Investigating Lebercilin Interacting Proteins TTC30A/B and SSNA1 in Cilia Related Processes

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Dokumentart: PhDThesis
Date: 2023-07-03
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Pharmazie
Advisor: Lukowski, Robert (Prof. Dr.)
Day of Oral Examination: 2023-04-05
DDC Classifikation: 000 - Computer science, information and general works
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Cilia are elongated compartments, emerging from the cellular surface of most eukaryotic cells. These organelles can be classified into motile and non-motile (primary or sensory) subtypes which are highly conserved across species [1-6]. Further, cilia consist of specific domains with distinct functions. The basal body derives from the centrosome, nucleates axonemal growth and is the starting point for intraciliary protein trafficking [7]. For cilium assembly, proteins are imported from the cytoplasm while free diffusion of proteins into the cilium is restricted by the transition zone [7]. The intraflagellar transport (IFT) system is a microtubule-based bidirectional transport process which moves cargo (e.g., tubulins) along the axoneme from the base to the tip of the cilium where they are incorporated into the growing axoneme [1, 8-10]. The process of IFT involves the movement of large protein complexes with the help of motor proteins [11]. The IFT particles are organized in two subcomplexes called complexes A and B consisting of 6 (A) and 16 (B) distinct proteins [8, 11, 12]. One of the core proteins of IFT-B is IFT70/TTC30, which comprises the two paralogues TTC30A and TTC30B [1, 8, 9, 13]. In the human retina, photoreceptors (rods, cones) have two major compartments: the inner segment (IS) and the outer segment (OS) [14]. The OS is a modified sensory cilium that is packed with membrane discs containing rhodopsin [14, 15]. The molecular transport between IS and OS is facilitated along the connecting cilium [16, 17]. Any biomolecule necessary for generation and maintenance of the photoreceptive outer segment is produced in the inner segment and needs to be transported by IFT via this route [17, 18]. A complete malfunction of the IFT leads to severe ciliogenesis defects, not only in the OS but in all cilia [2, 18, 19]. Defects in primary cilia structure and function are associated with a wide spectrum of inherited diseases involving various organs. Until now there are 35 of these so-called ciliopathies including Leber congenital amaurosis (LCA) [20, 21]. LCA is a rare hereditary retinal dystrophy which leads to photoreceptor degeneration in children already in the first year of life [22, 23]. To date 25 genes were identified to be associated with this disease, including LCA5 [24, 25]. This gene is coding for Lebercilin, which was previously described (by Boldt et al.) to interact with the IFT machinery, including TTC30A/B. This interaction was lost due to LCA-associated mutations, resulting in an impaired IFT mediated opsin transport in photoreceptors [26]. Further, Lebercilin was also described to interact with SSNA1 [26], which is essential for microtubule stability but is also involved in ciliary assembly and intraflagellar transport [27, 28]. In addition, both Lebercilin and SSNA1 were associated with regulators of cilia specific Sonic hedgehog (Shh) signaling [26]. This pathway regulates ciliogenesis and is dependent on intraflagellar transport [29, 30]. The study presented here investigated Lebercilin and its interacting proteins TTC30A and TTC30B as well as SSNA1 in cilia related processes. Initially, it was essential to create gene knockouts in human retinal pigment epithelium cell lines using the CRISPR/Cas9 system. Regarding TTC30, paralogue specific homozygous single knockouts as well as double knockouts were generated. By comparing KO and wildtype (wt) cell lines, structural features, differences in cilia assembly and protein function were assessed. In the TTC30A/B double knockout, the loss of cilia and tubulin polyglutamylation were observed. This phenotype resembled the previously described results of Takei et al. and Pathak et al. [31, 32]. These findings could be reversed by generating rescue cells, stably transfected with TTC30A or TTC30B wt constructs. In contrast, TTC30A/B single knockout cells had assembled cilia, but their length was reduced and they had less polyglutamylated tubulin. In addition to these generated knockout cell lines, a paralogue specific CRISPR/Cas9 based knock-in was performed to insert a FLAG-tag on endogenous level. These tagged TTC30A/B cell lines were used for mass spectrometry analysis to define paralogue specific interactomes. Both proteins were found to interact with all IFT-B complex proteins, which was in line with previous findings [26]. Additionally, it was revealed that a TTC30 single KO did not affect complex stability, possibly due to redundancy. But a TTC30A/B double knockout led to a destabilized IFT-B complex and eventually to a disrupted intraflagellar transport as well as ciliary assembly. Further, interactome analysis revealed so far unknown paralogue specific protein interactors. The proposed interaction of TTC30A with protein kinase A (PKA) catalytic subunit A was investigated. In TTC30A KO cells an increased PKA activity was observed plus a decreased localization of Smoothened (Smo) to the cilium. PKA and Smo are key regulators of cilia specific Shh and these findings indicate an inhibitory effect of TTC30A KO on this pathway. Next, the A375V TTC30B mutation was examined which was previously identified by Du et al. This mutation was described to influence Shh and possibly leading to synpolydactyly [33]. A MS analysis was performed, comparing wildtype (wt) and mutant TTC30A/B interactomes, to evaluate the impact of this mutation on the abundance of their protein interactors. The A375V mutation led to several paralogue specific differences in protein interaction. Especially, the decreased interaction of mutant TTC30A with IFT57 is interesting and needs further investigation. This finding and the involvement of TTC30A in Shh signaling, might be possible explanations of how TTC30 is connected to synpolydactyly. Regarding Lebercilin and SSNA1, the generation of respective knockouts in human retinal pigment epithelium cell lines was performed. LCA5 exon 3 and exon 6 as well as SSNA1 exon 1 and exon 3 knockouts were created. LCA5 and SSNA1 KO cells were compared to wt cells, to investigate the impact of these proteins on ciliary assembly and structure as well as their function. It was revealed, that LCA5 knockout led to a reduction of ciliary length and to overall less cells assembling cilia. In contrast, SSNA1 knockout led to an increase of ciliary length with no effect on the number of ciliated cells which is in line with previous findings [34]. The proposed involvement of Lebercilin and SSNA1 in Shh signaling could be partially verified as ciliary localization of Smo and Gli2 was decreased in SSNA1 KO cells [26, 28]. This result suggests an inhibitory effect of SSNA1 KO on this signaling pathway. Further, in Shh activated SSNA1 KO cells a ciliary accumulation of Lebercilin was observed, indirectly linking Lebercilin to Sonic hedgehog. These findings highlight the importance of Lebercilin-SSNA1 interaction in cilia related processes and imply that SSNA1 function is possibly connected to the misfunction of Lebercilin, which ultimately leads to photoreceptor degeneration. But to understand the protein function of Lebercilin and SSNA1 in cilia related processes and to uncover the underlying mechanism of Leber congenital amaurosis further investigations are needed.

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