Cross-reactivity between tumor MHC class I-restricted antigens and an enterococcal bacteriophage.

Intestinal microbiota have been proposed to induce commensal-specific memory T cells that cross-react with tumor-associated antigens. We identified major histocompatibility complex (MHC) class I-binding epitopes in the tail length tape measure protein (TMP) of a prophage found in the genome of the bacteriophage Mice bearing harboring this prophage mounted a TMP-specific H-2K-restricted CD8 T lymphocyte response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies. Administration of bacterial strains engineered to express the TMP epitope improved immunotherapy in mice.

Phosphorylation of eukaryotic initiation factor-2α (eIF2α) in autophagy.

The integrated stress response is characterized by the phosphorylation of eukaryotic initiation factor-2α (eIF2α) on serine 51 by one out of four specific kinases (EIF2AK1 to 4). Here we provide three series of evidence suggesting that macroautophagy (to which we refer to as autophagy) induced by a variety of distinct pharmacological agents generally requires this phosphorylation event. First, the induction of autophagic puncta by various distinct compounds was accompanied by eIF2α phosphorylation on serine 51.

Immunosuppression by Mutated Calreticulin Released from Malignant Cells.

Mutations affecting exon 9 of the CALR gene lead to the generation of a C-terminally modified calreticulin (CALR) protein that lacks the KDEL endoplasmic reticulum (ER) retention signal and consequently mislocalizes outside of the ER where it activates the thrombopoietin receptor in a cell-autonomous fashion, thus driving myeloproliferative diseases. Here, we used the retention using selective hooks (RUSH) assay to monitor the trafficking of CALR. We found that exon-9-mutated CALR was released from cells in response to the biotin-mediated detachment from its ER-localized hook, in vitro and in vivo.

A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary.

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated.

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus.

Acyl-CoA-Binding Protein Is a Lipogenic Factor that Triggers Food Intake and Obesity.

Autophagy facilitates the adaptation to nutritional stress. Here, we show that short-term starvation of cultured cells or mice caused the autophagy-dependent cellular release of acyl-CoA-binding protein (ACBP, also known as diazepam-binding inhibitor, DBI) and consequent ACBP-mediated feedback inhibition of autophagy. Importantly, ACBP levels were elevated in obese patients and reduced in anorexia nervosa.

Identification of pharmacological inhibitors of conventional protein secretion.

The retention using selective hooks (RUSH) system allows to withhold a fluorescent biosensor such as green fluorescent protein (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the biosensor from its hook. We constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and streptavidin within the endoplasmic reticulum (ER) lumen and then used this system to screen a compound library for inhibitors of the biotin-induced release of ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant.

Epigenetic anticancer agents cause HMGB1 release .

A systematic search for anticancer agents that may induce the release of high mobility group box 1 (HMGB1) protein from cells into the extracellular space has led to the identification of several drugs capable of elevating plasma HMGB1 levels , in mice. Such agents include bona-fide immunogenic cell death inducers such as oxaliplatin, as well as a series of epigenetic modifiers, namely azacitidine, decitabine, and suberoylanilide hydroxamic acid (SAHA).

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA).

Aspirin Recapitulates Features of Caloric Restriction.

The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs).

Identification of pharmacological agents that induce HMGB1 release.

The translocation of the protein high mobility group box 1 (HMGB1) from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane, constitutes a major cellular danger signal. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. Here, we developed a screening assay to identify pharmacological agents endowed with HMGB1 releasing properties.

Extracellular nucleosides and nucleotides as immunomodulators.

Some anticancer agents induce immunogenic cell death that is accompanied by the emission of danger signals into the tumor microenvironment, thus attracting and activating innate immune effectors and finally inducing anticancer immunity. The release of extracellular nucleosides such as adenosine triphosphate (ATP) from the tumor in response to anticancer therapy plays a pivotal role in the attraction of antigen presenting cells and the activation of inflammasome-mediated proinflammatory cascades. In contrast, the ectonucleotidase-catalyzed phosphohydrolysis of nucleotides to nucleosides reduces the extracellular availability of nucleotides, hence limiting the recruitment and activation of antigen-presenting cells.

FGF treatment of host embryos injected with ES cells increases rates of chimaerism.

In spite of the emergence of genome editing tools, ES cell mediated transgenesis remains the most controllable way of creating genetically modified animals. Although tetraploid (4N) complementation of 4N host embryos and ES cells, is the only method guaranteeing that offspring are entirely ES cell derived, this technique is challenging, not always successful and difficult to implement in some laboratory settings. The current study shows that pretreatment of host blastocysts with FGF4 prior to ES cell injection can provide an alternative method for the generation of animals displaying high rates of chimaerism.

Xist and Tsix Transcription Dynamics Is Regulated by the X-to-Autosome Ratio and Semistable Transcriptional States.

In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between X-linked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation.

Chromatin-Mediated Reversible Silencing of Sense-Antisense Gene Pairs in Embryonic Stem Cells Is Consolidated upon Differentiation.

Genome-wide gene expression studies have indicated that the eukaryotic genome contains many gene pairs showing overlapping sense and antisense transcription. Regulation of these coding and/or noncoding gene pairs involves intricate regulatory mechanisms. In the present study, we utilized an enhanced green fluorescent protein (EGFP)-tagged reporter plasmid cis linked to a doxycycline-inducible antisense promoter, generating antisense transcription that fully overlaps EGFP, to study the mechanism and dynamics of gene silencing after induction of noncoding antisense transcription in undifferentiated and differentiating mouse embryonic stem cells (ESCs).

Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation.

Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels.

Analysis of SHPRH functions in DNA repair and immunoglobulin diversification.

During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates.

The trans-activator RNF12 and cis-acting elements effectuate X chromosome inactivation independent of X-pairing.

X chromosome inactivation (XCI) in female placental mammals is a vital mechanism for dosage compensation between X-linked and autosomal genes. XCI starts with activation of Xist and silencing of the negative regulator Tsix, followed by cis spreading of Xist RNA over the future inactive X chromosome (Xi). Here, we show that XCI does not require physical contact between the two X chromosomes (X-pairing) but is regulated by trans-acting diffusible factors.

Characterization of three ammonium transporters of the glomeromycotan fungus Geosiphon pyriformis.

Members of the Glomeromycota form the arbuscular mycorrhiza (AM) symbiosis. They supply plants with inorganic nutrients, including nitrogen, from the soil. To gain insight into transporters potentially facilitating nitrogen transport processes, ammonium transporters (AMTs) of Geosiphon pyriformis, a glomeromycotan fungus forming a symbiosis with cyanobacteria, were studied.