RUFY3 regulates endolysosomes perinuclear positioning, antigen presentation and migration in activated phagocytes.

Endo-lysosomes transport along microtubules and clustering in the perinuclear area are two necessary steps for microbes to activate specialized phagocyte functions. We report that RUN and FYVE domain-containing protein 3 (RUFY3) exists as two alternative isoforms distinguishable by the presence of a C-terminal FYVE domain and by their affinity for phosphatidylinositol 3-phosphate on endosomal membranes. The FYVE domain-bearing isoform (iRUFY3) is preferentially expressed in primary immune cells and up-regulated upon activation by microbes and Interferons.

RUFY4 exists as two translationally regulated isoforms, that localize to the mitochondrion in activated macrophages.

We report here that RUFY4, a newly characterized member of the 'RUN and FYVE domain-containing' family of proteins previously associated with autophagy enhancement, is highly expressed in alveolar macrophages (AM). We show that RUFY4 interacts with mitochondria upon stimulation by microbial-associated molecular patterns of AM and dendritic cells. RUFY4 interaction with mitochondria and other organelles is dependent on a previously uncharacterized OmpH domain located immediately upstream of its C-terminal FYVE domain.

LAMP-5 is an essential inflammatory-signaling regulator and novel immunotherapy target for mixed lineage leukemia-rearranged acute leukemia.

Although great advances have been made in understanding the pathobiology of mixed lineage leukemia-rearranged (MLL-r) leukemias, therapies for this leukemia have remained limited, and clinical outcomes remain bleak. In order to identify novel targets for immunotherapy treatments, we compiled a lineage-independent MLL-r leukemia gene signature using publicly available data sets. Data from large leukemia repositories were filtered through the in silico human surfaceome, providing a list of highly predicted cell surface proteins overexpressed in MLL-r leukemias.

Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4.

In stressed cells, phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response. We show here that DCs are characterized by high eIF2α phosphorylation, mostly caused by the activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2α levels, DCs display active protein synthesis and no signs of a chronic integrated stress response.

Polymerase III transcription is necessary for T cell priming by dendritic cells.

Exposure to microbe-associated molecular patterns (MAMPs) causes dendritic cells (DCs) to undergo a remarkable activation process characterized by changes in key biochemical mechanisms. These enhance antigen processing and presentation, as well as strengthen DC capacity to stimulate naïve T cell proliferation. Here, we show that in response to the MAMPS lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (Poly I:C), RNA polymerase III (Pol lII)-dependent transcription and consequently tRNA gene expression are strongly induced in DCs.

Monitoring MHC Ubiquitination by MARCH Ubiquitin Ligases.

Ubiquitination is a reversible process that controls the intracellular transport of many transmembrane molecules. Ubiquitination of MHC I, MHC II, and CD1a by different members of the MARCH family of E3 ubiquitin ligases is a key event in the regulation of the potent immunostimulatory properties of activated dendritic cells. We describe here methods to monitor and quantify the ubiquitination levels of these different antigen presentation molecules and its impact on their cell surface accumulation.

Autophagy and MHC-restricted antigen presentation.

Major histocompatibility complex (MHC) molecules present peptide antigens to T lymphocytes and initiate immune responses. The peptides loaded onto MHC class I or MHC class II molecules can be derived from cytosolic proteins, both self and foreign. A variety of cellular processes, including endocytosis, vesicle trafficking, and autophagy, play critical roles in presentation of these antigens.

SunRiSE - measuring translation elongation at single-cell resolution by means of flow cytometry.

The rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues.

Guanabenz inhibits TLR9 signaling through a pathway that is independent of eIF2α dephosphorylation by the GADD34/PP1c complex.

Endoplasmic reticulum (ER) stress triggers or amplifies inflammatory signals and cytokine production in immune cells. Upon the resolution of ER stress, the inducible phosphatase 1 cofactor GADD34 promotes the dephosphorylation of the initiation factor eIF2α, thereby enabling protein translation to resume. Several aminoguanidine compounds, such as guanabenz, perturb the eIF2α phosphorylation-dephosphorylation cycle and protect different cell or tissue types from protein misfolding and degeneration.

At the crossway of ER-stress and proinflammatory responses.

