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1.  Target interaction profiling of midostaurin and its metabolites in neoplastic mast cells predicts distinct effects on activation and growth 
Leukemia  2015;30(2):464-472.
Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multi-kinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction-profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC50 values. Midostaurin and CGP62221 also produced growth-inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, that accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug-competition experiments revealed that midostaurin interacts with KIT and several additional kinase-targets. The key downstream-regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE-receptor-downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.
PMCID: PMC4896384  PMID: 26349526
mastocytosis; PKC412; drug profiling; KIT; histamine release
2.  Crystal structure of mammalian acid sphingomyelinase 
Nature Communications  2016;7:12196.
Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann–Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann–Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase.
Mutations in acid sphingomyelinase result in toxic accumulation of sphingomyelin and cause Niemann-Pick disease. Here, the authors report structures of mammalian acid sphingomyelinase, which reveal details of the molecular mechanism of acid sphingomyelinase function and regulation.
PMCID: PMC4961792  PMID: 27435900
3.  Structural basis for viral 5′-PPP-RNA recognition by human IFIT proteins 
Nature  2013;494(7435):60-64.
IFIT proteins are interferon-inducible, innate immune effector molecules that are thought to confer antiviral defence through disruption of protein-protein interactions in the host translation initiation machinery. However, recently it was discovered that IFITs could directly recognize viral RNA bearing a 5′-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here, we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an N-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double stranded viral PPP-RNA, RIG-I. Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence specific manner and requires approximately a 3-nucleotide 5′-overhang. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 were found to cause a defect in the anti-viral response by HEK cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA and lend insight into their downstream effector function.
PMCID: PMC4931921  PMID: 23334420 CAMSID: cams3927
innate immunity; TPR; IFIT1; ISG56; interferon; viral RNA; triphophosphate RNA; IFIT5; ISG58; crystal structure
4.  Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry 
Scientific Reports  2016;6:28107.
Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[18O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates.
PMCID: PMC4921819  PMID: 27346722
5.  The solute carrier SLC35F2 enables YM155-mediated DNA damage toxicity 
Nature chemical biology  2014;10(9):768-773.
Genotoxic chemotherapy is the most common cancer treatment strategy. However, its untargeted generic DNA-damaging nature and associated systemic cytotoxicity greatly limit the therapeutic applications. Here, we employed a haploid genetic screen in human cells to discover an absolute dependency of the clinically evaluated anti-cancer compound YM155 on SLC35F2, an uncharacterized member of the solute carrier protein family that is highly expressed in a variety of human cancers. YM155 generated DNA damage through intercalation, which was contingent on the expression of SLC35F2 and its drug importing activity. SLC35F2 expression and YM155 sensitivity correlated across a panel of cancer cell lines and targeted genome editing verified SLC35F2 as the main determinant of YM155-mediated DNA damage toxicity in vitro and in vivo. These findings suggest a novel route to targeted DNA damage by exploiting tumor and patient-specific import of YM155.
PMCID: PMC4913867  PMID: 25064833
6.  Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death 
ACS Chemical Biology  2015;10(7):1604-1609.
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.
PMCID: PMC4509420  PMID: 25965523
7.  The Tumor Suppressor Hace1 Is a Critical Regulator of TNFR1-Mediated Cell Fate 
Cell Reports  2016;15(7):1481-1492.
The HECT domain E3 ligase HACE1 has been identified as a tumor suppressor in multiple cancers. Here, we report that HACE1 is a central gatekeeper of TNFR1-induced cell fate. Genetic inactivation of HACE1 inhibits TNF-stimulated NF-κB activation and TNFR1-NF-κB-dependent pathogen clearance in vivo. Moreover, TNF-induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. Mechanistically, HACE1 is essential for the ubiquitylation of the adaptor protein TRAF2 and formation of the apoptotic caspase-8 effector complex. Intriguingly, loss of HACE1 does not impair TNFR1-mediated necroptotic cell fate via RIP1 and RIP3 kinases. Loss of HACE1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of RIP3 kinase and TNFR1. Thus, HACE1 controls TNF-elicited cell fate decisions and exerts tumor suppressor and anti-inflammatory activities via a TNFR1-RIP3 kinase-necroptosis pathway.
