Analysis of the effect of the controlled expression of Hsf1p on the function of human NAGLU variants involved in Sanfilippo syndrome

• Grant number: 24/19423-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: February 01, 2025
• End date: January 31, 2029
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Renata Castiglioni Pascon
• Grantee: Gabriela Maria Redondo da Silva
• Host Institution: Escola de Artes, Ciências e Humanidades (EACH). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 23/14056-0 - Analysis of the effect of the controlled expression of Hsf1p on the function of human NAGLU variants involved in Sanfilippo syndrome., AP.R

Abstract

Mucopolysaccharidoses comprise a subclass of lysosomal diseases caused by a deficiency in the enzymes that catabolize glycosaminoglycans; charged carbohydrates that accumulate in the lysosomes and cause pathologic bone, cardiopulmonary, auditive, and neurocognitive phenotypes. Sanfilippo syndrome is the mucopolysaccharidosis (MPS) whose treatment is more difficult, and this MPS involves severe cognitive impairment due to the accumulation of heparan-sulfate in the lysosomes, organelles that determine the development and homeostasis of neurons. This project aims to study mutations that affect the function of the ¿-N-acetylglucosaminidase (NAGLU, MPS IIIB) that contribute to ~30% of Sanfilippo syndrome cases. We intend to construct the first experimental model to study this disease in the budding yeast Saccharomyces cerevisiae, to conduct functional studies of NAGLU and its variants, that are known to be misfolded. NAGLU will be fused to fluorescent red tag mScarletI to observe the cellular localization of the protein; the enzymatic activity will be measured directly in cell extracts, in the presence of the fluorogenic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-¿-D-glucopyranoside; and protein stability will be studied in cells treated with cycloheximide to interrupt translation, and NAGLU band tracked by Western blot. The transcription factor Hsf1p of S. cerevisiae activates the expression of several proteins involved in protein folding. We will investigate if there is a Hsf1p expression level that can rescue NAGLU mutant protein folding. Later, similar experiments will be conducted in mammalian cells. If this proof of concept succeeds, this will indicate that control of human HSF1 expression level might be a potential treatment for the Sanfilippo syndrome.