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

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

Pathobiology of T-lymphoma

T–limfomi su tumori nastali od vlastitih izmijenjenih T–limfocita. Tipovi T–limfoma se međusobno teško razlikuju ukoliko se ne klasificiraju pomoću više, npr. razlike u morfološkoj strukturi zahvaćenog tkiva, imunofenotipu i histokemijske razlike. Poremećen regulatorni mehanizam rekombinacije VDJ gena je najvjerojatnije jedan od prvih i glavnih uzročnika razvoja T–limfoma, osim u malom broju slučajeva kada nastane funkcionalni triplet koji donosi veću varijabilanost TCR-a. U većini slučajeva nastane nefunkcionalni triplet ili stop kodon. T–limfome najčešće karakteriziraju translokacije, no mogu nastati i zbog delecije, ili mutacije u genomu čija je posljedica promijenjena ekspresija panT antigena. Takve stanice su nefunkcionalne i ukoliko ne nastupi programirana stanična smrt nastaju T–limfomi. Smatra se da virusi, npr. EBV, HIV i HTLV1, imaju značajan utjecaj na nastanak T–limfoma. Uočena je povezanost u učestalosti T–limfoma i geografskog položaja gdje je pojedini tip virusa zastupljeniji. Uzevši u obzir crnu, bijelu i žutu rasu, tipovi T–limfoma su različito zastupljeni, što se povezuje sa genetskim sličnostima unutar rase, odnosno razlikama između rasa.T–cell lymphoma is a type of cancer developed from it's own altered T-lymphocytes. Types of T–cell lymphoma are difficult to differentiate unless they are classified using multiple properties, e.g. differences in morphological structure of the affected tissue, immunophenotype and histochemical differences. Disrupted regulatory mechanism of VDJ genes recombination is probably one of the first and major causes of the T–cell lymphoma development. In rare cases the cause is the occurrence of a functional triplet, which brings in higher variability of the TCR, instead of a non-functional triplet or a stop codon. T–cell lymphoma is most often characterised by translocations, but it can be also developed by deletion or genome mutation resulting in changed expression of the panT antigen. These kind of cells are non-functional and if a programmed cellular death does not happen, the T–cell lymphomas develop. It is considered that viruses, e.g. EBV, HTLV1 and HIV have significant influence on the T–cell lymphoma formation. A correlation between the frequency of T–cell lymphoma appearance and geographic location is also perceived. Considering black, white and Asian races, T–cell lymphoma types have different occurrence that is linked to genetic similarities within a particular race as well as to differences between those races

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

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

NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix

Background NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho- histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. Results We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero- oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. Conclusions NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins