A multifaceted approach to investigate signal transduction at the receptor and post-receptor level focusing on the modulation of the free fatty acid receptor family

Knowledge about the function and dysfunction of cellular signal transduction is at the very heart of biomedical research since every physiological and pathophysiological process is intimately associated with adequate or defective signaling. The advent of innovative methodological techniques paved the way for major breakthroughs in deciphering cell signaling in the life sciences. Granting this, it is not technology per se but the proactive adjustment and attentive implementation of the various methodological approaches that lay the foundation for rewarding research efforts. It is becoming increasingly clear that data obtained from a single assay readout are generally insufficient to mirror the multidimensionality of biological signaling networks. Thus it was an aim of this study by synopsis of different technological, biochemical and pharmacological approaches to provide new insights into the anatomy of signaling events. G protein-coupled receptors (GPCRs) do not only represent the largest group of drug targets but are also prototypic as microprocessing units in the cellular signaling machinery and therefore ideally suited to study the biology of signal transduction. This class of receptors represent a cornucopia of signaling diversity since a plethora of signal transduction concepts are mirrored herein. Hence, GPCRs are supremely favored research objects and examined in the majority of the publications communicated in this thesis. Section I explained the principle of dynamic mass redistribution (DMR). The optical biosensor-based technique allows the detection of cellular events as an integrated whole cell readout and its label-free nature is capable to reflect signaling events in a non-invasive fashion without using any interfering labels. A set of different cellular backgrounds was used to demonstrate the versatility and applicability of the assay. The integration of recombinant, native and primary cells highlighted the potential of this technique to glean in vivo relevant biological information about investigated substances already in early stages of the drug discovery process. Since DMR provides unbiased insights into cell activation a testimony of multiple concurrent pathways triggered by a stimulus is accessible and helps to understand the underlying biology. Moreover, the first chapter described the possibility to untangle the GPCR signaling repertoire by means of selective pathway modulators and explained why the real time measuring mode allows the exploration of kinetic aspects in the cell response. Chapter 2 opens section II of this thesis, in which the biology of the free fatty acid receptor FFA2 is detailed. With a multifaceted approach exploiting the advantages of holistic label-free readouts it was possible to uncover an unprecedented mode-of-action of a GPCR modulating ligand. 4 CMTB previously classified as positive allosteric modulator and allosteric agonist (ago-PAM) at the FFA2 receptor was disclosed as the first sequentially activating ligand (SEAL) reported so far. This ligand binds and activates a first recognition site of the receptor and is then repositioned to another site, where a second signaling impulse is generated. The association of multiple binding events with multiple signaling events therein is a key difference between SEALs and classical bitopic ligands that simultaneously bind the receptor. This mechanism extends the possibility to encrypt biological information by a temporal dimension. In this concept, GPCRs could function as transceivers that de- and encode the information in an adjustable manner. Rationally correlating certain signaling events and the resulting phenotype within a complex biological environment remains a prime challenge in signal transduction research. The invention of designer receptors bettered the prospects to approach this question. By isolating a certain receptor signaling behavior and introduce this signaling machinery into an in vivo context scientists might be able to assess the relevance of a certain activation pattern for the individual phenotype. Chapter 3 reports on a strategy how to rationally design and generate a designer FFA2 receptor. A receptor activated solely by a synthetic ligand (RASSL) form of FFA2 was found based on species differences in agonist selectivity between the human and bovine FFA2 receptor and consequently did not respond to the endogenous short-chain fatty acid ligands but was selectively activated by stimulation with sorbic acid. Future studies involving designer receptors can help elucidating the in vivo role of the FFA2 receptor, since the ubiquitous availability of fatty acids in the body and overlapping ligand recognition pattern of FFA2 and FFA3 challenged a clear definition of FFA2 mediated phenotypes. Section III deals with the human free fatty acid receptor FFA1 and is introduced by Chapter 4 that presents a study that investigated the interplay between FFA1 and non-esterified fatty acids (NEFAs) in the disease context of diabetes type 2 (TD2). The deleterious effects of NEFAs on pancreatic beta cell function and survival were shown to be dependent of FFA1 activation, a hypothesis that has been claimed by previous reports. In the present study, however, the activation of FFA1 with a selective small molecule agonist proved even beneficial, while antagonists deteriorated β-cell health, a finding that is also corroborated by other studies. A single nuclear polymorphisms (SNP) in the gene of the FFA1 was identified to modulate the sensitivity against (gluco)lipotoxic effects of fasting levels of non-esterified fatty acid (NEFA) level in humans confirming a modulatory role of FFA1 in the pathophysiology of TD2. The second publication in chapter 4 focused again on a methodological issue and notes the importance to diligently validate the experimental tools used to determine localization and amount of protein expression, a prerequisite of reliable data acquisition. In doing so, several inconsistent results regarding the study of FFA1 receptor biology were questioned. In Chapter 5 we finally reported on the chemistry and optimization of synthetic FFA1 ligands. Because of the aim to develop highly selective and potent FFA1 agonists, the unbiased DMR assay proofed valuable to detect any off-target effects of the investigated compounds, as well as confirming their competence to activate the receptor with appropriate potency and efficacy. The three publications within this chapter present a strategic chemical approach to tailor the structures for their employment as pharmacologically validated substances. The availability of those ligands is inevitable to study receptor biology and was thus a prerequisite to conduct research as depicted in other chapters. In the last part of the thesis, section IV, two chapters describe the analysis of signal transduction at the post-receptor level. Chapter 6 provides further evidence for a bona fide second messenger role of the non-canonical cyclic nucleotides cCMP and cUMP by delineating the biological effectors. The label-free DMR assay disclosed cNMP target proteins and shed light into the signal transduction network of cNMPs in human cells, thereby emphasizing the applicability of this technique that is not restricted to the analysis of signal transduction generated from cell surface receptors but also initiated at the post-receptor level. Chapter 7, finally, characterized and defined the mode-of-action of a previously published pan-G protein inhibitor (BIM46187). By stabilizing the empty-pocket conformation of Gα subunits, which are in the activation process, BIM46187 is the first reported GTP entry inhibitor and was furthermore redefined as a compound that selectively targets Gαq proteins in a cell-type dependent manner. In conclusion, this thesis comprises several studies covering multiple aspects of cellular signal transduction providing a range from biological-pharmacological, technological and chemical perspectives on the subject. The bulk of the publications deals with the analysis of GPCR biology, especially the exploration of the free fatty acid receptor FFA1 and FFA2. These are complemented by a technology-centered, conceptual section and a section analyzing signal transduction at the post-receptor level

