A Sensitive Gel-Based Method Combining Distinct Cyclophellitol-Based Probes for the Identification of Acid/Base Residues in Human Retaining Beta-Glucosidases

Medical Biochemistr

In situ visualization of glucocerebrosidase in human skin tissue: zymography versus activity-based probe labeling

Drug Delivery Technolog

Stabilization of Glucocerebrosidase by Active Site Occupancy

Macromolecular Biochemistr

Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

Bio-organic SynthesisMedical Biochemistr

Towards broad spectrum activity-based glycosidase probes: synthesis and evaluation of deoxygenated cyclophellitol aziridines

Bio-organic SynthesisMedical Biochemistr

Novel defect in phosphatidylinositol 4-kinase type 2-alpha (PI4K2A) at the membrane-enzyme interface is associated with metabolic cutis laxa

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4-kinase type II alpha, caused by a homozygous missense mutation in thePI4K2Agene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4-phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl-chain pools of PI4P and PI(4,5)P(2)were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4-kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P(2)and PI(3,4,5)P-3. Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation inPI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.Medical Biochemistr

Activity-based probes for retaining β-glucosidases: Novel tools for research and diagnostics

The glycosylhydrolase glucocerebrosidase (GBA) cleaves the β-D-glucose moiety from one of the simplest glycosphingolipids, glucosylceramide, in the lysosome. Deficiency of GBA results in accumulations of glycosylceramide, primarily in macrophages, provoking Gaucher disease. The expression of this disease is remarkably variable, as heterogeneity among identical twins has even been reported. Moreover, correlation between in vitro GBA activity and the Gaucher phenotype is not very strict, suggesting additional factors influence the residual activity of GBA in situ. Registered treatments comprise of enzyme supplementation therapy employing exogenously produced GBA, and substrate reduction therapy, utilizing inhibitors for the glucosylceramide synthase. Recently, the use of small chemical chaperones, thought to stabilize mutant GBA in situ, has received major attention. The research described in this thesis addresses the present lack of suitable methods to visualize (active) GBA molecules in situ, as it proves a major limitation in fundamental research on GBA, Gaucher disease, the development of new treatments including chemical chaperone therapy, but also the creation of a technological fundament of similar methodologies for related glycosylhydrolases and their associated diseases

Activity-based probes for retaining β-glucosidases: Novel tools for research and diagnostics

The glycosylhydrolase glucocerebrosidase (GBA) cleaves the β-D-glucose moiety from one of the simplest glycosphingolipids, glucosylceramide, in the lysosome. Deficiency of GBA results in accumulations of glycosylceramide, primarily in macrophages, provoking Gaucher disease. The expression of this disease is remarkably variable, as heterogeneity among identical twins has even been reported. Moreover, correlation between in vitro GBA activity and the Gaucher phenotype is not very strict, suggesting additional factors influence the residual activity of GBA in situ. Registered treatments comprise of enzyme supplementation therapy employing exogenously produced GBA, and substrate reduction therapy, utilizing inhibitors for the glucosylceramide synthase. Recently, the use of small chemical chaperones, thought to stabilize mutant GBA in situ, has received major attention. The research described in this thesis addresses the present lack of suitable methods to visualize (active) GBA molecules in situ, as it proves a major limitation in fundamental research on GBA, Gaucher disease, the development of new treatments including chemical chaperone therapy, but also the creation of a technological fundament of similar methodologies for related glycosylhydrolases and their associated diseases