881 research outputs found
Correction to: When terminology hinders research: the colloquialisms of transitions of control in automated driving (Cognition, Technology & Work, (2022), 10.1007/s10111-022-00705-3)
No full textIn the original article, author affiliation published with error. The correct affiliations are: Davide Maggi—Institute for Transport Studies, Leeds, UK. Richard Romano—Institute for Transport Studies, Leeds, UK. Oliver Carsten—Institute for Transport Studies, Leeds, UK. Joost C. F. De Winter—Faculty of Mechanical, Maritime and Materials Engineering, Delft University of Technology, Delft, The Netherlands. The original article has been corrected.Human-Robot Interactio
Entry mechanism of natural killer cell derived granzyme B into target cells
No full textDuring elimination of virally infected or malignant cells, NK cells release lytic granules. These contain perforin and granzyme B (GrzB), which synergistically trigger apoptosis of affected cells. However, despite intensive research, the mechanism by which GrzB traverses membrane barriers to gain access to the target cell cytosol is controversial. Two main entry models have been described: I) perforin pores in the plasma membrane (PM) act as passageways for GrzB to allow direct diffusion into the cytosol or II) target cells internalize GrzB via endocytosis followed by a release from endosomes. Yet, mutual exclusivity is not necessarily a given; both pathways could also support each other. To investigate how GrzB enters target cells during NK cell-mediated cytotoxicity, we developed fluorescent localization reporters that can detect GrzB activity in living cells. We can localize these reporters at the PM, the endoplasmic reticulum (ER), or other intracellular compartments of target cells. Therefore, it becomes possible to assess the GrzB entry site over time using live cell imaging. Using the NK cell line NK92 or different human primary NK cells as effectors and HeLa cells as targets, we could observe a predominant GrzB activity at the PM of target cells; but low GrzB activity was also detected at the ER. This could indicate either a mutual supportive role of both pathways or diffusion of GrzB from the PM to the ER. However, we did not observe GrzB activity at mitochondria arguing against general diffusion of GrzB. By reducing the amount of active perforin with concanamycin A (CMA), we were able to show that the preferential entry through PM pores is independent of the available amount of perforin. However, in the absence of perforin, no GrzB activity was detectable in target cells, confirming the essential role of perforin for GrzB to enter target cells. Taken together, our results demonstrate that during NK cell-mediated killing GrzB enters HeLa directly via the PM. However, we found differences between target cells. K562 were almost exclusively killed without GrzB, while we found rare events in MDA-MB #468 cells where GrzB was predominantly delivered via the endocytic pathway, which may be enhanced by altering membrane tension
The activating receptors 2B4 and NTB-A, but not CRACC are subject to ligand-induced down-regulation on human natural killer cells
No full textActivation of natural killer cells can be mediated by different receptors. Stimulation of the receptors 2B4, NTB-A and CRACC, members of the SLAM-related receptor family, induces cytotoxicity and cytokine production. The surface expression of 2B4 and other activating natural killer cell receptors is down-modulated after receptor engagement, which results in a weaker response to consecutive stimulation. We tested whether this regulatory mechanism applies to all SLAM-related receptors expressed by primary human natural killer cells. After co-culture with target cells expressing the respective ligands different effects on receptor surface expression were observed. While 2B4 ex-pression was strongly reduced, NTB-A showed less prominent down-modulation and the expression level of CRACC remained unchanged. The expression levels of the receptor-proximal signaling molecules SAP, EAT-2 and FynT did not change after receptor engagement. Co-culture with target cells expressing the ligands for NTB-A or CRACC had no impact on subsequent NTB-A or CRACC-mediated NK cell activation
