UNRAVELLING THE FUNCTION OF THE UNIQUE N-TERMINAL DOMAIN OF TRYPANOSOMA CRUZI HIGH MOBILITY GROUP B PROTEIN.

TAVERNELLI LE; ALONSO VL; PEZZA A; SERRA EC; CRIBB P

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

High Mobility Group B (HMGBs) are a family of chromatin pro- teins capable of binding non-canonical DNA structures through one or more ?HMG box? domains. These proteins are highly conserved among eukaryotic organisms and are involved in key nuclear pro- cesses like transcription, replication, recombination and DNA repair. The HMGB from Trypanosoma cruzi (TcHMGB) contains two ?HMG box? domains and a unique N-terminal sequence present only in trypanosomatids? HMGBs. In silico studies showed that within this region there is a nuclear localization signal (NLS) and a DEK-C terminal domain, a putative third DNA-binding domain in trypano- somes ́ HMGBs.We obtained parasites capable of overexpressing truncated forms of TcHMGB (N-terminal domain and TcHMGBΔN) and the full length TcHMGB as well. Immuno uorescence assays showed that the wild type version is located in the nucleus but when we overexpressed TcHMGB lacking the N terminal domain (TcHMGBΔN) the protein is distributed along the whole parasite but excluded from the nucle- us. On the other hand, when we overexpressed only the N-terminal region, this fragment accumulated in the nucleus but also it was detected in the cytoplasm. These results suggest that the NLS is necessary but not suf cient to drive the protein to the nucleus.We previously determined that overexpression of TcHMGB results in changes in chromatin structure that is detrimental for the parasite tness. We now evaluated the effect of overexpressing the N-terminal region alone, which caused a dramatic decrease in epimastig- otes growth. In order to clarify this phenotype we analyzed the cell cycle by ow cytometry with synchronized cultures in the G1 phase, showing a clear delay in the cell progression compared to the neg- ative control. In vitro infection assays showed that amastigotes rep- lication can also be affected. We propose that the DEK-C terminal domain may be another contact point of TcHMGB with nuclear DNA contributing to chromatin structure remodeling.