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 glycosidases are acid hydrolases that fragment glycoconjugates in lysosomes. Their inherited deficiency in human is the cause of a number of lysosomal storage disorders (LSDs),... Show moreLysosomal glycosidases are acid hydrolases that fragment glycoconjugates in lysosomes. Their inherited deficiency in human is the cause of a number of lysosomal storage disorders (LSDs), showing characteristic lysosomal accumulation of undegraded glycoconjugates. In the past, activity-based probes (ABPs) based on cyclophellitol or cyclophellitol aziridine scaffold have emerged as powerful tools enabling sensitive quantification of a number of lysosomal glycosidases in extracts of cells and tissue, as well as in intact cells. This thesis describes the characterization of several novel ABP classes targeting α-glucosidase, β-glucuronidase, α-L-iduronidase, α-mannosidase, β-mannosidase, and β-galactosidase, as well as a broad scale of applications for ABPs in LSD research. Novel glucocerebrosidase inhibitors based on the cyclophellitol scaffold are also described, which are brain-permeable, selective, and potently inactivate the enzyme in adult zebrafish. Additionally, a protocol for gel-based and microscopy-based detection of glucocerebrosidase is described. Show less
Due to their central physiological roles in living organisms, retaining beta-glycosidases have been the subject of tremendous research efforts to examine their structure/function relation using... Show moreDue to their central physiological roles in living organisms, retaining beta-glycosidases have been the subject of tremendous research efforts to examine their structure/function relation using numerous biophysical and biochemical approaches. Since the proposition of the hydrolysis mechanism in the late fifties by Koshland1, the fundamental research on retaining b-glycosidases has been revolutionized by the discovery of multiple reversible and irreversible inhibitors. One of the most successful class of inhibitors are mechanism based inactivators, which were extensively used to identify the nucleophilic catalytic residues and to comprehend the catalytic mechanism and substrate itinerary. Subsequently, covalent inhibitors were used as warheads to synthesize chromogenic activity based probes (ABPs), which were widely used to selectively label and discover new retaining beta-glycosidases in complex biological samples. The organic synthesis and biological applications of these ABPs has become routine. Nevertheless, their binding mechanism and influences on protein conformation and dynamics remained unexplored. Therefore, this work is aimed to establish a bridge between the two research disciplines, using ABP technology to understand functional dynamics and conformational stability of retaining bglycosidases in solution and in vivo. The research relied on standard biochemistry and advanced NMR spectroscopy research approaches. Show less
This thesis describes biochemical investigations of glucocerebrosidase (GBA), the lysosomal β- glucosidase that is deficient in Gaucher disease (GD). Central in the performed research was the... Show moreThis thesis describes biochemical investigations of glucocerebrosidase (GBA), the lysosomal β- glucosidase that is deficient in Gaucher disease (GD). Central in the performed research was the examination of factors influencing the intralysosomal stability and half-life of GBA. The investigations made use of new chemical biology tools such as activity based probes (ABPs) and photo-activatable and clickable (PAC) lipids.The Discussion reviews the present insights into GBA in health and disease. In this connection, the molecular basis and clinical manifestation of Gaucher disease and Action Myoclonus Renal Failure syndrome are discussed, including the metabolic adaptations to GBA deficiency. Particular attention is paid to the lysosomal structural stability of GBA and associated resistance against proteolytic degradation by cysteine cathepsins. Literature findings and novel own results on this topic are discussed. New technology to study GBA by labeling with GlcCer and cyclophellitol derived probes is introduced and the application is described. Unresolved research questions on GBA and related disease conditions are identified. As future research objective the translation of fundamental knowledge on GBA to effective therapy of neuronopathic Gaucher disease and other disease conditions caused by enzyme reduction are discussed. Show less
Glycoconjugates (a carbohydrate connected to a lipid, protein or other carbohydrate) play a key role in great variety of biological processes. The synthesis of these constructs is tightly regulated... Show moreGlycoconjugates (a carbohydrate connected to a lipid, protein or other carbohydrate) play a key role in great variety of biological processes. The synthesis of these constructs is tightly regulated by enzymes. Defects in these enzymes may result in an impaired degradation of the glycoconjugate. Consequently, the levels of glycoconjugates are increased and this may eventually lead to storage disorders such as Gaucher disease. The research described in this thesis focuses on the synthesis of chemical tools (activity-based probes, ABPs) to study the enzymes involved in the degradation of glycoconjugates. Using these probes, it was demonstrated that the activity of peptide N-glycanase (the enzyme that is responsible for the hydrolysis of N-linked glycoproteins)inhibitors is determined by its reactive group. Furthermore, the activity-based protein profiling strategy was used to study degradation of glycosy lated proteins. It appeared that deglycosylation of O-GlcNAclated proteins is not a prerequisite for proteasomal degradation. To study beta-glucosidases (enzymes that catalyze the hydrolysis of O-glycosidic linkages), ABPs based on cyclophellitol have been developed. Especially fluorescently labeled probes bind efficiently and selectively to beta-glucosidases. These probes have been used to investigate Gaucher disease. Both wild-type and mutant forms of the enzyme could be labeled in vitro and in living cells which allowed rapid identification activity of this glucosidase. Show less
The study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and _-glucosidase 2 that... Show moreThe study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and _-glucosidase 2 that are enzymes involved in glucosylceramide metabolism. The study has resulted in many novel inhibitors of these three enzymes among which several that improve upon the inhibition profile of the lead compound in this study. The successful use of lipophilic iminosugars in type 2 diabetes models and the partial elucidation of their mechanism of action therein provide prospects for their development towards therapeutics for diabetes type 2. Show less
