Title Uloga ribosomskih proteina u aktivaciji supresora tumora p53 : doktorski rad Title (english) The role of ribosomal proteins in activation of tumor suppressor p53 Author Slađana Bursać Mentor Siniša Volarević (mentor)Committee member Ivana Weygand Đurašević (predsjednik povjerenstva) Committee member Biserka Mulac-Jeričević (član povjerenstva)Committee member Tihana Lenac Roviš (član povjerenstva)Committee member Siniša Volarević (član povjerenstva)Granter University of Rijeka Defense date and country 2013-01-01, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 576 - Cellular and subcellular biology. Cytology Abstract Ciljevi istraživanja:
Na temelju rezultata istraživanja u zadnjih desetak godina predložen je model u
kojem inhibicija sinteze ribosoma rezultira oštecenjem jezgrice i oslobaanjem
ribosomskih proteina (RP) RPL5, RPL11, RPL23, RPL26 i RPS7 iz jezgrice u jezgru,
gdje vežu negativni regulator p53, Mdm2 (engl. mouse double minute 2 homolog),
inhibiraju njegovu ubikvitin-ligaznu aktivnost, te posljedicno aktiviraju supresor tumora
p53.
Ciljevi su :
1. Odrediti specificnu ulogu RPL5, RPL11, RPL23, RPL26 i RPS7 u aktivaciji p53 nakon
inhibicije razlicitih koraka u sintezi ribosoma.
2. Razjasniti precizne molekularne mehanizme putem kojih ti RP aktiviraju p53.
Materijali i metode:
U stanicama A549 i U-2 OS sinteza ribosoma inhibirana je aktinomicinom D
(AktD), 5-fluorouracilom (5-FU) ili primjenom specificnih malih molekula RNA, siRNA
(utišavajucih RNA, engl. silencing RNA) protiv komponenti koje sudjeluju u sintezi
ribosoma. Podrijetlo, stabilnost i unutarstanicni smještaj novosintetiziranih RP te njihova
udruženost s drugim proteinima analizirana je brojnim metodama molekularne i stanicne
biologije te biokemije (tretman stanica inhibitorima sinteze proteina i proteasoma,
izolacija stanicnih odjeljaka, metoda Western blot, imunotaloženje, lancana reakcija
polimerazom i konfokalna mikroskopija).
Rezultati:
Dokazano je da su RPL5 i RPL11, a ne RPL23, RPL26 i RPS7, kljucni pozitivni
regulatori p53 nakon inhibicije sinteze ribosoma. Vecina RP sintetizira se i nakon
inhibicije sinteze ribosoma, ali se vrlo brzo razgrauju u proteasomima. Novosintetizirani
RPL5 i RPL11 ne razgrauju se u proteasomima, vec se nakupljaju u izvanribosomskim
odjeljcima gdje vežu negativni regulator p53, Mdm2. Sklop RPL5-RPL11-Mdm2-p53
transportira se u oštecenu jezgricu i udružuje s aktivatorom p53, proteinom PML (engl.
promyelocytic leucemia protein), što rezultira potpunom aktivacijom p53.
Zakljucci:
Dokazano je da su RPL5 i RPL11 kljucni aktivatori p53 nakon inhibicije sinteze
ribosoma te su razjašnjeni precizni molekularni mehanizmi ove regulacije. Naši rezultati
mogu pomoci u razumijevanju patogeneze bolesti uzrokovanih poremacajima sinteze
ribosoma.
Abstract (english) Objectives:
Overexpression experiments suggested that perturbation of ribosome biogenesis
causes nucleolar disruption and translocation of a number of ectopically expressed
ribosomal proteins (RPs), including RPL5, RPL11, RPL23, RPL26, and RPS7, from the
nucleolus to the nucleoplasm, where they bind to Mdm2 and inhibit its ubiquitin ligase
function toward p53, leading to p53 up-regulation.
Our objectives are:
1. To determine the role of specific endogenous RPs in p53 activation upon impairments
of various steps of ribosome biogenesis.
2. To elucidate the molecular mechanisms by which specific RPs activate p53 under
these conditions.
Material and Methods:
Ribosome biogenesis was inhibited with actinomycin D, 5- fluorouracil or siRNAmediated
depletion of RPs. A number of biochemical as well as molecular and cellular
biological methods were used to analyze the source, stability and intracellular
localization of newly synthesized RPL5 and RPL11 upon impairment of ribosome
biogenesis, including treatment with protein synthesis or proteasome inhibitors, Western
blot analysis, qRT-PCR, immunoprecipitation, cellular fractionations and live-cell imaging
microscopy.
Results:
We showed that RPL5 and RPL11, but not RPL23, RPL26 and RPS7, are
required for p53 activation upon inhibition of ribosome biogenesis. Whereas several
other newly synthesized RP are degraded by proteasomes upon inhibition of Pol I
activity by actinomycin D, RPL5 and RPL11 accumulate in the ribosome-free fraction
where they bind to Mdm2. This selective accumulation of free RPL5 and RPL11 is due
to their mutual protection from proteasomal degradation. Furthermore, the endogenous,
newly synthesized RPL5 and RPL11 continue to be imported into nucleoli even after
nucleolar disruption and colocalize with Mdm2, p53 and PML.
Conclusions:
We demonstrate the key role of RPL5 and RPL11 in p53 activation by
impairments of ribosome biogenesis and uncover the molecular mechanisms of this
regulation. Our findings may have important implications with respect to understanding
the pathogenesis of diseases caused by impaired ribosome biogenesis.
Keywords
Inhibicija sinteze ribosoma
Jezgrica
Ribosomski proteini
Proteasom
Sinteza ribosoma
Supresor tumora p53.
Keywords (english)
Inhibition of ribosome biogenesis
Nucleolus
p53 tumor suppressor
Proteasome
Ribosome biogenesis
Ribosomal proteins.
Language croatian URN:NBN urn:nbn:hr:188:247520 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL http://libraries.uniri.hr/cgi-bin/ucat/unilib.cgi?form=D1130410067 Type of resource Text Extent 125 str File origin Born digital Access conditions Open access Terms of use Created on 2017-01-19 18:53:43
