Protein arginylation in amyotrophic lateral sclerosis (ALS): a link between ATE1 and TDP-43

• Grant number: 22/15734-9
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: May 10, 2023
• End date: April 09, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Giuseppe Palmisano
• Grantee: Janaína Macedo da Silva
• Supervisor: Albert Lee
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Macquarie University, Australia
• Associated to the scholarship: 21/00140-3 - Understanding the role of protein arginylation in health and disease through analytical and biological approaches, BP.DR

Abstract

Amyotrophic lateral sclerosis (ALS) occurs due to the degeneration of both upper and lower motor neurons in the cerebral cortex and spinal cord. ALS has a prevalence of 5 individuals in 100,000 each year worldwide, most being classified as sporadic, therefore, of unknown cause, making it challenging to adopt palliative strategies. The causes of ALS are only partially understood; however, it is known that changes in more than 40 genes and loci are associated with disease progression, including TDP-43. About 10% of those diagnosed have an autosomal dominant mendelian inheritance pattern. The remaining 90% are classified as sporadic ALS, as they do not have genetic ancestry for the development of the disease. Mutations in TDP-43 may show some link between familial and sporadic ALS, as cytosolic aggregates are identified independently of disease genesis. The different mutations already identified in the protein are associated with an increase in the propensity to aggregate, change in the subcellular location, increase in stability, and resistance to proteases. Previous studies reported in cytoplasmic aggregates from patients with ALS, the identification of endogenous TDP-43 fragments naturally generated by the action of proteases. These fragments are substrates of the ATE1 in the N-degron pathway (degradation of proteins pathway). Comparison between the half-life of TDP-43 with an N-terminal destabilizing residue (ATE1 substrate) and an identical protein with an N-terminal non-destabilizing residue showed that fragments susceptible to arginylation or arginylated have a shorter half-life. Very encouragingly, the results performed in vitro on 293T cells showed that the Arg-Asp247-TDP43 and Asp247-TDP43 fragments (ATE1 substrate) are degraded within the first 5 minutes (~90%). The role of arginylation in the degradation of TDP-43 fragments, nerve regeneration, pathological degradation of pro-apoptotic fragments, autophagy modulation, and cell homeostasis encourages to study of the functions of the arginylation in ALS. The proposed project aims to identify and elucidate the role of ATE1 on the stability, localization, and interactome of TDP-43 (wild-type and mutants) using human cells (in vitro) and zebrafish animal models (in vivo). We will use molecular biology strategies combined with quantitative mass spectrometry and bioinformatic analyses to elucidate the role of ATE1 in ALS. In particular, ATE1 expression will be modulated in human cell lines with TDP-43 wild-type or mutant to determine the enzyme interactome. ATE1 levels will be evaluated in both cells with different TDP-43 mutations and overexpressed TDP-43. Protein arginylation will be studied also in zebrafish models containing wild-type and mutated TDP-43. This project will shed light on the relationship between arginylation and TDP-43. In addition, we intend to promote knowledge transfer between the laboratories involved and strengthen research collaboration between groups from the University of São Paulo (USP) and Macquarie University.