The research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in... Show moreThe research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in depth characterization of lysosome function in health and disease. An integrative approach was used to study the physiological role of the lysosome, characterizing the function of lysosomal hydrolases and signalling on a cellular level as well as within the context of tissue. Show less
Lysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A... Show moreLysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A common LSD is Gaucher disease, which is characterized by a defect in glucocerebrosidase (GCase) degrading glucosylceramide (GlcCer) in lysosomes. In this thesis, the zebrafish is evaluated as vertebrate animal model for the investigation of lysosomal storage disorders, in particular Gaucher disease. Zebrafish are an appealing model organism to study genetic disorders with a high evolutionary conservation of genes and proteins compared to humans, easy maintenance and simple genetic and pharmacological manipulation. Zebrafish larvae are of particular use as zebrafish can generate hundreds of off-spring which have a rapid embryonal development, are transparent and fit in a 96-wells plate. In this thesis several biochemical and genetic techniques have been developed in order to 1) compare the catalytic features of zebrafish GCase with human GCase, 2) investigate the consequences of its defect in zebrafish larvae and adults as well as a concomitant defect in non-lysosomal GBA2 and 3) study the potential toxicity of excessive glucosylsphingosine during GCase deficiency as consequence of a defect in lysosomal acid ceramidase. GCase-deficient zebrafish showed similar symptoms and affected molecular mechanisms as patients and mouse models. Therefore the zebrafish offers exciting new possibilities to study molecular mechanisms underlying pathological processes during lysosomal hydrolase deficiencies. Show less
This thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an... Show moreThis thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an understudied modification—UFM1. While the first chapter describes the attempt to introduce an unnatural amino acid into proteins to enable covalent substrate capture, the subsequent chapter reports on a unique cascading activity-based probe capable of being relayed through the enzyme cascade. The repertoire of activity-based probes was later expanded to include the Ubiquitin-like modifiers SUMO and UFM1. Additionally, the enigmatic role of UFM1 was deciphered by adapting a proteomics method previously used for SUMO, to uncover UFM1-modifed substrates. This approach enabled the dissection of a relevant pathway governed by UFMylation—ribosomal function during SRP-mediated protein translocation. Show less
This thesis describes the use of an activity-based proteomics method to study the endocannabinoid system. A protocol for label-free chemical proteomics to measure serine hydrolase activity in mouse... Show moreThis thesis describes the use of an activity-based proteomics method to study the endocannabinoid system. A protocol for label-free chemical proteomics to measure serine hydrolase activity in mouse tissue is described. This method is used to compare a Niemann-Pick Type C mouse model to healthy mice. Additionally, several novel activity-based probes for the enzymes diacylglycerol lipase alpha and a/b-hydrolase domain containing protein 6 are described. Show less
The thesis describes the design, synthesis and application of chemical tools to study the physiological roles of DAGLa/b and MAGL in vitro and in vivo. Chapter2 reports on the design, synthesis... Show moreThe thesis describes the design, synthesis and application of chemical tools to study the physiological roles of DAGLa/b and MAGL in vitro and in vivo. Chapter2 reports on the design, synthesis and in vitro characterization of DH376 as a new dual DAGL inhibitors. In Chapter 3 the discovery of DH379 as a tailor-made activity-based probe for DAGLa/b and the effects of acute pharmacological blockade of DAGLa/b by DH376 in healthy and lipopolysaccharide-treated mice on brain lipid networks and neuroinflammation is described. Chapter 4 reports the efficacy of DH376 in refeeding behavior of fasted mice. In Chapter 5 the development of the first DAGL PET ligand [18F]DH439 is disclosed. The structure-activity relationship of disubstituted piperidinyl ureas as DAGL inhibitors is reported in Chapter 6. The design, synthesis and application of a highly selective tailor-made activity-based imaging probe for MAGL is discussed in Chapter 7. Show less
Glycoside hydrolases (GHs), enzymes that catalyze the hydrolytic cleavage of glycosidic bonds, receive continuing interest both in fundamental and applied biology and biomedicine. Lysosomal... Show more Glycoside hydrolases (GHs), enzymes that catalyze the hydrolytic cleavage of glycosidic bonds, receive continuing interest both in fundamental and applied biology and biomedicine. Lysosomal storage disorders (LSDs) are caused by inborn metabolic errors due to deficiency in specific lysosomal enzymes, most commonly GHs. Diagnosis and treatment of LSDs require regular quantification of the active lysosomal enzymes in patient tissues. Activity-based protein profiling (ABPP) has emerged in the past decades as a powerful technique to study enzyme families in cell extracts and living tissues. Originally developed for serine hydrolases and cysteine proteases, various enzyme classes can be studied by means of ABPP today, including retaining GHs. The research described in this thesis focused on expanding the field of activity-based glycosidase profiling through the development of functional and configurational analogues of cyclophellitol aziridine as activity-based probes (ABPs) for various retaining glycoside hydrolases (GHs), namely α-L-fucosidases, β-glucosidases, α-glucosidases and β-glucuronidases. Attention is focused on the design and synthesis of the cyclophellitol aziridine derivatives and their application in chemical biology studies of various retaining GHs. Show less
