incorporation into proteins. In both cases, we were able to demonstrate efficient incorporation of 12C into the newly synthesized proteins, opening the way for original approaches in quantitative proteomics.

Rabies virus P protein binds to TBK1 and interferes with the formation of innate immunity-related liquid condensates.

Viruses must overcome the interferon-mediated antiviral response to replicate and propagate into their host. Rabies virus (RABV) phosphoprotein P is known to inhibit interferon induction. Here, using a global mass spectrometry approach, we show that RABV P binds to TBK1, a kinase located at the crossroads of many interferon induction pathways, resulting in innate immunity inhibition. Mutations of TBK1 phosphorylation sites abolish P binding. Importantly, we demonstrate that upon RABV infection or detection of dsRNA by innate immunity sensors, TBK1 and its adaptor proteins NAP1 and SINTBAD form dynamic cytoplasmic condensates that have liquid properties. These condensates can form larger aggregates having ring-like structures in which NAP1 and TBK1 exhibit locally restricted movement. P binding to TBK1 interferes with the formation of these structures. This work demonstrates that proteins of the signaling pathway leading to interferon induction transiently form liquid organelles that can be targeted by viruses.

The adaptive response to alternative carbon sources in the pathogen Candida albicans involves a remodeling of thiol- and glutathione-dependent redox status.

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases known as candidiasis. During infection in vivo, Candida albicans must adapt to host microenvironments and this adaptive response is crucial for the survival of this organism, as it facilitates the effective assimilation of alternative carbon sources others than glucose. We performed a global proteomic analysis on the global changes in protein abundance in response to changes in micronutrient levels, and, in parallel, explored changes in the intracellular redox and metabolic status of the cells. We show here that each of the carbon sources considered - glucose, acetate and lactate - induces a unique pattern of response in C. albicans cells, and that some conditions trigger an original and specific adaptive response involving the adaptation of metabolic pathways, but also a complete remodeling of thiol-dependent antioxidant defenses. Protein S-thiolation and the overproduction of reduced glutathione are two components of the response to high glucose concentration. In the presence of acetate, glutathione-dependent oxidative stress occurs, reduced thiol groups bind to proteins, and glutathione is exported out of the cells, these changes probably being triggered by an increase in glutathione-S-transferases. Overall, our results suggest that the role of cellular redox status regulation and defenses against oxidative stress, including the thiol- and glutathione-dependent response, in the adaptive response of C. albicans to alternative carbon sources should be reconsidered.

Capillary liquid chromatography coupled with mass spectrometry for analysis of nanogram protein quantities on a wide-pore superficially porous particle column in top-down proteomics.

In top-down proteomics experiments, intact protein ions are subjected to gas-phase fragmentation for MS analysis without prior digestion. This approach is used to characterize post-translational modifications and clipped forms of proteins, avoids several "inference" problems associated with bottom-up proteomics, and is well suited to the study of proteoforms. In the past decade, top-down proteomics has progressed rapidly, taking advantage of MS instrumentation improvements and the efforts of pioneering groups working to improve sample handling and data processing. The potential of this technology has been established through its successful use in a number of important biological studies. However, many challenges remain to be addressed like improving protein separation capabilities such that it might become possible to expand the dynamic range of whole proteome analysis, address co-elution and convoluted mass spectral data, and aid final data processing from peak identification to quantification. In this study, we investigated the use of a wide-pore silica-based superficially porous media with a high coverage phenyl bonding, commercially packed into customized capillary columns for the purpose of top-down proteomics. Protein samples of increasing complexity were tested, namely subunit digests of a monoclonal antibody, components of purified histones and proteins extracted from eukaryotic ribosomes. High quality mass spectra were obtained from only 100 ng of protein sample while using difluoroacetic acid as an ion pairing agent to improve peak shape and chromatographic resolution. A peak width at half height of about 15 s for a 45 min gradient time was observed on a complex mixture giving an estimated peak capacity close to 100. Most importantly, efficient separations were obtained for highly diverse proteins and there was no need to make method specific adjustments, suggesting this is a highly versatile and easy-to-use setup for top-down proteomics.

Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction.

Orthohantaviruses are rodent-borne emerging viruses that may cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses-Puumala virus (PUUV), responsible for a mild to moderate form of hemorrhagic fever with renal syndrome in humans, Tula virus (TULV) with low pathogenicity, and non-pathogenic Prospect Hill virus (PHV)-with human and rodent host cell lines. Besides the fact that cell susceptibility to virus infection was shown to depend on the cell type and virus strain, the three orthohantaviruses were able to infect Vero E6 and HuH7 human cells, but only the former secreted infectious particles. In cells derived from PUUV reservoir, the bank vole (Myodes glareolus), PUUV achieved a complete viral cycle, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting differences in host specificity. A search for mature virions by electron microscopy (EM) revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. We described differential interactions of orthohantaviruses with cellular factors, as supported by the cellular distribution of viral nucleocapsid protein with cell compartments, and proteomics identification of cellular partners. Our results also showed that interferon (IFN) dependent gene expression was regulated in a cell and virus species dependent manner. Overall, our study highlighted the complexity of the host-virus relationship and demonstrated that orthohantaviruses are restricted at different levels of the viral cycle. In addition, the study opens new avenues to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how it depends on tissue type and host species.

