Interactions between protein Itch, p63/p73 and mutated p53 in tumor cells

Abstract

Tumor-supresorski gen TP53 mutiran je u više od 50% tumora čovjeka. Zbog promjena u konformaciji, mutirani protein p53mut veže se za p53 divljeg tipa te p63 i p73, članove porodice p53 i tako ih inaktivira. Nisku razinu proteina p63 i p73 u stanicama održava ubikvitinska ligaza ITCH koja omogućuje njihovu razgradnju putem proteasoma. Metodom koimunoprecipitacije i analizom metodom Western blot određivala sam interakcije između proteina ITCH, p63/p73 i p53mut u tumorskim stanicama. Potvrdila sam da dolazi do stvaranja interakcija između egzogenog ITCH (divljeg tipa i ITCHmut) i izoformi TAp63α, ΔNp63α, TAp73β i ΔNp73α u stanicama koje ne eksprimiraju p53 kao i u stanicama koje eksprimiraju p53mut (p53R248W). Plazmidne vektore s ugrađenim genima za TAp63α, ΔNp63α, TAp73β i ΔNp73α kotransfecirala sam s vektorom za ITCHmut u stanice koje eksprimiraju p53R248W kako bih utvrdila utječe li ubikvitinska ligaza ITCH na stvaranje heterokompleksa između p53mut i drugih članova porodice p53. Pokazala sam da ITCHmut ne utječe na stvaranje heterokompleksa između p53mut i izoformi TAp63α, ΔNp63α i TAp73β, dok je intenzitet vezanja za ΔNp73α bio jači u prisutnosti ITCHmut. Iako je ITCH glavni regulator razine p63 i p73 u stanicama, moguće je da ima zanemarivo djelovanje na interakcije između p53mut i p63/p73.Tumor suppressor gene TP53 is mutated in over 50% of human tumors. Altered conformation enables mutated p53, p53mut, to form heterocomplex with wild type p53 as well as with p63 and p73, the members of p53 family, making them inactive. Low levels of p63 and p73 are maintained by ubiquitin ligase ITCH and subsequent proteasomal degradation. Performing coimmunoprecipitation assays followed by Western blot analysis I have determined protein interactions between ITCH, p63/p73 and p53mut in tumor cells. I confirmed the interactions between exogenous protein ligase ITCH (both wild type and functional mutant) and TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms in p53 null and p53 mutant cell line (p53R248W). Expression vectors encoding for TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms were cotransfected with mutated protein ITCHmut into the cells expressing p53R248W in order to analyze the effect of ubiquitin ligase ITCH on interactions between p53mut and p63/p73. I have shown that ITCHmut does not affect heterocomplex formation between p53mut and TAp63α, ΔNp63α and TAp73β, while ΔNp73α forms stronger interactions when ITCHmut was present. Although ubiquitin ligase ITCH is the main regulator of p63/p73 protein levels in cells, it has negligible effect on p53mut and p63/p73 interactions