The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

Abstract

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

NME/NM23/NDPK and Histidine Phosphorylation

“…The study of NME1-NME2 interaction in vivo by the use of FRET/FLIM revealed that NME1 and NME2 co-localize, with irradiation-induced DNA damage causing a small shift in NME1/NME2 homo-and hetero-isoenzyme ratios [83]. In agreement with this, it has been observed more recently that NME3 stimulates mitochondrial elongation dependent on oligomerization activity [84].…”

Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA damage responses

“…Numerous studies have confirmed a possible role in DNA repair for NN23-H1 and NM23-H2, human orthologous of TcNDPK1, through the base excision repair and nucleotide excision repair pathways (BER and NER, respectively) and also in DSB repair. (6,28) Consistent with this role, NM23-H1 was reported to be peri-nuclear and nuclear and rapidly translocate to sites of DNA damage Fig. 4: TcPARP activity.…”

“…Numerous studies have confirmed a possible role in DNA repair for NN23-H1 and NM23-H2, human orthologous of TcNDPK1, through the base excision repair and nucleotide excision repair pathways (BER and NER, respectively) and also in DSB repair. 6 , 28 Consistent with this role, NM23-H1 was reported to be peri-nuclear and nuclear and rapidly translocate to sites of DNA damage in the nucleus. 4 , 5 The mechanism underlying nuclear translocation is poorly understood because these enzymes lack a canonical nuclear localisation signal, although passive diffusion is also expected due to their small molecular weight.…”

“…For example, NME-H1 and NMEH-2 have been iden-tified as potential transcription factors through their DNA-binding activities; (3) and NME-H1 is also involved in the repair of DNA damages induced by UV and gamma radiation, bleomycin, cisplatin or etoposide in different cell lines, which is associated with an increased in its nuclear localisation. (4,5,6) The DNA related functions of NDPKs are also being investigated in different organisms other than mammals, such as bacteria, plants, yeasts and protozoan parasites. A plenty of reports associate them to DNA processing activities because, like human ones, they are able to interact and cleave DNA.…”

“…For example, NME-H1 and NMEH-2 have been identified as potential transcription factors through their DNA-binding activities; 3 and NME-H1 is also involved in the repair of DNA damages induced by UV and gamma radiation, bleomycin, cisplatin or etoposide in different cell lines, which is associated with an increased in its nuclear localisation. 4 , 5 , 6 …”