Restoring NKX6-2 function by protein complementation: a proof-of-concept

• Autor(a) principal: Peralta, Pedro Emanuel Ferreira
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/138619
• topic: NKX6-2; Spastic Ataxia 8; rotein complementation; transcriptional factor; pRL miniTK-luciferase system; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

NKX6-2 is a transcriptional repressor involved in the regulation of various genes associated with cell fate determination of neurons, oligodendrocytes, or pancreatic cells, as well as glucagon production, among other phenomena. Loss-of-function mutations in NKX6-2 are the cause of Spastic Ataxia 8 (SPAX8), a rare recessive hereditary disease that leads to an early-onset hypomyelinating leukodystrophy due to the lack of oligodendrocyte maturation. The most common disease-associated NKX6-2 mutations involve the formation of premature termination codons (PTCs). These PTCs will render the nascent mRNA a potential target for degradation via nonsense-mediated mRNA decay (NMD). The small fraction of mRNA that survives NMD and is eventually translated to protein will produce truncated, non-functional NKX6-2 proteins. We aimed to overcome these two issues to lay the groundwork for the development of new therapies for SPAX8. Using site-directed mutagenesis, we were able to recreate SPAX8-related mutations on a cDNA-based NKX6-2-Venus fusion protein, but not in a NKX6-2 minigene including its introns, exons, and 5 ́and 3 ́UTR regions. The effects of a SPAX8-related mutation, in the amino acid 41, was verified by microscopy, western blot, and a home-made luciferase activity assay where the NKX6-2 DNA response element was inserted in a pRL miniTK-luciferase system. We tried unsuccessfully to restore the normal phenotypes by means of a complementary NKX6- 2-Flagtag-P2A-T2A-mCherry fusion protein, that contains the amino acids 35 to 277. Normal, full-length NKX6-2 had an exclusively nuclear localization, and its overexpression induced a change in the morphology of cell nuclei. SPAX8-causing mutations in NKX6-2 led to an even distribution in both nuclei and cytoplasm, indicating a loss of the nuclear localization signal, and in one case protein aggregation. Transfection of cells with SPAX8-mutant NKX6-2 and its complementary fragment showed a partial increase in truncated NKX6-2 in the nucleus. Our NKX6-2 luciferase assay was able to detect NKX6-2 transcriptional repression, which was lost in a SPAX8 mutant and not recovered by the complementary fragment. We have created new cellular models for the study of SPAX8 cellular and molecular alterations. Our attempt to restore mutant NKX6-2 function by means of a complementary fragment was functionally ineffective, but the fact that we could increase the nuclear localization of the mutant protein suggests that other complementary fragments could be more effective. Furthermore, we obtained basic information about the domains involved in the nuclear localization and transcriptional repressor activity of NKX6-2. Although our attempts to study NMD in this context were unsuccessful, we are still trying to sort out its potential role in SPAX8, and the minigene we designed could be a good starting point. If we succeed, our efforts could be applied to other genetic disorders involving PTCs, which are associated to one-third of inherited human diseases.