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Insight into TRAPPII assembly and function through characterization of naturally occurring TRAPP gene variants


Insight into TRAPPII assembly and function through characterization of naturally occurring TRAPP gene variants

Almousa, Hashem (2023) Insight into TRAPPII assembly and function through characterization of naturally occurring TRAPP gene variants. PhD thesis, Concordia University.

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Proteins and other macromolecules are sorted throughout the cell in membrane bound vesicles in a process known as membrane trafficking. Defects in this process are associated with various human disorders. The transport protein particle (TRAPP) complexes, including TRAPPII and TRAPPIII, are highly conserved multi-subunit protein complexes involved in subcellular membrane trafficking pathways.

In my thesis, I assessed the effect of biallelic variants in TRAPPC10 and TRAPPC6B, two TRAPPII components, in individuals with a neurodevelopmental disorder characterized by microcephaly, intellectual disability, and brain atrophy. Here, I present comprehensive genetic, clinical, and functional data to assess the impact of these variants on TRAPPII stability, membrane trafficking, and ciliogenesis.

Molecular studies revealed disruption of protein-protein interactions between mutant TRAPPC10 and its adaptor protein TRAPPC2L, and between mutant TRAPPC6B and TRAPPC3, affecting TRAPPII complex-specific members that lead to the disruption of TRAPPII assembly. Individuals with an alteration in the TRAPPC10 gene have no functional TRAPPC10 protein, which resulted in a reduction in levels of another TRAPPII component, TRAPPC9. Additionally, TRAPPC10-/- knockout cells exhibited a membrane trafficking defect that can be rescued by wild type- but not mutant TRAPPC10. Similarly, TRAPPC6B patient-derived fibroblasts displayed reduced levels of TRAPPC6B and TRAPPII complex-specific members, suggesting that variants in TRAPPC6B may preferentially affect TRAPPII functions compared to TRAPPIII functions. This observation was consistent with co-immunoprecipitation experiments that revealed significant enrichment of TRAPPC6B in TRAPPII, while its homologue, TRAPPC6A, co-precipitated equally with TRAPPII and TRAPPIII. Additionally, the TRAPPC6B patient-derived fibroblasts displayed reduced trafficking into the Golgi apparatus and Golgi fragmentation. These defects could be rescued by wild type TRAPPC6B. Furthermore, my studies also revealed that TRAPPC10-/- knockout and TRAPPC6B patient-derived fibroblasts showed reduced levels of ciliation.

Finally, I demonstrated that translational read-through inducing drugs like Ataluren have the potential to rescue cellular defects attributed to nonsense mutations in the TRAPPC6B gene. This promising drug may serve as a viable treatment for TRAPPopathies, a group of neurodevelopmental, skeletal, and muscular disorders collectively associated with genetic variations in TRAPP genes.

Overall, my research confirms the crucial role of TRAPPC10 and TRAPPC6B in brain development and function, and provides insights into the potential disease pathomolecular mechanism, which involves the disruption of TRAPPII assembly and functions. The findings suggest that the use of new therapeutic compounds like Ataluren could effectively address these disruptions in a specific patient population.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Thesis (PhD)
Authors:Almousa, Hashem
Institution:Concordia University
Degree Name:Ph. D.
Date:27 March 2023
Thesis Supervisor(s):Sacher, Michael
ID Code:992246
Deposited By: Hashem Almousa
Deposited On:14 Nov 2023 19:25
Last Modified:14 Nov 2023 19:25
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