Tesis doctorales

Neuronal ceroid lipofuscinoses (NCLs) are a group of rare, fatal lysosomal storage disorders that are genetically inherited and primarily affect the nervous system. There is no cure, and affected children experience premature death. Fourteen types of NCLs have been identified, with mutations in CLN1 and CLN3 genes being the most prevalent. 1

S-acylation is the reversible attachment of lipids to cysteine residues in cellular proteins via a thioester linkage.2 This lipid modification has a key role in the regulation of protein function. It is a reversible process controlled by enzymes and the degradation of S-acylated proteins occurs in the lysosome. Previous reports have linked NCLs, particularly CLN1 and CLN3, with the metabolism of S-acylated proteins. Hence, CLN1 is associated with a deficient activity of palmitoyl-protein thioesterase 1 (PPT1), an enzyme crucial for the lysosomal degradation of S-acylated proteins.3,4 Another lysosomal protein, whose dysfunction is the primary cause of the most common type of NCL, is CLN3. Its precise biological role remains elusive, though it has been hypothesized to act as a fatty acid desaturase specifically on S-acylated proteins.5 A deeper understanding of the molecular mechanism behind these two proteins may reveal new biomarkers, diagnostic tools and therapeutic targets for these currently untreatable diseases.

For a long time, it has been thought that S-acylation was restricted to the attachment of palmitic acid. However, evidence now suggests a broader spectrum of saturated, monounsaturated, and polyunsaturated fatty acids. Our first goal will be to profile the substrate specificity of PPT1. To accomplish this, we will design a new substrate and set up a novel assay to determine the preference of PPT1 for different fatty acids. Moreover, the established assay will be employed to identify PPT1 inhibitors.

The second objective is to profile the lysosomal S-acylome. Previous research in the group demonstrated the utility of hydroxylamine in selectively cleaving thioester bonds and facilitating mass spectrometric analysis of the released fatty acids. 6 Building on this, hydroxylamine derivatives with lysosomal targeting groups will be synthesized to specifically analyze fatty acid composition of the proteins within this organelle. The third objective seeks to deploy the previously developed tools to investigate the proposed role of CLN3 as a fatty acid desaturase in cells overexpressing this protein.

All in all, this research aims to advance our understanding of NCL diseases and the proteins involved, potentially facilitating the identification of new treatment strategies for children suffering from these rare, fatal disorders.