CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

The dysregulated expression of immune checkpoint molecules enables cancer cells to evade immune destruction. While blockade of inhibitory immune checkpoints like PD-L1 forms the basis of current cancer immunotherapies, a deficiency in costimulatory signals can render these therapies futile. CD58, a costimulatory ligand, plays a crucial role in antitumor immune responses, but the mechanisms controlling its expression remain unclear. Using two systematic approaches, we reveal that CMTM6 positively regulates CD58 expression. Notably, CMTM6 interacts with both CD58 and PD-L1, maintaining the expression of these two immune checkpoint ligands with opposing functions. Functionally, the presence of CMTM6 and CD58 on tumor cells significantly affects T cell-tumor interactions and response to PD-L1-PD-1 blockade. Collectively, these findings provide fundamental insights into CD58 regulation, uncover a shared regulator of stimulatory and inhibitory immune checkpoints, and highlight the importance of tumor-intrinsic CMTM6 and CD58 expression in antitumor immune responses

The cohesin acetylation cycle controls chromatin loop length through a PDS5A brake mechanism

Cohesin structures the genome through the formation of chromatin loops and by holding together the sister chromatids. The acetylation of cohesin’s SMC3 subunit is a dynamic process that involves the acetyltransferase ESCO1 and deacetylase HDAC8. Here we show that this cohesin acetylation cycle controls the three-dimensional genome in human cells. ESCO1 restricts the length of chromatin loops, and of architectural stripes emanating from CTCF sites. HDAC8 conversely promotes the extension of such loops and stripes. This role in controlling loop length turns out to be distinct from the canonical role of cohesin acetylation that protects against WAPL-mediated DNA release. We reveal that acetylation controls the interaction of cohesin with PDS5A to restrict chromatin loop length. Our data support a model in which this PDS5A-bound state acts as a brake that enables the pausing and restart of loop enlargement. The cohesin acetylation cycle hereby provides punctuation in the process of genome folding

