Structural studies on lysosomal proteins
Year: 2014
Type: dissertation
Abstract: Lysosomes are membrane enclosed acidic cell organelles found ubiquitously in higher eukaryotes. The lysosomal lumen contains more than 60 soluble lysosomal hydrolases, which degrade and recycle cellular macromolecules. Mutations in genes encoding lysosomal or lysosome related proteins result in over 50 different lysosomal storage disorders (LSDs) affecting 1 out of every 7700 newborn children. For instance, the first described LSD, Pompe disease, is caused by a mutation that impairs the function of lysosomal α-glucosidase (GAA) and that results in lysosomal accumulation of glycogen. In this study, several lysosomal proteins were studied via a variety of techniques to increase the knowledge of lysosomal function and correspondingly, the lysosome associated diseases. In order to better understand its function, the previously not well characterised phospholipase B-like protein 1 (PLBD1) was purified from bovine kidneys. It was crystallised and the structure solved by X-ray crystallography to a 1.9 Å resolution. The structure showed that PLBD1 is a member of the N-terminal nucleophile aminohydrolases superfamily. This would imply that PLBD1 is not an esterase as the name suggests, but an amidase. The finding that the hydrophobic tail of the potential phospholipid substrate does not fit into the acyl binding cavity also argues against phosphoesterase function. As a first step in the protein transport pathway to lysosomes mannose-6- phosphate-tag is added to lysosomal proteins. This is initiated by N- acetylglucosamine-1-phosphotransferase (GlcNAc phosphotransferase), which requires a recognition signal on the folded surface of the lysosomal proteins. In this study, the conservation of the signal in four lysosomal proteins was analysed. The phosphorylated N-glycosylation sites and the lysine residues on the GlcNAc phosphotransferase recognition site are well conserved at the sequence level in orthologous proteins, but not necessarily in the protein family. Based on surface analysis of PLBD1 and comparison to the paralogous PLBD2, the most likely recognition site for the GlcNAc phosphotransferase for the PLBD1 could be suggested. LSD associated mutations affect protein function through several mechanism. Several disease-associated missense mutations disturb the protein fold. A general analysis of four enzymes associated with LSD showed that the disease-associated missense mutations are not equally distributed among the 20 amino acids. Glycine, arginine and proline are clearly over-represented among the mutations comparedto their abundance in protein sequences. The hydrophobic amino acids tend to be under-represented among disease-associated mutations. The amino acids where mutation frequently involves a disease have unique properties that contribute to the protein structure in a way that cannot be compensated by other amino acids. Enzyme enhancement therapy with chemical chaperones is a novel treatment for LSDs and has shown potential also for Pompe disease. In this study, the stabilisation capacity of potential chemical chaperones for GAA were tested. Most of the compounds stabilised rhGAA against thermal unfolding and some stabilised even better than would be expected from their binding affinity. In addition, the compounds were modelled to the active site of a GAA structural model and based on this three factors to be considered in chemical chaperone design were defined. Firstly, the ligand size can vary, but the four OH-groups in the ligand are critical in orienting the molecule and making the binding specific. Last but most importantly, a positive charge and its location determine the strength of binding to GAA. This thesis with its structural studies of lysosomal proteins provides molecular understanding of lysosomal protein biology, which is critical for full understanding lysosome function and its involvement in diseases.
Source: Työväentutkimus Vuosikirja
Author: Heidi Repo
Language: English
Subfield: Genetics (clinical)
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2213016787