Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.     

Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.     

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny,Physiology, and Niche Adaptation on Human Skin

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin’s carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.     

Influence of Granulocyte-Macrophage Colony-Stimulating Factor or Influenza Vaccination on HLA-DR,Infection and Delirium Days in Immunosuppressed Surgical Patients: Double Blind,Randomised Controlled Trial

Purpose

Surgical patients are at high risk for developing infectious complications and postoperative delirium. Prolonged infections and delirium result in worse outcome. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and influenza vaccination are known to increase HLA-DR on monocytes and improve immune reactivity. This study aimed to investigate whether GM-CSF or vaccination reverses monocyte deactivation. Secondary aims were whether it decreases infection and delirium days after esophageal or pancreatic resection over time.

Methods

In this prospective, randomized, placebo-controlled, double-blind, double dummy trial setting on an interdisciplinary ICU of a university hospital 61 patients with immunosuppression (monocytic HLA-DR [mHLA-DR] <10,000 monoclonal antibodies [mAb] per cell) on the first day after esophageal or pancreatic resection were treated with either GM-CSF (250 μg/m²/d), influenza vaccination (Mutagrip 0.5 ml/d) or placebo for a maximum of 3 consecutive days if mHLA-DR remained below 10,000 mAb per cell. HLA-DR on monocytes was measured daily until day 5 after surgery. Infections and delirium were followed up for 9 days after surgery. Primary outcome was HLA-DR on monocytes, and secondary outcomes were duration of infection and delirium.

Results

mHLA-DR was significantly increased compared to placebo (p < 0.001) and influenza vaccination (p < 0.001) on the second postoperative day. Compared with placebo, GM-CSF-treated patients revealed shorter duration of infection (p < 0.001); the duration of delirium was increased after vaccination (p = 0.003).

Conclusion

Treatment with GM-CSF in patients with postoperative immune suppression was safe and effective in restoring monocytic immune competence. Furthermore, therapy with GM-CSF reduced duration of infection in immune compromised patients. However, influenza vaccination increased duration of delirium after major surgery.

Trial Registration

www.controlled-trials.com ISRCTN27114642