Graphical Abstract
•Hace1 deficiency impairs TNF-driven NF-κB activation and apoptosis•Necroptosis via RIP1/RIP3/MLKL is still functional in the absence of Hace1•Hace1–/– animals show enhanced severity of colitis and colon cancer•Genetic inactivation of RIP3 and TNFR1 reverts the phenotype of hace1–/– mice
Tortola et al. report that the E3 ubiquitin ligase HACE1 is a gatekeeper of TNFR1-mediated cell fate. Hace1 deficiency impairs TNF-driven NF-κB activation and apoptosis and predisposes cells to necroptosis. Consequently, hace1–/– mice show enhanced colitis and colon cancer, which can be reverted by inactivation of pro-necroptotic kinase RIP3 and TNFR1.
PMCID: PMC4893156  PMID: 27160902
8.  Proteome-wide small molecule and metabolite interaction mapping 
Nature methods  2015;12(11):1055-1057.
Thermal stabilization of proteins upon ligand binding provides an efficient means to assess binding of small molecules to proteins. We show here that in combination with quantitative mass spectrometry the approach allows for the systematic survey of protein engagement by cellular metabolites and drugs. The profiling of methotrexate, (S)-crizotinib and 2′3′-cGAMP in intact cells identified the respective cognate targets including the transmembrane receptor STING involved in innate immune signalling.
PMCID: PMC4629415  PMID: 26389571
target deconvolution; proteomics; drug discovery; metabolite; network
9.  JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia 
Nature genetics  2014;46(9):1021-1027.
Analysis of patients with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling differentiation, maintenance, and decay of neutrophils. We identify 9 distinct homozygous mutations in the gene encoding Jagunal homolog 1 (JAGN1) in 14 SCN patients. JAGN1-mutant granulocytes are characterized by ultrastructural defects, paucity of granules, aberrant N-glycosylation of multiple proteins, and increased apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte-colony stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor necessary in differentiation and survival of neutrophils.
PMCID: PMC4829076  PMID: 25129144
10.  Complement factor H binds malondialdehyde epitopes and protects from oxidative stress 
Nature  2011;478(7367):76-81.
Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.
PMCID: PMC4826616  PMID: 21979047
11.  Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia 
Nature chemical biology  2015;11(8):571-578.
The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short 30 kDa C/EBPα translational isoform, termed p30, represents the most common type of CEBPA mutations in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as its down-regulation inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a novel small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30-cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.
PMCID: PMC4511833  PMID: 26167872
12.  The promise and peril of chemical probes 
Nature chemical biology  2015;11(8):536-541.
Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice.
PMCID: PMC4706458  PMID: 26196764
13.  Superoxide Dismutase 1 Protects Hepatocytes from Type I Interferon-Driven Oxidative Damage 
Immunity  2015;43(5):974-986.
Tissue damage caused by viral hepatitis is a major cause of morbidity and mortality worldwide. Using a mouse model of viral hepatitis, we identified virus-induced early transcriptional changes in the redox pathways in the liver, including downregulation of superoxide dismutase 1 (Sod1). Sod1−/− mice exhibited increased inflammation and aggravated liver damage upon viral infection, which was independent of T and NK cells and could be ameliorated by antioxidant treatment. Type I interferon (IFN-I) led to a downregulation of Sod1 and caused oxidative liver damage in Sod1−/− and wild-type mice. Genetic and pharmacological ablation of the IFN-I signaling pathway protected against virus-induced liver damage. These results delineate IFN-I mediated oxidative stress as a key mediator of virus-induced liver damage and describe a mechanism of innate-immunity-driven pathology, linking IFN-I signaling with antioxidant host defense and infection-associated tissue damage.
Graphical Abstract
In Brief
Bergthaler and colleagues show that superoxide dismutase 1 protects the liver from type I interferon-driven oxidative damage in viral hepatitis. Liver damage was mediated by hepatocyte-intrinsic IFNAR1-STAT1 signaling.
PMCID: PMC4658338  PMID: 26588782
14.  Mutations in the heat-shock protein A9 (HSPA9) gene cause the EVEN-PLUS syndrome of congenital malformations and skeletal dysplasia 
Scientific Reports  2015;5:17154.
We and others have reported mutations in LONP1, a gene coding for a mitochondrial chaperone and protease, as the cause of the human CODAS (cerebral, ocular, dental, auricular and skeletal) syndrome (MIM 600373). Here, we delineate a similar but distinct condition that shares the epiphyseal, vertebral and ocular changes of CODAS but also included severe microtia, nasal hypoplasia, and other malformations, and for which we propose the name of EVEN-PLUS syndrome for epiphyseal, vertebral, ear, nose, plus associated findings. In three individuals from two families, no mutation in LONP1 was found; instead, we found biallelic mutations in HSPA9, the gene that codes for mHSP70/mortalin, another highly conserved mitochondrial chaperone protein essential in mitochondrial protein import, folding, and degradation. The functional relationship between LONP1 and HSPA9 in mitochondrial protein chaperoning and the overlapping phenotypes of CODAS and EVEN-PLUS delineate a family of “mitochondrial chaperonopathies” and point to an unexplored role of mitochondrial chaperones in human embryonic morphogenesis.
