Publications by authors named "Manuele Rebsamen"

21 Publications

  • Page 1 of 1

Epistasis-driven identification of SLC25A51 as a regulator of human mitochondrial NAD import.

Nat Commun 2020 12 1;11(1):6145. Epub 2020 Dec 1.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

About a thousand genes in the human genome encode for membrane transporters. Among these, several solute carrier proteins (SLCs), representing the largest group of transporters, are still orphan and lack functional characterization. We reasoned that assessing genetic interactions among SLCs may be an efficient way to obtain functional information allowing their deorphanization. Here we describe a network of strong genetic interactions indicating a contribution to mitochondrial respiration and redox metabolism for SLC25A51/MCART1, an uncharacterized member of the SLC25 family of transporters. Through a combination of metabolomics, genomics and genetics approaches, we demonstrate a role for SLC25A51 as enabler of mitochondrial import of NAD, showcasing the potential of genetic interaction-driven functional gene deorphanization.
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http://dx.doi.org/10.1038/s41467-020-19871-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708531PMC
December 2020

A substrate-based ontology for human solute carriers.

Mol Syst Biol 2020 07;16(7):e9652

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Solute carriers (SLCs) are the largest family of transmembrane transporters in the human genome with more than 400 members. Despite the fact that SLCs mediate critical biological functions and several are important pharmacological targets, a large proportion of them is poorly characterized and present no assigned substrate. A major limitation to systems-level de-orphanization campaigns is the absence of a structured, language-controlled chemical annotation. Here we describe a thorough manual annotation of SLCs based on literature. The annotation of substrates, transport mechanism, coupled ions, and subcellular localization for 446 human SLCs confirmed that ~30% of these were still functionally orphan and lacked known substrates. Application of a substrate-based ontology to transcriptomic datasets identified SLC-specific responses to external perturbations, while a machine-learning approach based on the annotation allowed us to identify potential substrates for several orphan SLCs. The annotation is available at https://opendata.cemm.at/gsflab/slcontology/. Given the increasing availability of large biological datasets and the growing interest in transporters, we expect that the effort presented here will be critical to provide novel insights into the functions of SLCs.
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http://dx.doi.org/10.15252/msb.20209652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374931PMC
July 2020

TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9.

Nature 2020 05 13;581(7808):316-322. Epub 2020 May 13.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus.
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http://dx.doi.org/10.1038/s41586-020-2282-0DOI Listing
May 2020

Insights into the transport side of the human SLC38A9 transceptor.

Biochim Biophys Acta Biomembr 2019 09 8;1861(9):1558-1567. Epub 2019 Jul 8.

Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036 Arcavacata di Rende, Italy; CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnology, via Amendola 165/A, 70126 Bari, Italy. Electronic address:

The lysosomal amino acid transporter SLC38A9 is referred to as transceptor, i.e. a transporter with a receptor function. The protein is responsible for coupling amino acid transport across the lysosomal membrane according to the substrate availability to mTORC1 signal transduction. This process allows cells to sense amino acid level responding to growth stimuli in physiological and pathological conditions triggering mTOR regulation. The main substrates underlying this function are glutamine and arginine. The functional and kinetic characterization of glutamine and arginine transport was performed using human SLC38A9 produced in E. coli, purified by affinity chromatography and reconstituted in liposomes. A cooperative behaviour for the wild type protein was revealed for both the substrates. A novel Na binding site, namely T453, was described by combined approaches of bioinformatics, site-directed mutagenesis and transport assay. Stimulation by cholesterol of glutamine and arginine transport was observed. The biological function of SLC38A9 relies on the interaction between its N-terminus and components of the mTOR complex; a deletion mutant of the N-terminus tail was produced and transport of glutamine was assayed revealing that this portion does not play any role in the intrinsic transport function of the human SLC38A9. Different features for glutamine and arginine transport were revealed: human SLC38A9 is competent for glutamine efflux, while that of arginine is negligible. In line with these results, imposed ∆pH stimulated glutamine, not arginine transport. Arginine plays, on the contrary, a modulatory function and is able to stimulate glutamine efflux. Interestingly, reciprocal inhibition experiments also supported by bioinformatics, suggested that glutamine and arginine may bind to different sites in the human SLC38A9 transporter.
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http://dx.doi.org/10.1016/j.bbamem.2019.07.006DOI Listing
September 2019

LZTR1 is a regulator of RAS ubiquitination and signaling.

Science 2018 12 15;362(6419):1171-1177. Epub 2018 Nov 15.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 () gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the ortholog resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for involvement in a variety of inherited and acquired human disorders.
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http://dx.doi.org/10.1126/science.aap8210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794158PMC
December 2018

Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking.

Cell Death Differ 2019 06 20;26(6):1138-1155. Epub 2018 Sep 20.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090, Vienna, Austria.

Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map such essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-of-function and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor surface levels. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~400 SLC genes, for TNFR1-mediated and FAS-mediated but not TRAIL-R1-mediated responses. Mechanistically, we demonstrate that loss of SLC39A7 resulted in augmented ER stress and impaired receptor trafficking, thereby globally affecting downstream signaling. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9-based synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1, which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-of-function and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting further investigation of their individual contribution and potential role in pathological conditions.
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http://dx.doi.org/10.1038/s41418-018-0192-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748104PMC
June 2019

The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.

