Title ULOGA SUPRESORA TUMORA
p53 U REGULACIJI HOMEOSTAZE PROTEINA U STANICAMA HETEROZIGOTNIMA ZA GEN ZA RIBOSOMSKI PROTEIN L24 Title (english) THE ROLE OF p53 TUMOR SUPPRESSOR IN PROTEIN HOMEOSTASIS REGULATION IN CELLS HETEROZYGOUS FOR RIBOSOMAL PROTEIN GENE L24 Author Deana Jurada Mentor Siniša Volarević (mentor)Committee member Berislav Lisnić (predsjednik povjerenstva)Committee member Tihomir Balog (član povjerenstva)Committee member Ivana Mikolašević (član povjerenstva)Committee member Siniša Volarević (član povjerenstva)Granter University of Rijeka Defense date and country 2020, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences 577 - Biochemistry. Molecular biology. Biophysics ThesaurusMESH - Medical Subject Headings )
Ribosomes
genetics
Ribosomes
metabolism
Tumor Suppressor Protein p53
Ribosomal Proteins
Neoplasms
genetics
Abstract Cilj istraživanja: Heterozigotne mutacije RP u sisavaca uzrokuju razvojne anomalije i zloćudne tumore. Inaktivacija supresora tumora p53 u tim uvjetima sprječava nastanak razvojnih anomalija i potiče razvoj zloćudnih tumora. Međutim, molekularni mehanizmi putem kojih smanjeni izražaj RP uzrokuje promjene u stanicama i uloga p53 u tom kontekstu nepoznati su. Ključna pretpostavka ovog istraživanja je da heterozigotna inaktivacija RPL24 uzrokuje poremećaj homeostaze proteina te da p53 umanjuje negativne učinke tih promjena putem regulacije proteostatskih mehanizama. Ciljevi istraživanja su: 1. Izolirati fibroblaste iz wt, RPL24+/-, p53-/-, RPL24+/-:p53-/- embrija miševa (MEF, od engl. mouse embryonic fibroblasts); 2. Odrediti posljedice heterozigotnosti za RPL24 u MEF-ovima; 3. Odrediti ulogu p53 u RPL24+/- MEF-ovima.
Materijal i metode: Istraživanja su provedena na MEF-ovima izoliranima iz wt, RPL24+/-, p53-/- i RPL24+/-:p53-/- embrija. Za određivanje posljedica heterozigotnosti za RPL24 u MEF-ovima i uloge p53 u tim procesima korištene su metode za analizu diobe stanica, sinteze proteina, signalnog puta mTORC1, proteasoma, autofagije, reaktivnih kisikovih radikala (ROS), agregata proteina te morfologije i funkcije mitohondrija.
Rezultati: U RPL24+/- MEF-ovima smanjena je sinteza proteina ovisna o signalnom putu mTORC1, a povećana je aktivnost proteasoma i mTORC1-ovisne autofagije. Ti mehanizmi umanjuju proteotoksični stres u RPL24+/- MEF-ovima, ali nisu dostatni za njegovo potpuno sprječavanje, što se očituje nakupljanjem agregata proteina i ROS-a. Inaktivacija p53 u RPL24+/- MEF-ovima sprječava inhibiciju Sesn2/mTORC1-ovisne sinteze proteina i inhibira mTORC1-ovisnu autofagiju, što rezultira dodatnim nakupljanjem agregata proteina. Povećana sinteza proteina, smanjena količina ROS-a i p21 uslijed inaktivacije p53 u RPL24+/- MEF-ovima omogućuju njihovu ubrzanu diobu. Inaktivacija p53 u RPL24+/- MEF-ovima remeti normalnu morfologiju i smještaj mitohondrija i uzrokuje energetski stres.
Zaključak: Razjašnjenje mehanizama putem kojih RPL24+/- uzrokuje promjene u MEF- ovima te uloge p53 u njihovoj regulaciji važne su za razumijevanje patogeneze bolesti karakteriziranih smanjenim izražajem RP poput anemije Diamond i Blackfan i zloćudnih tumora te mogu doprinijeti identifikaciji ciljeva za njihovo liječenje.
Abstract (english) Aim of the study: Mutations of genes encoding RPs in mammals cause congenital malformations and malignant tumors. Inactivation of p53 tumor supressor under this conditions prevents congenital malformations and facilitates the development of malignant tumors. However, molecular mechanisms underlying these processes are unknown. I hypothesized that heterozygous inactivation of RPL24 leads to perturbation of cellular proteostasis and that p53 modulates the pathological phenotype by regulating proteostatic mechanisms. Specific aims of this research are: 1.To isolate fibroblasts from wt, RPL24+/-
, p53-/-, RPL24+/-:p53-/- mouse embryos (MEFs), 2. To identify the consequences of RPL24 heterozygosity in MEFs, 3. To determine the role of p53 in RPL24+/- MEFs.
Materials and methods: wt, RPL24+/-, p53-/-, RPL24+/-:p53-/- MEFs are used in this study. To determine the consequences of RPL24 heterozygosity on protein homeostatsis and the role of p53 in that processes, I used a number of methods to assess cell division, protein synthesis, reactive oxygen species (ROS), protein aggregates and mitochondria morfology and function as well as activity of mTORC1 signaling pathway, proteasomes and autophagy.
Results: In RPL24+/- MEFs mTORC1-dependant protein synthesis is downregulated, whereas proteasomal and autophagosomal degradation are upregulated. However, activation of these proteostatic mechanisms are not sufficient to fully prevent formation of protein aggregates and ROS in these MEFs. Inactivation of p53 in RPL24+/- MEFs prevents Sesn2/mTORC1-dependant inhibition of protein synthesis and inhibits autophagy, which enhances the accumulation of protein aggregates. Despite the presence of protein aggregates, increased protein synthesis, reduced amount of ROS and p21 upon p53 inactivation in RPL24+/- MEFs enable accelaration of their proliferation. Finally, inactivation of p53 in RPL24+/- MEFs disrupts normal mitochondria morphology and cellular localization leading to the energetic stress.
Conclusion: These results will enable better understanding of the molecular mechanisms by which perturbations in ribosome biogenesis lead to Diamond and Blackfan anemia and malignant tumors and they may reveal therapeutic liabilities in these diseases.
Keywords
RPL24
p53
dioba stanica
mTORC1
autofagija
poremećaj proteostaze
Keywords (english)
RPL24
p53
cell division
mTORC1
autophagy
dysregulated proteostatic mechanisms
Language croatian URN:NBN urn:nbn:hr:184:715800 Promotion 2021 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2021-03-03 00:07:28