PMCID: PMC4657157  PMID: 26598328
15.  Superoxide Dismutase 1 Protects Hepatocytes from Type I Interferon-Driven Oxidative Damage 
Immunity  2015;43(5):974-986.
Tissue damage caused by viral hepatitis is a major cause of morbidity and mortality worldwide. Using a mouse model of viral hepatitis, we identified virus-induced early transcriptional changes in the redox pathways in the liver, including downregulation of superoxide dismutase 1 (Sod1). Sod1−/− mice exhibited increased inflammation and aggravated liver damage upon viral infection, which was independent of T and NK cells and could be ameliorated by antioxidant treatment. Type I interferon (IFN-I) led to a downregulation of Sod1 and caused oxidative liver damage in Sod1−/− and wild-type mice. Genetic and pharmacological ablation of the IFN-I signaling pathway protected against virus-induced liver damage. These results delineate IFN-I mediated oxidative stress as a key mediator of virus-induced liver damage and describe a mechanism of innate-immunity-driven pathology, linking IFN-I signaling with antioxidant host defense and infection-associated tissue damage.
Video Abstract
Graphical Abstract
•Viral infection leads to redox dysregulation including the downregulation of SOD1•Sod1−/− mice exhibit aggravated viral hepatitis, which is rescued by antioxidants•IFN-I signaling via STAT1 drives SOD1 downregulation and early liver damage•Ablation of IFN-I signaling ameliorates viral hepatitis in Sod1−/− and WT mice
Bergthaler and colleagues show that superoxide dismutase 1 protects the liver from type I interferon-driven oxidative damage in viral hepatitis. Liver damage was mediated by hepatocyte-intrinsic IFNAR1-STAT1 signaling.
PMCID: PMC4658338  PMID: 26588782
16.  Identification of kinase inhibitor targets in the lung cancer microenvironment by chemical and phosphoproteomics 
Molecular cancer therapeutics  2014;13(11):2751-2762.
A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. Whereas these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib) and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes and signaling pathways that are simultaneously engaged by multi-targeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments.
PMCID: PMC4221415  PMID: 25189542
Kinase inhibitor; chemical proteomics; phosphoproteomics; non-small cell lung cancer; microenvironment
17.  SLC38A9: A lysosomal amino acid transporter at the core of the amino acid-sensing machinery that controls MTORC1 
Autophagy  2015;12(6):1061-1062.
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.
PMCID: PMC4922434  PMID: 26431368
amino acid transport; cancer; metabolism; MTOR; nutrient sensing; solute carrier proteins
18.  SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1 
Nature  2015;519(7544):477-481.
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 responses1-3. 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 4-8. 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, while 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.
PMCID: PMC4376665  PMID: 25561175
mTOR; solute carrier proteins; amino acid transport; metabolism; cancer
19.  Phosphatase and tensin homolog (PTEN) in antigen-presenting cells controls Th17-mediated autoimmune arthritis 
Autoreactive T cells are a central element in many systemic autoimmune diseases. The generation of these pathogenic T cells is instructed by antigen-presenting cells (APCs). However, signaling pathways in APCs that drive autoimmune diseases, such as rheumatoid arthritis, are not understood.
We measured phenotypic maturation, cytokine production and induction of T cell proliferation of APCs derived from wt mice and mice with a myeloid-specific deletion of PTEN (myeloid PTEN-/-) in vitro and in vivo. We induced collagen-induced arthritis (CIA) and K/BxN serum transfer arthritis in wt and myeloid-specific PTEN-/- mice. We measured the cellular composition of lymph nodes by flow cytometry and cytokines in serum and after ex vivo stimulation of T cells.
We show that myeloid-specific PTEN-/- mice are almost protected from CIA. Myeloid-specific deletion of PTEN leads to a significant reduction of cytokine expression pivotal for the induction of systemic autoimmunity such as interleukin (IL)-23 and IL-6, leading to a significant reduction of a Th17 type of immune response characterized by reduced production of IL-17 and IL-22. In contrast, myeloid-specific PTEN deficiency did not affect K/BxN serum transfer arthritis, which is independent of the adaptive immune system and solely depends on innate effector functions.