Cell Host Microbe 2018 06 17;23(6):766-774.e5. Epub 2018 May 17.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna 1090, Austria; Center for Physiology and Pharmacology, Medical University of Vienna, Vienna 1090, Austria. Electronic address:

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.
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http://dx.doi.org/10.1016/j.chom.2018.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002608PMC
June 2018

LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning.

J Cell Biol 2017 12 9;216(12):4199-4215. Epub 2017 Oct 9.

Division of Cell Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.
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http://dx.doi.org/10.1083/jcb.201703061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716276PMC
December 2017

A time-resolved molecular map of the macrophage response to VSV infection.

NPJ Syst Biol Appl 2016 27;2:16027. Epub 2016 Oct 27.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Studying the relationship between virus infection and cellular response is paradigmatic for our understanding of how perturbation changes biological systems. Immune response, in this context is a complex yet evolutionarily adapted and robust cellular change, and is experimentally amenable to molecular analysis. To visualize the full cellular response to virus infection, we performed temporal transcriptomics, proteomics, and phosphoproteomics analysis of vesicular stomatitis virus (VSV)-infected mouse macrophages. This enabled the understanding of how infection-induced changes in host gene and protein expression are coordinated with post-translational modifications by cells in time to best measure and control the infection process. The vast and complex molecular changes measured could be decomposed in a limited number of clusters within each category (transcripts, proteins, and protein phosphorylation) each with own kinetic parameter and characteristic pathways/processes, suggesting multiple regulatory options in the overall sensing and homeostatic program. Altogether, the data underscored a prevalent executive function to phosphorylation. Resolution of the molecular events affecting the RIG-I pathway, central to viral recognition, reveals that phosphorylation of the key innate immunity adaptor mitochondrial antiviral-signaling protein (MAVS) on S328/S330 is necessary for activation of type-I interferon and nuclear factor κ B (NFκB) pathways. To further understand the hierarchical relationships, we analyzed kinase-substrate relationships and found RAF1 and, to a lesser extent, ARAF to be inhibiting VSV replication and necessary for NFκB activation, and AKT2, but not AKT1, to be supporting VSV replication. Integrated analysis using the omics data revealed co-regulation of transmembrane transporters including SLC7A11, which was subsequently validated as a host factor in the VSV replication. The data sets are predicted to greatly empower future studies on the functional organization of the response of macrophages to viral challenges.
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http://dx.doi.org/10.1038/npjsba.2016.27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516859PMC
October 2016

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

Mol Cell Proteomics 2016 Mar;15(3):1139-50

From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; ‖Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.
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http://dx.doi.org/10.1074/mcp.o115.055350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813694PMC
March 2016

SLC38A9: A lysosomal amino acid transporter at the core of the amino acid-sensing machinery that controls MTORC1.

Autophagy 2016 06 2;12(6):1061-2. Epub 2015 Oct 2.

a CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , Vienna , Austria.

The mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) acts as a crucial regulator of cellular metabolism by integrating growth factor presence, energy and nutrient availability to coordinate anabolic and catabolic processes, and controls cell growth and proliferation. Amino acids are critical for MTORC1 activation, but the molecular mechanisms involved in sensing their presence are just beginning to be understood. We recently reported that the previously uncharacterized amino acid transporter SLC38A9 is a member of the lysosomal sensing machinery that signals amino acid availability to MTORC1. SLC38A9 is the first component of this complex shown to physically engage amino acids, suggesting a role at the core of the amino acid-sensing mechanism.
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http://dx.doi.org/10.1080/15548627.2015.1091143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922434PMC
June 2016

The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Cell Rep 2015 Jun 18;11(12):1919-28. Epub 2015 Jun 18.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria; Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria. Electronic address:

Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.
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http://dx.doi.org/10.1016/j.celrep.2015.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4508342PMC
June 2015

Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death.

ACS Chem Biol 2015 Jul 15;10(7):1604-9. Epub 2015 May 15.

∥CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, 1090 Vienna, Austria.

Little is known about the regulation of nonapoptotic cell death. Using massive insertional mutagenesis of haploid KBM7 cells we identified nine genes involved in small-molecule-induced nonapoptotic cell death, including mediators of fatty acid metabolism (ACSL4) and lipid remodeling (LPCAT3) in ferroptosis. One novel compound, CIL56, triggered cell death dependent upon the rate-limiting de novo lipid synthetic enzyme ACC1. These results provide insight into the genetic regulation of cell death and highlight the central role of lipid metabolism in nonapoptotic cell death.
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http://dx.doi.org/10.1021/acschembio.5b00245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4509420PMC
July 2015

SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1.

Nature 2015 Mar 7;519(7544):477-81. Epub 2015 Jan 7.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
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http://dx.doi.org/10.1038/nature14107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4376665PMC
March 2015

A reversible gene trap collection empowers haploid genetics in human cells.