The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

Cisplatin causes covalent inhibition of protein-tyrosine phosphatase 1B (PTP1B) through reaction with its active site cysteine: Molecular, cellular and in vivo mice studies.

Protein tyrosine phosphatase 1B (PTP1B) is a critical regulator of different signalling cascades such as the EGFR pathway. The biological importance of PTP1B is further evidenced by knockout mice studies and the identification of recurrent mutations/deletions in PTP1B linked to metabolic and oncogenic alterations. Cisplatin is among the most widely used anticancer drug. The biological effects of cisplatin are thought to arise primarily from DNA damaging events involving cisplatin-DNA adducts. However, increasing evidence indicate that the biological properties of cisplatin could also rely on the perturbation of other processes such as cell signalling through direct interaction with certain cysteine residues in proteins. Here, we provide molecular, cellular and in vivo evidence suggesting that PTP1B is a target of cisplatin. Mechanistic studies indicate that cisplatin inhibited PTP1B in an irreversible manner and binds covalently to the catalytic cysteine residue of the enzyme. Accordingly, experiments conducted in cells and mice exposed to cisplatin showed inhibition of endogenous PTP1B and concomitant increase in tyrosine phosphorylation of EGFR. These findings are consistent with previous studies showing tyrosine phosphorylation-dependent activation of the EGFR pathway by cisplatin and with recent studies suggesting PTP1B inhibition by cisplatin and other platinum complexes. Importantly, our work provides novel mechanistic evidence that PTP1B is a protein target of cisplatin and is inhibited by this drug at molecular, cellular and in vivo levels. In addition, our work may contribute to the understanding of the pathways undergoing modulation upon cisplatin administration beyond of the established genotoxic effect of cisplatin.

Quantitative Proteomics in Yeast : From bSLIM and Proteome Discoverer Outputs to Graphical Assessment of the Significance of Protein Quantification Scores.

Simple light isotope metabolic labeling (bSLIM) is an innovative method to accurately quantify differences in protein abundance at the proteome level in standard bottom-up experiments. The quantification process requires computation of the ratio of intensity of several isotopologs in the isotopic cluster of every identified peptide. Thus, appropriate bioinformatic workflows are required to extract the signals from the instrument files and calculate the required ratio to infer peptide/protein abundance. In a previous study (Sénécaut et al., J Proteome Res 20:1476-1487, 2021), we developed original open-source workflows based on OpenMS nodes implemented in a KNIME working environment. Here, we extend the use of the bSLIM labeling strategy in quantitative proteomics by presenting an alternative procedure to extract isotopolog intensities and process them by taking advantage of new functionalities integrated into the Minora node of Proteome Discoverer 2.4 software. We also present a graphical strategy to evaluate the statistical robustness of protein quantification scores and calculate the associated false discovery rates (FDR). We validated these approaches in a case study in which we compared the differences between the proteomes of two closely related yeast strains.

Paramecium Polycomb repressive complex 2 physically interacts with the small RNA-binding PIWI protein to repress transposable elements.

Polycomb repressive complex 2 (PRC2) maintains transcriptionally silent genes in a repressed state via deposition of histone H3K27-trimethyl (me3) marks. PRC2 has also been implicated in silencing transposable elements (TEs), yet how PRC2 is targeted to TEs remains unclear. To address this question, we identified proteins that physically interact with the Paramecium enhancer-of-zeste Ezl1 enzyme, which catalyzes H3K9me3 and H3K27me3 deposition at TEs. We show that the Paramecium PRC2 core complex comprises four subunits, each required in vivo for catalytic activity. We also identify PRC2 cofactors, including the RNA interference (RNAi) effector Ptiwi09, which are necessary to target H3K9me3 and H3K27me3 to TEs. We find that the physical interaction between PRC2 and the RNAi pathway is mediated by a RING finger protein and that small RNA recruitment of PRC2 to TEs is analogous to the small RNA recruitment of H3K9 methylation SU(VAR)3-9 enzymes.

Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation.

BACKGROUND & AIMS: NADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process. METHODS: The NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed. RESULTS: We identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid-induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage. CONCLUSIONS: The ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD.

Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis.

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.