These data demonstrate that the presence of PTEN in myeloid cells is required for the development of CIA. Deletion of PTEN in myeloid cells inhibits the development of autoimmune arthritis by preventing the generation of a pathogenic Th17 type of immune response.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0742-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4549861  PMID: 26307404
20.  A Conserved Circular Network of Coregulated Lipids Modulates Innate Immune Responses 
Cell  2015;162(1):170-183.
Lipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems.
Graphical Abstract
•Coregulation between membrane lipid species is organized in a circular network•The lipid network is conserved and reflects metabolism, localization, and adaptation•Sphingolipid metabolism regulates TLR trafficking, signaling, and cytokine release•Network-wide functional lipid annotations predict TLR responses in patient cells
Combining lipidomics with genetic perturbations in immune cells reveals the logic of inter-lipid regulatory structure and enables the functional assignment of lipids to different steps of Toll-like receptor signaling. Moreover, quantitative lipidomics alone can predict the inflammatory response of patient-derived cells.
PMCID: PMC4523684  PMID: 26095250
21.  The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity 
Cell Reports  2015;11(12):1919-1928.
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.
Graphical Abstract
•Identification of SMPDL3B as lipid-modulating phosphodiesterase on macrophages•Negative regulatory role for SMPDL3B in Toll-like receptor function•Strong influence of SMPDL3B on membrane lipid composition and fluidity•Smpdl3b-deficient mice show enhanced responsiveness in TLR-dependent peritonitis
Heinz et al. identify the lipid-modulating phosphodiesterase SMPDL3B as negative regulator of Toll-like receptor function. Smpdl3b-deficiency strongly affected macrophage lipid composition and fluidity and led to higher responsiveness to TLR stimulation. Peritonitis models in Smpdl3b-deficient mice confirmed the negative regulatory role in vivo.
PMCID: PMC4508342  PMID: 26095358
22.  Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network 
mBio  2015;6(3):e00712-15.
Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction.
Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.
PMCID: PMC4453011  PMID: 26037124
23.  Targeting a cell state common to triple-negative breast cancers 
Molecular Systems Biology  2015;11(2):789.
Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their ‘basal-like’ transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.
PMCID: PMC4358660  PMID: 25699542
breast cancer; cell state; small-molecule screen
24.  Biallelic loss-of-function mutation in NIK causes a primary immunodeficiency with multifaceted aberrant lymphoid immunity 
Nature Communications  2014;5:5360.
Primary immunodeficiency disorders enable identification of genes with crucial roles in the human immune system. Here we study patients suffering from recurrent bacterial, viral and Cryptosporidium infections, and identify a biallelic mutation in the MAP3K14 gene encoding NIK (NF-κB-inducing kinase). Loss of kinase activity of mutant NIK, predicted by in silico analysis and confirmed by functional assays, leads to defective activation of both canonical and non-canonical NF-κB signalling. Patients with mutated NIK exhibit B-cell lymphopenia, decreased frequencies of class-switched memory B cells and hypogammaglobulinemia due to impaired B-cell survival, and impaired ICOSL expression. Although overall T-cell numbers are normal, both follicular helper and memory T cells are perturbed. Natural killer (NK) cells are decreased and exhibit defective activation, leading to impaired formation of NK-cell immunological synapses. Collectively, our data illustrate the non-redundant role for NIK in human immune responses, demonstrating that loss-of-function mutations in NIK can cause multiple aberrations of lymphoid immunity.
Primary immunodeficiency disorders can be used to identify key immune functions. Here, the authors identify a biallelic mutation in the gene encoding NF-κB-inducing kinase in a family suffering a range of infections, and show that it causes defects in NK and T-cell function and has broad effects on B-cell function.
PMCID: PMC4263125  PMID: 25406581
25.  Stereospecific targeting of MTH1 by (S)-crizotinib as anticancer strategy 
Nature  2014;508(7495):222-227.
Activated Ras GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small-molecules, such as SCH51344, but their molecular mechanism of action remains generally enigmatic. Here, using a chemical proteomic approach we identify the target of SCH51344 as the human mutT homologue MTH1, a nucleotide pool sanitising enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells whereas MTH1 overexpression mitigated sensitivity toward SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys, and MTH1 co-crystal structures of both enantiomers provided a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation and small molecule inhibitors of MTH1 in general as a promising novel class of anti-cancer agents.
PMCID: PMC4150021  PMID: 24695225
DNA repair; stereoselectivity; drug; MTH1; crizotinib; cancer

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