Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation.

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Endolysosomal TRPML1 channel regulates cancer cell migration by altering intracellular trafficking of E-cadherin and ß1-integrin.

Metastasis still accounts for 90% of all cancer-related death cases. An increase of cellular mobility and invasive traits of cancer cells mark two crucial prerequisites of metastasis. Recent studies highlight the involvement of the endolysosomal cation channel TRPML1 in cell migration. Our results identified a widely antimigratory effect upon loss of TRPML1 function in a panel of cell lines in vitro and reduced dissemination in vivo. As mode-of-action, we established TRPML1 as a crucial regulator of cytosolic calcium levels, actin polymerization, and intracellular trafficking of two promigratory proteins: E-cadherin and ß1-integrin. Interestingly, KO of TRPML1 differentially interferes with the recycling process of E-cadherin and ß1-integrin in a cell line-dependant manner, while resulting in the same phenotype of decreased migratory and adhesive capacities in vitro. Additionally, we observed a coherence between reduction of E-cadherin levels at membrane site and phosphorylation of NF-κB in a ß-catenin/p38-mediated manner. As a result, an E-cadherin/NF-κB feedback loop is generated, regulating E-cadherin expression on a transcriptional level. Consequently, our findings highlight the role of TRPML1 as a regulator in migratory processes and suggest the ion channel as a suitable target for the inhibition of migration and invasion.

Cell specification and functional interactions in the pig blastocyst inferred from single-cell transcriptomics and uterine fluids proteomics.

The embryonic development of the pig comprises a long in utero pre- and peri-implantation development, which dramatically differs from mice and humans. During this peri-implantation period, a complex series of paracrine signals establishes an intimate dialogue between the embryo and the uterus. To better understand the biology of the pig blastocyst during this period, we generated a large dataset of single-cell RNAseq from early and hatched blastocysts, spheroid and ovoid conceptus and proteomic datasets from corresponding uterine fluids. Our results confirm the molecular specificity and functionality of the three main cell populations. We also discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent progenitor cells, and the other, expressing pro-apoptotic markers, which could correspond to the Rauber's layer. Our work provides new insights into the biology of these populations, their reciprocal functional interactions, and the molecular dialogue with the maternal uterine environment.

Proteome profile of the cerebellum from α7 nicotinic acetylcholine receptor deficient mice.

The alpha7 nicotinic acetylcholine receptor (α7 nAChR; CHRNA7) is expressed in the nervous system and in non-neuronal tissues. Within the central nervous system, it is involved in various cognitive and sensory processes such as learning, attention, and memory. It is also expressed in the cerebellum, where its roles are; however, not as well understood as in the other brain regions. To investigate the consequences of absence of CHRNA7 on the cerebellum proteome, we performed a quantitative nano-LC-MS/MS analysis of samples from CHRNA7 knockout (KO) mice and corresponding wild type (WT) controls. Liver, an organ which does not express this receptor, was analyzed, in comparison. While the liver proteome remained relatively unaltered (three proteins more abundant in KOs), 90 more and 20 less abundant proteins were detected in the cerebellum proteome of the KO mice. The gene ontology analysis of the differentially abundant proteins indicates that the absence of CHRNA7 leads to alterations in the glutamatergic system and myelin sheath in the cerebellum. In conclusion, our dataset provides new insights in the role of CHRNA7 in the cerebellum, which may serve as a basis for future in depth-investigations.

The cuticle proteome of a planktonic crustacean.

The cuticles of arthropods provide an interface between the organism and its environment. Thus, the cuticle's structure influences how the organism responds to and interacts with its surroundings. Here, we used label-free quantification proteomics to provide a proteome of the moulted cuticle of the aquatic crustacean Daphnia magna, which has long been a prominent subject of studies on ecology, evolution, and developmental biology. We detected a total of 278 high-confidence proteins. Using protein sequence domain and functional enrichment analyses, we identified chitin-binding structural proteins and chitin-modifying enzymes as the most abundant protein groups in the cuticle proteome. Structural cuticular protein families showed a similar distribution to those found in other arthropods and indicated proteins responsible for the soft and flexible structure of the Daphnia cuticle. Finally, cuticle protein genes were also clustered as tandem gene arrays in the D. magna genome. The cuticle proteome presented here will be a valuable resource to the Daphnia research community, informing genome annotations and investigations on diverse topics such as the genetic basis of interactions with predators and parasites.

Prolonged exposure to dexamethasone alters the proteome and cellular phenotype of human testicular peritubular cells.