Nat Methods 2013 Oct 25;10(10):965-71. Epub 2013 Aug 25.

Haplogen GmbH, Vienna, Austria.

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-β, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.
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http://dx.doi.org/10.1038/nmeth.2609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342250PMC
October 2013

Protein interaction networks in innate immunity.

Trends Immunol 2013 Dec 2;34(12):610-9. Epub 2013 Jul 2.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

The immune response to pathogens is controlled by complex and tightly regulated molecular networks. Recent technological advances have empowered approaches to investigate innate immune signaling and monitor host-pathogen interactions at a systems level. Protein complexes are key players in pathogen recognition and integrate much of the host molecular responses that occur at the transcriptional and translational level. The ability to monitor protein complex abundance, dynamics, and composition is therefore important to understand the ability of cells to mount the appropriate immune response. Here, we focus on current proteomics technologies applied to identify the protein complexes involved, and highlight recent studies illustrating the power of these approaches to unravel how the dedicated molecular machinery is integrated with other cellular processes to safeguard homeostasis.
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http://dx.doi.org/10.1016/j.it.2013.05.002DOI Listing
December 2013

NLRC5 deficiency selectively impairs MHC class I- dependent lymphocyte killing by cytotoxic T cells.

J Immunol 2012 Apr 12;188(8):3820-8. Epub 2012 Mar 12.

Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland.

Nucleotide-binding oligomerization domain-like receptors (NLRs) are intracellular proteins involved in innate-driven inflammatory responses. The function of the family member NLR caspase recruitment domain containing protein 5 (NLRC5) remains a matter of debate, particularly with respect to NF-κB activation, type I IFN, and MHC I expression. To address the role of NLRC5, we generated Nlrc5-deficient mice (Nlrc5(Δ/Δ)). In this article we show that these animals exhibit slightly decreased CD8(+) T cell percentages, a phenotype compatible with deregulated MHC I expression. Of interest, NLRC5 ablation only mildly affected MHC I expression on APCs and, accordingly, Nlrc5(Δ/Δ) macrophages efficiently primed CD8(+) T cells. In contrast, NLRC5 deficiency dramatically impaired basal expression of MHC I in T, NKT, and NK lymphocytes. NLRC5 was sufficient to induce MHC I expression in a human lymphoid cell line, requiring both caspase recruitment and LRR domains. Moreover, endogenous NLRC5 localized to the nucleus and occupied the proximal promoter region of H-2 genes. Consistent with downregulated MHC I expression, the elimination of Nlrc5(Δ/Δ) lymphocytes by cytotoxic T cells was markedly reduced and, in addition, we observed low NLRC5 expression in several murine and human lymphoid-derived tumor cell lines. Hence, loss of NLRC5 expression represents an advantage for evading CD8(+) T cell-mediated elimination by downmodulation of MHC I levels-a mechanism that may be exploited by transformed cells. Our data show that NLRC5 acts as a key transcriptional regulator of MHC I in lymphocytes and support an essential role for NLRs in directing not only innate but also adaptive immune responses.
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http://dx.doi.org/10.4049/jimmunol.1102671DOI Listing
April 2012

Viruses under the control of RIP kinases.

Cell Cycle 2010 Feb 2;9(3):438-9. Epub 2010 Mar 2.

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http://dx.doi.org/10.4161/cc.9.3.10776DOI Listing
February 2010

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Nat Immunol 2010 Jan 15;11(1):63-9. Epub 2009 Nov 15.

III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.
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http://dx.doi.org/10.1038/ni.1824DOI Listing
January 2010

DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB.

EMBO Rep 2009 Aug 10;10(8):916-22. Epub 2009 Jul 10.

Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland.

Detection of viral nucleic acids is central to antiviral immunity. Recently, DAI/ZBP1 (DNA-dependent activator of IRFs/Z-DNA binding protein 1) was identified as a cytoplasmic DNA sensor and shown to activate the interferon regulatory factor (IRF) and nuclear factor-kappa B (NF-kappaB) transcription factors, leading to type-I interferon production. DAI-induced IRF activation depends on TANK-binding kinase 1 (TBK1), whereas signalling pathways and molecular components involved in NF-kappaB activation remain elusive. Here, we report the identification of two receptor-interacting protein (RIP) homotypic interaction motifs (RHIMs) in the DAI protein sequence, and show that these domains relay DAI-induced NF-kappaB signals through the recruitment of the RHIM-containing kinases RIP1 and RIP3. We show that knockdown of not only RIP1, but also RIP3 affects DAI-induced NF-kappaB activation. Importantly, RIP recruitment to DAI is inhibited by the RHIM-containing murine cytomegalovirus (MCMV) protein M45. These findings delineate the DAI signalling pathway to NF-kappaB and suggest a possible new immune modulation strategy of the MCMV.
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http://dx.doi.org/10.1038/embor.2009.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726668PMC
August 2009

TRADD protein is an essential component of the RIG-like helicase antiviral pathway.

Immunity 2008 May 24;28(5):651-61. Epub 2008 Apr 24.

Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland.

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses.
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http://dx.doi.org/10.1016/j.immuni.2008.03.013DOI Listing
May 2008