# name
Relevant deubiquitinating enzymes in cilium formation and function in the retina: USP48

# metadata.dc.contributor.other[].value
Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística

# metadata.dc.description.abstract[].value
[eng] Ciliopathies encompass a broad group of heterogeneous inherited disorders associated with dysfunction of the cilium, a ubiquitous microtubule-based organelle that translates extracellular stimuli into cellular responses. The retina is one of the most affected tissues by mutations in ciliary genes due to the highly specialised neurosensory cilium that photoreceptors possess, known as outer segment, where photoreception and phototransduction occur. To date, mutations in more than 100 ciliary genes have been associated with retinal degeneration, accounting for almost 25 % of inherited retinal dystrophy (IRD) cases. Proteins related to the ubiquitin-proteasome system play a pivotal role during retinal differentiation and ciliogenesis of photoreceptor cells. In fact, mutations in several genes involved in ubiquitination and proteostasis have been identified as underlying causes of IRDs and/or ciliopathies. USP48 is a deubiquitinating enzyme whose role in the retina is still unexplored. However, previous reports reveal its relevance for neurosensory organs since dominant mutations in this gene are causative of hearing loss. Building upon prior findings regarding USP48 expression in the retina, we sought to uncover USP48 involvement in retinal development and cilium function with the ultimate aim to gather evidences to nominate it as a new candidate gene for unsolved cases of retinal ciliopathies. We demonstrated that USP48 is highly expressed in cones in mouse retinas, in agreement with previous RNA-seq data. Furthermore, we described for the first time that a pool of endogenous USP48 localises to the basal body in retinal cell lines, independently of the cell cycle phase. Intriguingly, the overexpressed full-length and Cterminal isoforms fail to localise to the basal body in serum-starved hTERT-RPE1 cells, raising questions about which USP48 protein isoform precisely localises to the basal body and centrosome. Moreover, neither USP48 overexpression nor siRNA-based silencing affects ciliogenesis or cilium length. However, proteomic analysis of mouse retinas revealed that several USP48 interactors had cilium-related functions. Among USP48 partners, ARL3 and UNC119a drew our attention due to their association with retinal ciliopathies and involvement in protein transport in photoreceptor cilia. Subsequent in vitro analysis indicated that USP48 increases ARL3 and UNC119a protein levels through different mechanisms and interacts with them via distinct protein domains. Furthermore, USP48 is also able to increase the protein levels of different ARL3 mutants. These findings collectively indicate that USP48 could play a role in regulating key ciliary proteins, thereby modulating intracellular protein transport and ciliary trafficking to the photoreceptor outer segment, crucial to maintain photoreceptor homeostasis and function. Due to limitations of cell-based studies – which involve transient transgenesis with a short time frame for system manipulation – and the necessity for a retinal tissue context, we further aimed to generate a USP48-/- human induced pluripotent stem cell (iPSC) line by CRISPR/Cas9. The subsequent differentiation into retinal organoids would allow the study of the retinal and cilium alterations caused by the absence of USP48 during retinal development and differentiation. Unfortunately, we could not identify a homozygous iPSC colony for USP48 deletion, suggesting the deletion of both USP48 loci might lethal in iPSCs. Notably, USP48+/- iPSCs displayed a severe ciliary phenotype, indicating that USP48 haploinsufficiency is disrupting key cellular pathways for cilium formation and further hinting at the potential lethality due to the USP48 knockout. Finally, the differentiation for the USP48+/- iPSC line alongside the BJ iPSC control line was initiated and the phenotypical characterisation of the retinal organoids is still ongoing. In conclusion, our investigation demonstrates that USP48 is a new ciliary regulator that potentially modulates intracellular and ciliary protein transport in the retina. Consequently, we posit USP48 as a new candidate gene for retinal ciliopathies. Finally, the studies in retinal organoids promise to shed light on USP48 role in the retina and photoreceptor cilia.

# metadata.dc.description.abstract[].value
[cat] Les ciliopaties són un grup heterogeni de trastorns hereditaris causats per la disfunció del cili, un orgànul constituït de microtúbuls que tradueix estímuls externs a respostes cel·lulars. La retina és un dels teixits més afectats per mutacions a gens ciliars degut al cili neurosensorial altament especialitzat que tenen els fotoreceptors. S’han identificat mutacions en més de 100 gens ciliars en pacients amb degeneració de la retina, comptabilitzant gairebé el 25 % dels casos de distròfies hereditàries de la retina (IRDs). Cal remarcar que el sistema ubiquitinaproteasoma és rellevant durant la diferenciació de la retina i la ciliogènesi i, de fet, mutacions a diversos gens implicats en la proteostasi s’han associat tant a IRDs com a ciliopaties. USP48 és un enzim deubiquitinant de funció desconeguda a la retina. Basant-nos en estudis previs, aquesta tesi s’enfoca a determinar el paper d’USP48 a la retina i al cili per tal de proposar-lo com un nou gen candidat per casos sense resoldre de ciliopaties de retina. USP48 localitza a la base del cili a cèl·lules humanes de la retina i hem identificat les proteïnes ciliars ARL3 i UNC119a com a interactors. Aquests resultats indiquen que USP48 podria estar modulant el transport intracel·lular i ciliar, necessari per l’homeòstasi i funció dels fotoreceptors, a través de la regulació de proteïnes ciliars clau. Un dels objectius de la tesi consisteix en generar una línia de cèl·lules mare pluripotents induïdes (iPSCs) amb la deleció del gen USP48 (USP48-/-), per després diferenciar-les a organoids de retina i realitzar anàlisis fenotípiques de les alteracions ciliars causades per la manca d’USP48. Cal remarcar que no s’ha identificat cap colònia USP48-/-, però sí USP48+/-, suggerint que la deleció dels dos al·lels d’USP48 podria ser letal en iPSCs. La caracterització de les iPSCs USP48+/- ha revelat que l’haploinsuficiència d’USP48 causa defectes en la formació del cili, ressaltant la importància d’USP48 en el fenotip ciliar i evidenciant la potencial letalitat de la deleció d’USP48 en homozigosi. En el moment del dipòsit d’aquesta tesi, la diferenciació dels organoids USP48+/- estava en progrés. En resum, la nostra recerca demostra que USP48 és un nou regulador ciliar que potencialment modula el transport intracel·lular i ciliar a la retina. Així doncs, proposem USP48 com un nou gen candidat de ciliopaties de retina. Finalment, els estudis en curs als organoids de retina permetran dilucidar el rol d’USP48 a la retina i al cili dels fotoreceptors.

# metadata.dc.rights[].value
cc by-nc (c) Sánchez Bellver, Laura, 2024

# metadata.dc.source[].value
Tesis Doctorals - Departament - Genètica, Microbiologia i Estadística

# metadata.dc.title[].value
Relevant deubiquitinating enzymes in cilium formation and function in the retina: USP48