Human testicular peritubular cells (HTPCs) are smooth muscle cells, which in the testis form a small compartment surrounding the seminiferous tubules. Contractions of HTPCs are responsible for sperm transport, HTPCs contribute to spermatogenesis, have immunological roles and are a site of glucocorticoid receptor expression. Importantly, HTPCs maintain their characteristics in vitro, and thus can serve as an experimental window into the male gonad. Previously we reported consequences of 3-day treatment with Dexamethasone (Dex), a synthetic glucocorticoid and multi-purpose anti-inflammatory drug. However, as glucocorticoid therapies in man often last longer, we now studied consequences of a prolonged 7-day exposure to 1 µM Dex. Combining live cell imaging with quantative proteomics of samples taken from men, we confirmed our recent findings but more importantly, found numerous novel proteomic alterations induced by prolonged Dex treatment. The comparison of the 7-day treatment with the 3-day treatment dataset revealed that extracellular matrix- and focal adhesion-related proteins become more prominent after 7 days of treatment. In contrast, extended stimulation is, for example, associated with a decrease of proteins related to cholesterol and steroid metabolism. Our dataset, which describes phenotypic and proteomic alterations, is a valuable resource for further research projects investigating effects of Dex on human testicular cells.

A Novel Interaction of Slug (SNAI2) and Nuclear Actin.

Actin is a protein of central importance to many cellular functions. Its localization and activity are regulated by interactions with a high number of actin-binding proteins. In a yeast two-hybrid (Y2H) screening system, snail family transcriptional repressor 2 (SNAI2 or slug) was identified as a yet unknown potential actin-binding protein. We validated this interaction using immunoprecipitation and analyzed the functional relation between slug and actin. Since both proteins have been reported to be involved in DNA double-strand break (DSB) repair, we focused on their interaction during this process after treatment with doxorubicin or UV irradiation. Confocal microscopy elicits that the overexpression of actin fused to an NLS stabilizes complexes of slug and γH2AX, an early marker of DNA damage repair.

Influence of the polymer type of a microplastic challenge on the reaction of murine cells.

Due to global pollution derived from plastic waste, the research on microplastics is of increasing public interest. Until now, most studies addressing the effect of microplastic particles on vertebrate cells have primarily utilized polystyrene particles (PS). Other studies on polymer microparticles made, e.g., of polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), or poly (ethylene terephthalate) (PET), cannot easily be directly compared to these PS studies, since the used microparticles differ widely in size and surface features. Here, effects caused by pristine microparticles of a narrow size range between 1 - 4 µm from selected conventional polymers including PS, PE, and PVC, were compared to those of particles made of polymers derived from biological sources like polylactic acid (PLA), and cellulose acetate (CA). The microparticles were used to investigate cellular uptake and assess cytotoxic effects on murine macrophages and epithelial cells. Despite differences in the particles' properties (e.g. ζ-potential and surface morphology), macrophages were able to ingest all tested particles, whereas epithelial cells ingested only the PS-based particles, which had a strong negative ζ-potential. Most importantly, none of the used model polymer particles exhibited significant short-time cytotoxicity, although the general effect of environmentally relevant microplastic particles on organisms requires further investigation.

Phosphorylation of PFKL regulates metabolic reprogramming in macrophages following pattern recognition receptor activation.

Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1ß than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1ß. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.

Multi-omics analysis of diabetic pig lungs reveals molecular derangements underlying pulmonary complications of diabetes mellitus.

Growing evidence shows that the lung is an organ prone to injury by diabetes mellitus. However, the molecular mechanisms of these pulmonary complications have not yet been characterized comprehensively. To systematically study the effects of insulin deficiency and hyperglycaemia on the lung, we combined proteomics and lipidomics with quantitative histomorphological analyses to compare lung tissue samples from a clinically relevant pig model for mutant INS gene-induced diabetes of youth (MIDY) with samples from wild-type littermate controls. Among others, the level of pulmonary surfactant-associated protein A (SFTPA1), a biomarker of lung injury, was moderately elevated. Furthermore, key proteins related to humoral immune response and extracellular matrix organization were significantly altered in abundance. Importantly, a lipoxygenase pathway was dysregulated as indicated by 2.5-fold reduction of polyunsaturated fatty acid lipoxygenase ALOX15 levels, associated with corresponding changes in the levels of lipids influenced by this enzyme. Our multi-omics study points to an involvement of reduced ALOX15 levels and an associated lack of eicosanoid switching as mechanisms contributing to a proinflammatory milieu in the lungs of subjects with diabetes mellitus.