Role of the ESCRT complex in Trypanosomatids: functional characterization of Vps32.

NADIA M. BARRERA; TAMARA STERNLIEB; PATRICIO D. GENTA; MILENA MASSIMINO STEPÑICKA; ALEJANDRA C. SCHOIJET; GUILLERMO D. ALONSO

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB). XXIX Reunión Anual de la Sociedad Argentina de Protozoología (SAP).; 2017

CONJUNTO DE SOCIEDADES DE BIOCIENCIAS

The ESCRT (Endosomal Sorting Complex Required for Transport) is a machinery that drives a diverse collection of membrane remodeling events such as endocytosis, multivesicular body biogenesis, autophagy, release of enveloped viruses, reorganization of the nuclear envelope and cytokinetic abscission during mitosis exit. This complex is composed of four subcomplexes (0-III) been ESCRTIII the effector of the complex. Vps32 is the most abundant protein in ESCRTIII and the abscission capability is given for its molecular structure alternating between a monomeric-closed state to polymeric-open state. Here we investigate the conservation of Vps32 in Trypanosoma cruzi and Trypanosoma brucei. The Saccharomyces cerevisiae gene corresponding to the Vps32 (Snf7) was used to screen TriTrypDB databases. We found the T. cruzi orthologue (TcVps32) which have two alleles in CL Brener strain: TcCLB.511589.250 (Esmeraldo) and TcCLB.511229.100 (Non Esmeraldo). These sequences were used to screen T. brucei database resulting in a high-scored target: Tb427tmp.01.1390. Protein domains, secondary structure and isoelectric point were determinated showing the presence of Snf7 motif, alpha helices with the MIM motif in the last helix and basic N-terminal and acid C-terminal which implies a structure conservation among eukaryotic cells. To perform a functional characterization, TcVps32 coding sequence was amplified by PCR fused to a hemagglutinin tag at N-terminal (HA-TcVps32) or without tag (TcVps32). The PCR products were subcloned into pRIBOTEX and the constructs (pRibo-HA-TcVps32 and pRibo-TcVps32) were transfected in T. cruzi epimastigote cells. Simultaneously, in T. brucei procyclic form we designed an RNAi strategy where a 405bp of TbVps32 was cloned into the p2T7 vector (TbVps32-RNAi) which allow a tetracycline inducible silencing of this protein. The TcVps32 overexpressing T. cruzi epimastigotes and the procyclic form of T. brucei transfected with TbVps32-RNAi are under evaluation.