Immune cells detect specific microbes or damage to tissue integrity in order to initiate efficient immune responses. Abnormal accumulation of proteins in the endoplasmic reticulum (ER) can be seen as a sign of cellular malfunction and stress that triggers a collection of conserved emergency rescue programs. These different signaling cascades, which favor ER proteostasis and promote cell survival, are collectively known as the unfolded protein response (UPR).

BAD-LAMP controls TLR9 trafficking and signalling in human plasmacytoid dendritic cells.

Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1 late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection.

Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method.

Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive, and laborious.

Guanabenz Prevents d-Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality.

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression.

MARCH9-mediated ubiquitination regulates MHC I export from the TGN.

Given the heterogeneous nature of antigens, major histocompatibility complex class I (MHC I) intracellular transport intersects with multiple degradation pathways for efficient peptide loading and presentation to cytotoxic T cells. MHC I loading with peptides in the endoplasmic reticulum (ER) is a tightly regulated process, while post-ER intracellular transport is considered to occur by default, leading to peptide-bearing MHC I delivery to the plasma membrane. We show here that MHC I traffic is submitted to a previously uncharacterized sorting step at the trans Golgi network (TGN), dependent on the ubiquitination of its cytoplasmic tail lysine residues.

Protein synthesis inhibition and GADD34 control IFN-β heterogeneous expression in response to dsRNA.

In innate immune responses, induction of type-I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type-I IFN production is linked to cell's ability to enter dsRNA-activated PKR-dependent translational arrest and then overcome this inhibition by decreasing eIF2α phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression.

Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement.

LAMP5 Fine-Tunes GABAergic Synaptic Transmission in Defined Circuits of the Mouse Brain.

LAMP5 is member of the LAMP family of membrane proteins. In contrast to the canonical members of this protein family, LAMP1 and LAMP2, which show widespread expression in many tissues, LAMP 5 is brain specific in mice. In C.

Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis.

Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2).

Unfolded protein response gene GADD34 is overexpressed in rheumatoid arthritis and related to the presence of circulating anti-citrullinated protein antibodies.

Growth arrest and DNA damage-inducible gene 34 (GADD34) is an inducible cofactor of protein phosphatase 1, which has an important role in the unfolded protein response. GADD34 has been shown to be necessary for type I interferon and proinflammatory cytokine production in response to viral infection in murine models. We investigate the expression of GADD34 in rheumatoid arthritis (RA), in which proinflammatory cytokines have an important pathogenic role.

RUFY4: Immunity piggybacking on autophagy?

Although autophagy is a highly conserved mechanism among species and cell types, few are the molecules involved with the autophagic process that display cell- or tissue- specific expression. We have unraveled the positive regulatory role on autophagy of RUFY4 (RUN and FYVE domain containing 4), which is expressed in subsets of immune cells, including dendritic cells (DCs). DCs orchestrate the eradication of pathogens by coordinating the action of the different cell types involved in microbe recognition and destruction during the immune response.

RUN and FYVE domain-containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4.

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain-containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs).

Integration of PKR-dependent translation inhibition with innate immunity is required for a coordinated anti-viral response.

Viral triggering of the innate immune response in infected cells aims at delaying viral replication and prevents tissue spreading. Viral replication is delayed by host protein synthesis inhibition and infected cell apoptosis on one hand, while infection spreading is controlled by the synthesis of specific proteins like type-I interferons (IFNs) and pro-inflammatory cytokines on the other hand. How do these two apparent conflicting responses cooperate within the same infected cells to mount effective defenses against pathogens? What are the molecules or the complexes resolving this contradiction over time? Some recent studies reveal unanticipated connections between innate immunity and stress pathways, giving important clues on how the cellular responses are orchestrated to limit infection efficiently.

In vivo imaging of the spatiotemporal activity of the eIF2α-ATF4 signaling pathway: Insights into stress and related disorders.

The eIF2α-ATF4 pathway is involved in cellular adaptation to stress and is dysregulated in numerous diseases. Activation of this pathway leads to phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and the recruitment of the transcription factor ATF4 (activating transcription factor 4) to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of target genes. To monitor the spatiotemporal modulation of this pathway in living animals, we generated a novel CARE-driven luciferase mouse model (CARE-LUC).