Impact of Disease-associated Missense Mutations on Protein Interaction Networks in Ciliopathies

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Zitierfähiger Link (URI): http://hdl.handle.net/10900/141563
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1415633
http://dx.doi.org/10.15496/publikation-82910
Dokumentart: Dissertation
Erscheinungsdatum: 2023-05-31
Sprache: Englisch
Fakultät: 7 Mathematisch-Naturwissenschaftliche Fakultät
Fachbereich: Biochemie
Gutachter: Schwarzer, Dirk (Prof. Dr.)
Tag der mündl. Prüfung: 2023-05-23
Schlagworte: Zilie , Signaling , Proteomanalyse , Augenheilkunde
Lizenz: http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=de http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=en
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Abstract:

Primary cilia are antenna-shaped organelles protruding from the cellular surface of most eukaryotic cells. They are highly conserved through evolution and serve as signaling hub for a variety of crucial signaling pathways, such as sonic hedgehog signaling. Since cilia are biosynthetically inactive, cargo needs to be actively targeted to and transported along the cilium. This so called intraflagellar transport (IFT) is facilitated by motor proteins in conjunction with large protein complexes, which serve as binding hubs for cargo. The retrograde transport of cargo towards the ciliary base is facilitated by dynein-2 and the IFT-A protein complex. Due to the important role of cilia in many cellular processes defects in genes coding for ciliary components are associated with a variety of severe diseases, which are commonly referred to as ciliopathies. The impact of disease-associated mutations on protein-protein interactions (PPIs) is not fully understood yet. One lead hypothesis is the concept of edgetic mutations, meaning that a mutation in a protein leads to the loss of specific interactions rather than a total loss of function. In addition, it was hypothesized that missense mutations are hypomorph and have a quantitative effect on the native protein function. To illuminate these questions missense mutations from previous publications were selected in four (IFT43, IFT121, IFT122 and IFT140) of the six components of the IFT-A protein complex. Affinity purification in combination with LC-MS/MS analysis was used to investigate the protein interactome of the wildtype proteins as well as the changes induced by disease-associated missense mutations. The generated data creates valuable new insights into the protein interactions of the wildtype forms of IFT43, IFT121, IFT122 and IFT140. The comparison of 37 mutants with the corresponding wildtype conditions revealed a variety of specific perturbations in protein interaction networks caused by the disease-associated missense mutations, further strengthening the concept of edgetic effects of disease-associated mutations. Der Dekan In addition, the analysis of the mutants revealed quantitative effects on specific PPIs, causing loss of binding to interaction partners to a varying degree. Single nucleotide polymorphisms in IFT140 did not impair complex integrity of IFT-A, further strengthening the disease relevance of the previously observed perturbations in PPIs caused by ciliopathy-associated missense mutations. Analysis of the allelic combination and the reported clinical features in patients indicated a correlation between genotype and phenotype in the analysed patients. The mass spectrometric analysis revealed the ciliary protein TULP3 as interactor of IFT140, that is affected by a subset of missense mutations in IFT140. TULP3 was reported to link the IFT-A complex to membrane-bound cargo, including several GPCRs involved in the negative regulation of hedgehog signaling. For functional analysis of IFT140 a CRISPR/Cas9-mediated knockout of IFT140 in hTERTRPE1 cells was generated. Consistent with our expectations real-time PCR revealed that the expression of the hedgehog target gene GLI1 is increased in IFT140 knockout cell as compared to the wildtype. Defects in hedgehog signaling lead to defects which are consistent with some of the observed phenotypes in ciliopathy-associated patients. Since hedgehog signaling can be pharmacologically targeted this may create a rationale for future treatment.

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