Identification and characterization of small-molecule modulators that promote cancer cell elimination
No full textIn this doctoral thesis, two strategies were used to fight cancer. The first strategy aimed to eliminate cancer via natural killer (NK) cells. Within the tumor microenvironment (TME), cancer cells and the cancer-supporting tissue hijack immunosuppressive pathways, leading to the increased secretion of immune inhibitory factors that aid tumor progression, ultimately leading to the evasion of NK-cell mediated eradication of cancer cells. A phenotypic co-culture assay was optimized to mimic the TME. The assay consists of a human lung adenocarcinoma cells A549, primary lymphocytes, and immunomodulatory factors. This setup was used to screen a library of 29,502 small molecules in search of compounds that can restore NK cell-mediated cancer cell elimination. The second strategy was focused on eliminating cancer through indirect targeting of the small GTPase Kirsten rat sarcoma (KRas). KRas is mutated in approximately 17 % of all solid tumors, and current FDA-approved drugs solely target its G12C mutant. The activity of this oncoprotein depends on its localization to the plasma membrane, and within the cell, its translocation within the cell is facilitated by its interaction with the prenyl group-binding chaperone retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ). Therefore, instead of directly targeting KRas, the covalent inhibitor Deltacovalin was designed to target PDEδ.Im Rahmen dieser Doktorarbeit wurden zwei Strategien zur Bekämpfung von Krebs verwendet. Der erste Ansatz zielte darauf ab, Krebs durch NK Zellen zu eliminieren. In der Tumormikroumgebung (TMU), nutzen Krebszellen und das umgebende unterstützende Gewebe immuninhibierende Signalwege, die das Wachstum von Tumoren fördern und dadurch die NK Zell-vermittelte Eliminierung von Krebszellen inhibieren. Um eine Substanzbibliothek bestehend aus 29,502 Substanzen zu testen wurde ein phänotypisches TMU-nachahmendes Testverfahren bestehend aus der humanen Lungenkrebszelllinie A549, primären Lymphozyten und immunmodulierenden Faktoren optimiert. Ziel dieses Testverfahrens war es Verbindungen zu identifizieren, die die NK Zell-vermittelte Krebszelleliminierung begünstigt. Im Fokus der zweiten Strategie lag die Bekämpfung von Krebs über die kleine GTPase KRas, die in etwa 17 % aller Tumoren mutiert ist. Gegenwärtig sind die von der FDA zugelassenen Medikamente ausschließlich auf die G12C Mutante fokussiert. Die Aktivität von KRas wird durch seine Lokalisierung an der Plasmamembran ermöglicht. Für die Translokation von KRas innerhalb der Zelle ist die Interaktion mit PDEδ essentiell. Daher wurde eine kovalente Substanz mit dem Namen Deltacovalin entwickelt, um die Aktivität von KRas indirekt über die Inhibierung des Interaktionspartners PDEδ zu hemmen
CRISPR-based screening & functional characterization of long non-coding RNAs in melanoma
No full textMelanoma, the most lethal form of skin cancer, is increasingly prevalent in Western populations and is characterized by its high metastatic potential. This cancer type is distinguished by a significant accumulation of somatic mutations, primarily due to UV radiation, leading to a mutation rate that exceeds that of most other solid tumors. The progression of melanoma involves the uncontrolled proliferation and spread of malignant melanocytes, with notable disruptions in the MAPK-ERK and PI3K-AKT-mTOR signalling pathways contributing to the development of advanced therapeutic strategies. Recent research has highlighted the importance of the non-coding regions of the genome, previously considered "junk DNA", for their regulatory functions. Long non-coding RNAs (lncRNAs), which are typically longer than 200 nucleotides, have been identified as key players in cellular development, differentiation, and cancer progression. Numerous studies have established a link between lncRNAs and the growth and progression of melanoma, with elevated lncRNA expression levels observed in melanoma cases. Herein, the present study attempted to elucidate the involvement of a specific set of lncRNAs in mechanisms underlying cell growth and proliferation. These lncRNAs, which had been observed to exhibit increased expression in melanoma cell lines and in short-term cultures derived from brain and lymph node metastasis, are examined by employing CRISPRi screening. Furthermore, functional characterization analyses were conducted on the most promising lncRNA candidates, including BDNF-AS, GMDS-AS1, and a novel, non-annotated lncRNA named XLOC030781 based on preliminary data that validate their importance in melanoma. Suppression of these lncRNAs led to apoptosis and inhibited cell cycle progression, with XLOC030781 playing a notable role in melanoma migration, broadening its functional scope in relation to this malignancy. Finally, the implementation of fluorescence in situ hybridization technique provided data on their subcellular localization, offering complementary information on their functionality
Kristallisation und strukturbiologische Charakterisierung klinisch-relevanter Mutationsvarianten der Rezeptor-Tyrosinkinasen EGFR und Her2
No full textLungenkrebs ist die häufigste Ursache für krebsbedingte Todesfälle weltweit.[1] In den letzten Jahren hat jedoch die sogenannte Präzisionsmedizin die Krebstherapie von Nicht-kleinzelligen Lungenkrebs-Patienten (NSCLC-Patienten) revolutioniert. Die Identifizierung von prädikativen Biomarkern sowie das detaillierte genetische Verständnis ermöglichte die Entwicklung niedermolekularer Verbindungen zur gezielten Inhibierung der aberranten Zielstrukturen genetisch definierter Patientengruppen. Durch die gezielte Adressierung der onkogenen Zielproteine können die Nebenwirkungen verringert werden, wodurch die Präzisionsmedizin eine vielversprechende Alternative zur platinbasierten Chemotherapie darstellt.[2,3]
Genetische Mutationen, die zur Entstehung und zur Progression von NSCLC führen, sind häufig in den Rezeptor-Tyrosinkinasen zu finden. Hierbei sind im Rahmen dieser Arbeit vor allem der epidermale Wachstumsfaktor-Rezeptor (EGFR) sowie der humane epidermale Wachstumsfaktor-Rezeptor 2 (Her2) zu nennen.[4,5] Die Entstehung von Resistenzmutationen innerhalb der Kinase-Domäne von EGFR, während der Behandlung mit zielgerichteten Tyrosinkinase-Inhibitoren, erfordert das stetige Aufklären der Resistenzmechanismen sowie die Entwicklung neuer Wirkstoffe.[6] Daher ist ein detailliertes Verständnis der Mutanten sowie der Protein-Inhibitor-Interaktionen auf molekularer Ebene für die Entwicklung neuartiger Verbindungen erforderlich.
Die Proteinkristallographie stellt eine wichtige Methode zur Strukturaufklärung dar. [7,8] Allerdings sind bis zur finalen Kristallstruktur viele Herausforderungen wie das Konstruktdesign, die Proteinexpression in unterschiedlichen Expressionssystemen sowie die Proteinreinigung und die Identifizierung geeigneter Kristallisation-bedingungen zu meistern.
Nach Optimierung der genannten Arbeitsschritte konnten im Rahmen dieser Arbeit verschiedene Kristallisationssysteme Krebs-relevanter EGFR-Varianten etabliert werden, mit deren Hilfe ein verlässliches Wachstum qualitativ hochwertiger Proteinkristalle in Komplex mit niedermolekularen Verbindungen für die Strukturaufklärung ermöglicht werden konnte. Neben dem Interaktionsnetzwerk der Verbindungen und es Zielproteins konnten wichtige strukturelle Einblicke für weitere strukturbasierte Designansätze neuartiger Inhibitoren sowie Optimierungen bereits vorliegende Inhibitoren identifiziert werden.
Im Rahmen dieser Arbeit konnten insgesamt 47 finale Kristallstrukturen der EGFR-Mutationsvarianten generiert werden, von denen bisher zwölf in der Protein Data Bank publiziert wurden.
[1] F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre, A. Jemal. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. Ca: Cancer J. Clin. 2018, 68, 394-424. [2] A. Thomas, S. V. Liu, D. S. Subramaniam, G. Giaccone. Refining the Treatment of NSCLC According to Histological and Molecular Subtypes. Nat. Rev. Clin. Oncol. 2015, 12, 511-526. [3] S. O. Dolly, D. C. Collins, R. Sundar, S. Popat, T. A. Yap. Advances in the Development of Molecularly Targeted Agents in Non-Small-Cell Lung Cancer. Drugs 2017, 77, 813-827. [4] S. V. Sharma, D. W. Bell, J. Settleman, D. A. Haber. Epidermal Growth Factor Receptor Mutations in Lung Cancer. Nat. Rev. Cancer 2007, 7, 169-181. [5] J. Mendelsohn, J. Baselga. The EGF Receptor Family as Targets for Cancer Therapy. Oncogene 2000, 19, 6550-6565. [6] J. Lategahn, M. Keul, D. Rauh. Lessons To Be Learned: The Molecular Basis of Kinase-Targeted Therapies and Drug Resistance in Non-Small Cell Lung Cancer. Angew. Chem., Int. Ed. 2018, 57, 2307-2313. [7] M. W. Parker. Protein Structure from X-Ray Diffraction. J. Biol. Phys. 2003, 29, 341-362. [8] Z. Sayers, B. Avsar, E. Cholak, I. Karmous. Application of Advanced X-Ray Methods in Life Sciences. Biochim. Biophys. Acta. Gen. Subj. 2017, 1861, 3671-3685
Identification of small molecule inhibitors of the kynurenine pathway
No full textThe immune system functions as an effective barrier against tumor development. However, malignant cells can avoid elimination by the immune system by acquiring certain characteristics that alter the immune system in the tumor microenvironment (TME). For instance, cancer cells can actively induce immune tolerance by expression of the immuno-suppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 expression results in degradation of the essential amino acid tryptophan (Trp) and production of kynurenines (Kyn) via the Kyn pathway. Targeting the Kyn pathway has emerged to be an attractive target in immuno-oncology.
In this thesis, two compound classes were discovered through a cell-based screening that reduce Kyn levels in cancer cells upon stimulation with the cytokine interferon-γ (IFN-γ) by two distinct mechanisms. The indole-tetrahydropyridine pseudo-natural products (PNPs) called apoxidoles inhibit IDO1 directly by binding and stabilizing apo-IDO1 in vitro and in cells. The displacement of the IDO1 cofactor heme catalytically inactivates the enzyme and thereby decreases Kyn levels. The second small molecule class of N-substituted indoles called epoxykynins represents an alternative approach to modulate the Kyn pathway by not directly targeting IDO1. Instead, epoxykynin inhibits the catalytic activity of the C-terminal fatty acid hydrolase domain of the soluble epoxide hydrolase (sEH-H). As part of the arachidonic acid (AA) cascade, the highly active enzyme sEH-H plays an important role in the hydrolysis of CYP epoxygenase-derived fatty acid epoxides. Thereby, it contributes to the regulation of bioavailable epoxides and controls a variety of biological processes, such as inflammation, vasodilation, angiogenesis, neuropsychiatries and pain. Thereby, it contributes to the regulation of bioavailable epoxides and controls a variety of biological processes, such as inflammation, vasodilation, angiogenesis, neuropsychiatries and pain. The results obtained throughout this thesis uncover a cross-talk between sEH-H and the Kyn pathway.
The discovery of new Kyn pathway inhibitors is in high demand, since the most advanced holo-IDO1 inhibitor epacadostat has recently failed in clinical trials. Additionally, the identification of epoxykynins has deepened the understanding of the functional link between the Kyn pathway and the AA metabolism. These findings might enable novel strategies to design immunotherapies and enhance the host’s immune system to overcome cancer-induced immune tolerance
β2 adrenergic receptor desensitization through chronic stimulation of Natural Killer cells
No full textStress is a ubiquitous phenomenon that impacts the health of populations across all social strata. While acute stress can be beneficial, chronic stress elevates the risk for various diseases. A major part of the body’s stress response operates through the Sympathetic-Adrenal-Medullary (SAM) axis, leading to the release of epinephrine. This stress mediator can impact the immune system and alter the immune defense response. Natural Killer (NK) cells play a critical role in early immune responses, defending against pathogens and malignant cells. During stressful situations, NK cells are recruited to the blood circulation within minutes. The expression of adrenergic receptors, particularly β2 adrenergic receptors (β2AR), renders them sensitive to epinephrine.
This study analyzed the response of NK cells to acute and chronic β2AR stimulation. We confirmed that acute β2AR stimulation inhibits NK cell functions in vitro. Epinephrine showed potent inhibitory effects, suppressing the activation of NK cells independent of the activation signal. The β2AR stimulation reduced NK cell adhesion, IFNγ secretion, degranulation, and cytotoxicity. We could show that β2AR stimulation effectively blocked the LFA-1 activity and led to the detachment from its ligand ICAM-1. The effect was rescued through the addition of Propranolol, a beta blocker, or ADRB2 knock out implicating LFA-1 inhibition as critical role for NK cell mobilization. The metabolic analysis revealed that β2AR agonists influenced the metabolic profile upon NK cell activation, leading to a prolonged glycolysis activity displayed by Seahorse ECAR values.
In contrast, chronic β2AR stimulation nullified NK cell inhibition. After five days of β2AR treatment, NK cells were no longer responsive to a β2AR agonist. Chronic β2AR stimulation did not alter protein translation but led to receptor phosphorylation through the PKA feedback loop, initiating a G-protein switch and receptor desensitization. The β2AR treatment with long-acting β2 agonist (LABA) Indacaterol and epinephrine displayed different properties. While epinephrine inhibited NK cells only transiently as long as the β2AR agonist was abundant, Indacaterol could not be washed away and continuously stimulated the receptor. For this reason, a single LABA treatment was sufficient to induce NK cell desensitization.
However, peripheral NK cells from LABA-treated patients remained responsive to epinephrine. They did not exhibit inhibition but showed an overall correlation in NK cell fitness with asthma severity.
Taken together, the transient, inhibitory effect of epinephrine indicate that the hormone plays a crucial role in NK cell recruitment during acute stressful situations while repeated β2AR stimulation leads to desensitization
Regulation of natural killer cell cytotoxic pathways during serial killing activity
No full textNatural Killer (NK) cells act as the front line of the body´s defense in the immune system and they are key players in efficiently recognizing and eliminating virus-infected and tumor cells. Individual NK cells can eliminate multiple target cells in a sequential manner during a process named serial killing. There are two major pathways NK cells use to induce apoptosis in their target cells: By releasing the content of cytotoxic granules containing the serine protease granzyme B (GrzB) and the pore-forming protein perforin or by the engagement of death receptors, which initiates caspase cascades via Caspase-8 (Casp8). The contribution of both cell death processes to NK cell cytotoxicity and serial killing remains poorly understood. Therefore, investigating the interplay between these pathways in more detail is important for a better understanding of NK cell cytotoxicity. To visualize the GrzB and death receptor-mediated target cell death in a time-dependent manner, we used fluorescent localization reporters that enabled us to simultaneously measure the activities of GrzB and of Casp8 in target cells upon contact with NK cells by life cell imaging. In this study we observed that NK cells kill their initial targets via the fast GrzB-induced pathway and switch to a slow death receptor-mediated killing for the final target. During the target cell contact NK cells lost GrzB and perforin, whereas the expression of CD95L, a main death cell ligand, was increased on the NK cell surface. The reduction of the lytic granules can be efficiently restored by the stimulation with different cytokines such as IL- 15, IL-2 or IL-21. Perforin deficient NK cells or ILC3 cells were unable to perform GrzB-mediated killing and no serial killing could be mediated without this pathway. In contrast, the absence of death receptor CD95 on the target cell and/or the absence of death receptor ligands on the NK cell had no direct influence on the GrzB-mediated serial killing. This demonstrates that the use of GrzB vs. death receptor-mediated target cell killing is differentially regulated during the serial killing activity of NK cells. Taken together, we observed a rapid target cell death which was induced by GrzB and originated from early established NK : target contacts. In contrast, cell death mediated by Casp-8 was a result of later target cell engagements and took much longer from NK : target cell contact to target cell death
- …