Transparent Flexible Thermoelectric Material Based on Non-toxic Earth-Abundant p-Type Copper Iodide Thin Film

Thermoelectric devices that are flexible and optically transparent hold unique promise for future electronics. However, development of 'invisible' thermoelectric elements is hindered by the lack of p-type transparent thermoelectric materials. Here we present the superior room-temperature thermoelectric performance of p-type transparent copper iodide (CuI) thin films deposited by industrially applicable room-temperature sputtering techniques. The optical transmittance is 60–85% in the visible and near-infrared regions. Large Seebeck coefficients and power factors of the obtained CuI thin films are analyzed based on a single-band model. The low thermal conductivity of the CuI films is attributed to a combined effect of the heavy element iodine and strong phonon scattering. Accordingly, we achieve a large thermoelectric figure of merit of ZT = 0.21 at 300 K for the CuI films, which is three orders of magnitude higher compared with state-of-the-art p-type transparent materials. A transparent and flexible CuI-based thermoelectric element is demonstrated

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq

Program FFlexCom — High frequency flexible bendable electronics for wireless communication systems

Today, electronics are implemented on rigid substrates. However, many objects in daily-life are not rigid — they are bendable, stretchable and even foldable. Examples are paper, tapes, our body, our skin and textiles. Until today there is a big gap between electronics and bendable daily-life items. Concerning this matter, the DFG Priority Program FFlexCom aims at paving the way for a novel research area: Wireless communication systems fully integrated on an ultra-thin, bendable and flexible piece of plastic or paper. The Program encompasses 13 projects led by 25 professors. By flexibility we refer to mechanical flexibility, which can come in flavors of bendability, foldability and, stretchability. In the last years the speed of flexible devices has massively been improved. However, to enable functional flexible systems and operation frequencies up to the sub-GHz range, the speed of flexible devices must still be increased by several orders of magnitude requiring novel system and circuit architectures, component concepts, technologies and materials

A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be “frozen” pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors

microbeMASST: A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms’ role in ecology and human health

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms’ role in ecology and human health

Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators

The physiological function of free fatty acids (FFAs) has long been regarded as indirect in terms of their activities as educts and products in metabolic pathways. The observation that FFAs can also act as signaling molecules at FFA receptors (FFARs), a family of G protein-coupled receptors (GPCRs), has changed the understanding of the interplay of metabolites and host responses. Free fatty acids of different chain lengths and saturation statuses activate FFARs as endogenous agonists via binding at the orthosteric receptor site. After FFAR deorphanization, researchers from the pharmaceutical industry as well as academia have identified several ligands targeting allosteric sites of FFARs with the aim of developing drugs to treat various diseases such as metabolic, (auto)inflammatory, infectious, endocrinological, cardiovascular, and renal disorders. GPCRs are the largest group of transmembrane proteins and constitute the most successful drug targets in medical history. To leverage the rich biology of this target class, the drug industry seeks alternative approaches to address GPCR signaling. Allosteric GPCR ligands are recognized as attractive modalities because of their auspicious pharmacological profiles compared to orthosteric ligands. While the majority of marketed GPCR drugs interact exclusively with the orthosteric binding site, allosteric mechanisms in GPCR biology stay medically underexploited, with only several allosteric ligands currently approved. This review summarizes the current knowledge on the biology of FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120), and GPR84, including structural aspects of FFAR1, and discusses the molecular pharmacology of FFAR allosteric ligands as well as the opportunities and challenges in research from the perspective of drug discovery

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13.

Frizzleds (FZDs) are unconventional G protein-coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD-G protein coupling remain poorly understood. Previously, we showed that FZD6 assembles with Gαi1/Gαq (but not with Gαs, Gαo and Ga12/13), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD4, a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD4 assembles-in a DVL-independent manner-with Gα12/13 but not representatives of other heterotrimeric G protein subfamilies, such as Gαi1, Gαo, Gαs, and Gαq The FZD4-G protein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD4 green fluorescent protein-transfected cells depend on Gα12/13 Furthermore, expression of FZD4 and Gα12 or Gα13 in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of Gα12/13 signaling, underlining the functionality of an FZD4-Gα12/13-RHO signaling axis. In summary, Gα12/13-mediated WNT/FZD4 signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD4 signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development