Subcellular localisation of the NME6 protein and anti-NME6 antibody testing

Abstract

Nukleozid-difosfatske kinaze (NDPK/Nm23/NME) čine obitelj evolucijski očuvanih enzima, koji prenose fosfatnu skupinu γ s andenozin trifosfata na nukleozid difosfate i na taj način održavaju ravnotežu nukleotida u stanci, a smatra se da imaju i neke druge biokemijske funkcije. Nukleozid-difosfatske kinaze sudjeluju u brojnim biološkim procesima kao što su proliferacija, diferencijacija i razvoj, a imaju značajnu ulogu i u razvoju tumora i metastaziranju. Unatoč brojnim istraživanjima, njihova biološka funkcija nije do danas u potpunosti razjašnjena. Deset članova obitelji proteina NME (od NME1 do NME10) podijeljeni su u dvije skupine. Skupinu I čine proteini od NME1 do NME4 i svi posjeduju aktivnost kinaze NDP, dok u skupinu II ubrajamo NME5 do NME10, od kojih većina ne pokazuje aktivnost kinaze NDP. Strukturno, NME6 dijeli svega 34-41% homologije s proteinima iz prve skupine, dok je njegov izražaj relativno nizak u većini tkiva. Malobrojna istraživanja upućuju na to da je NME6 smješten u mitohondrijima i u citoplazmi. Cilj diplomskog rada bio je odrediti lokalizaciju proteina NME6 u humanim tumorskim stanicama HeLa uz pomoć fluorescentnog reporterskog sustava, testirati antitijelo anti-NME6 te optimizirati metodu Western blot. U tu svrhu, koristili smo metode transformacije, transfekcije, fluorescentnu imunocitokemiju, analizu Western blot te konfokalnu mikroskopiju.Nucleoside diphosphate kinases are evolutionary conserved enzymes which transfer the γ phosphate from adenosine triphosphates to nucleoside diphosphates consequently maintaining the nucleotide homeostasis in the cell. Additionally, they are also considered to have other biochemical functions. NDP kinases are involved in several biological processes such as proliferation, differentiation and development. Moreover, they are proven to have a significant role in tumour development and metastasis. However, despite intensive research in the field, all of their biological functions still aren’t completely resolved. The NME family consists of 10 genes/proteins (from NME1 to NME10), divided into two groups. Group I encompasses proteins from NME1 to NME4 which all exhibit the NDP kinase. Group II consists of proteins from NME5 to NME10 most of which show no NDP kinase activity. Structurally, NME6 shares only 34-41% homology with Group I proteins, while its expression is relatively low in most tissues. Scarce data on NME6 indicates that it is located in the mitochondria and the cytoplasm. The aim of this thesis was to determine the localization of the NME6 protein in HeLa tumour cells using GFP reporter system, to test the anti-NME6 antibody, and optimise the Western blot method. For that purpose, we used transformation, transfection and fluorescent immunocytochemistry methods, Western blot analysis and confocal microscopy