Identification and molecular characterization of inhibitors of necroptosis

Das Gleichgewicht zwischen Zellproliferation und Zelltod ist von entscheidender Bedeutung für die Homöostase multizellulärer Organismen. Die Beseitigung obsolet gewordener, geschädigter oder infizierter Zellen kann durch genetisch kodierte Zelltodprogramme bewerkstelligt werden. Es gibt mehrere regulierte Zelltod-Signalwege, und deren Störung kann zur Entstehung von Krankheitspathologie und Gewebeschäden führen. Nekroptose ist eine entzündungsfördernde, programmierte Form nekrotischen Zelltods und trägt zur Pathophysiologie verschiedener menschlicher Erkrankungen, unter anderem Herzinfarkt, Schlaganfall und Arteriosklerose, bei. Nekroptotischer Zelltod kann durch Ligandenbindung an Toll-like- oder Todesrezeptoren, bakterielle und virale Infektionen, sowie im Rahmen steriler Entzündungen ausgelöst werden. Die Signaltransduktion erfolgt über die Proteine RIPK3 und MLKL, wobei die Kinase RIPK3 durch eines der vorgeschalteten Adapterproteine RIPK1, TRIF oder DAI aktiviert wird. Während Nekroptose im Rahmen der angeborenen Immunantwort gegen intrazelluläre Erreger einen vorteilhaften Effekt hat, verstärkt sie die schädlichen Auswirkungen steriler Entzündungen. Die Eingrenzung pathologischer Nekrose durch pharmakologische Nekroptose-Inhibition stellt somit eine vielversprechende Behandlungsstrategie dar, jedoch sind gegenwärtig keine Nekroptose-Inhibitoren für den klinischen Gebrauch verfügbar. Ich setzte mir daher zum Ziel, pharmakologische Wirkstoffe zu identifizieren, welche den nekroptotischen Zelltod blockieren. Im Zuge meiner Doktorarbeit habe ich eine repräsentative Auswahl zugelassener, und damit bezüglich Pharmakokinetik und Sicherheit erprobter, Arzneimittel auf Nekroptose-Inhibition getestet. Ich zeige, dass die beiden in der Krebstherapie eingesetzten Kinase-Inhibitoren Ponatinib und Pazopanib den nekroptotischen Zelltod spezifisch und mit hoher Wirksamkeit blockieren. Um den zugrundeliegenden molekularen Mechanismus aufzuklären, identifizierte ich die zellulären Angriffspunkte von Ponatinib mittels chemischer Proteomik und liefere damit die erste umfassende Aufstellung der molekularen Wirkorte dieses Medikaments. Bemerkenswerterweise finden sich die wichtigsten Proteine des Nekroptose-Signaltransduktionswegs unter den Wirkstoffzielen. Mittels verschiedenere biochemischer Tests bestätige ich, dass Ponatinib sowohl RIPK1 als auch RIPK3 bindet und hemmt, während Pazopanib bevorzugt RIPK1 zum Angriffspunkt hat. Ponatinib ist somit der erste beschriebene duale RIPK1/RIPK3 Inhibitor. Da die detaillierte Beschreibung des Nekroptose-assoziierten Protein-Netzwerks die Identifizierung zusätzlicher Angriffspunkte für die gezielte Modulation und therapeutische Intervention erwarten lässt, setzte ich mir des Weiteren zum Ziel, die Voraussetzungen für die Massenspektrometrie-basierte Kartierung des Nekroptose-Signaltransduktionswegs zu schaffen. Zu diesem Zweck wurde ein induzierbares, retrovirales Expressionssystem, welches sich für Protein-Interaktionsstudien auf Basis von Affinitätsreinigung im Zweischritt-Verfahren eignet und im jeweils physiologisch relevanten zellulären Zusammenhang angewendet werden kann, etabliert. Das System eignet sich weiter für die Analyse von Proteinen deren Expression zytotoxische Effekte hervorruft, wie ich anhand einer konstitutiv aktiven Form von MLKL demonstriere. Im Rahmen dieser Experimente entdeckte und charakterisierte ich die Bedeutung des molekularen Chaperon HSP90 für die Stabilität und Funktionalität von MLKL. Zusammengenommen identifiziere ich in der vorliegenden Arbeit neue Nekroptose-Inhibitoren unter bereits zugelassenen sowie derzeit in klinischer Testung befindlichen Medikamenten und beschreibe den jeweils zugrundeliegenden molekularen Wirkmechanismus. Diese Ergebnisse schaffen somit eine konsolidierte chemische Basis für die Entwicklung klinisch relevanter Inhibitoren des nekroptotischen Zelltods.The balance between cell proliferation and cell death is crucial for the homeostasis of multicellular organisms. Cells that have become obsolete, damaged or infected can be eliminated by genetically encoded death programs. Multiple regulated cell death pathways exist, and disease pathologies and tissue damage ensue when these processes are disturbed. Necroptosis is a pro-inflammatory form of programmed necrotic cell death that has been shown to be involved in the pathophysiology of several human diseases, including myocardial infarction, stroke, and atherosclerosis. Necroptotic cell death depends on receptor interacting serine/threonine kinase (RIPK)3 and its substrate mixed-lineage kinase domain-like (MLKL). RIPK3 activation is mediated by the upstream adaptor proteins RIPK1, TIR domain-containing adapter inducing interferon-beta (TRIF) or DNA-dependent activator of interferon regulator factors (DAI) upon death receptor or Toll-like receptor (TLR) stimulation, bacterial or viral infection, and in the context of sterile inflammation. Necroptotic cell death has a beneficial role in innate immune responses against intracellular pathogens. In contrast, it exacerbates sterile-injury induced inflammation. Limiting pathological necrosis by interfering with programmed necrotic cell death thus represents a promising therapeutic strategy. However, no necroptosis inhibitors are currently available for clinical use. For this reason, I aimed at identifying pharmacological agents that block necroptotic cell death. In the course of this thesis, I performed a phenotypic screen for necroptosis inhibitors on a representative panel of drugs with Food and Drug Administration (FDA)-approval, implying established pharmacokinetic and safety profiles. I identified the kinase inhibitors ponatinib and pazopanib, both used as anti-cancer therapeutics, to potently and specifically block necroptotic cell death. In order to unravel the precise molecular mode of action effectuating necroptosis inhibition, I chose to apply an unbiased chemical proteomics-based target deconvolution approach to ponatinib. This study provides the first comprehensive description of the cellular target spectrum of this clinically used drug, notably revealing key members of the necroptosis signaling pathway. By a series of complementary, biochemical assays, I showed that pazopanib preferentially targets RIPK1, whereas ponatinib directly binds and blocks both RIPK1 and RIPK3. This work thus establishes ponatinib as the first dual RIPK1/RIPK3 inhibitor. As detailed understanding of the protein network underlying necroptosis signaling is expected to reveal novel entry points for targeted modulation and therapeutic intervention, I furthermore aimed at providing the basis for mass spectrometry (MS)-based mapping of the necroptosis pathway. To this end, an inducible, retroviral expression system enabling tandem affinity purification (TAP)-based interaction studies in the respective physiologically relevant cellular setting was established and characterized. I demonstrated the feasibility of studying cell death-inducing proteins using this vector system by a constitutively active mutant form of MLKL, leading to the identification of MLKL as a novel heat shock protein 90 (HSP90) client protein. Collectively, the work I present here has identified necroptosis inhibitors among compounds currently undergoing clinical development and already approved drugs, as well as unraveled their molecular mode of action. These findings thus provide a consolidated basis for medicinal chemistry approaches aimed at the development of anti-necroptosis agents for future clinical application in necroptosis-associated human diseases.submitted by Astrid FausterZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)171473

Interbacterial Macromolecular Transfer by the Campylobacter fetus subsp. venerealis Type IV Secretion System ▿

We report here the first demonstration of intra- and interspecies conjugative plasmid DNA transfer for Campylobacter fetus. Gene regions carried by a Campylobacter coli plasmid were identified that are sufficient for conjugative mobilization to Escherichia coli and C. fetus recipients. A broader functional range is predicted. Efficient DNA transfer involves the virB9 and virD4 genes of the type IV bacterial secretion system encoded by a pathogenicity island of C. fetus subsp. venerealis. Complementation of these phenotypes from expression constructions based on the promoter of the C. fetus surface antigen protein (sap) locus was temperature dependent, and a temperature regulation of the sap promoter was subsequently confirmed under laboratory conditions. Gene transfer was sensitive to surface or entry exclusion functions in potential recipient cells carrying IncPα plasmid RP4 implying functional relatedness to C. fetus proteins. The virB/virD4 locus is also known to be involved in bacterial invasion and killing of cultured human cells in vitro. Whether specifically secreted effector proteins contribute to host colonization and infection activities is currently unknown. Two putative effector proteins carrying an FIC domain conserved in a few bacterial type III and type IV secreted proteins of pathogens were analyzed for secretion by the C. fetus or heterologous conjugative systems. No evidence for interbacterial translocation of the Fic proteins was found

A conserved circular network of coregulated lipids modulates innate immune responses

SummaryLipid 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

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

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.ISSN:1535-9476ISSN:1535-948

The lipid-modifying enzyme SMPDL3B negatively regulates innate immunity

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

Glutaminyl cyclase is an enzymatic modifier of the CD47- SIRPα axis and a target for cancer immunotherapy

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells 1 . The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a ‘don’t eat me’ signal on tumor cells by binding to SIRPα expressed on myeloid cells 2–5 . Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer