INTECH   27907
INSTITUTO TECNOLOGICO DE CHASCOMUS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
TGFBR2-SE, a new soluble human TGF-β type II receptor isoform, is sumoylated, interacts with cytoplasmic and nuclear proteins, and is secreted on exosomes
Autor/es:
BERTOLIO, MS; PERONE, MJ; RODRIGUEZ, TM; DEWEY, RA; VELASCO ZAMORA, J
Lugar:
Mar del Plata
Reunión:
Congreso; ReuniĆ³n Conjunta de SAIC, SAI y SAFIS; 2018
Resumen:
We have recently described in human cells a new splicing variant of TGF-β type II receptor lacking the last 63 nucleotides of exon II and the first 86 nucleotides of exon III. This deletion of 149 nucleotides causes a frameshift with the appearance of an early stop codon rendering a truncated protein of 80 amino acids lacking the transmembrane domain, known as TGFBR2 soluble endogenous (TGFBR2-SE). Based on a computational sumoylation analysis we found that TGFBR2-SE has three potential non-concensus sumoylation lysines (K76, 77 and 78). To test whether TGFBR2?SE is actually sumoylated, we performed protein immunoprecipitation of 293T cell lysates with a specific -TGFBR2-SE pAb, followed by immunoblotting with mAbs (-TGFBR2?SE, and -SUMO1). In this way we found that the new isoform is posttranslationally modified by sumo addition. For further protein characterization, and to allow increased expression in mammalian cells, we codon optimized the TGFBR2-SE cDNA. Furthermore, to ease protein purification and enhanced in vivo half-life, we fused it in frame with the human IgG1 Fc domain, and expressed it in 293T cells by means of a lentiviral vector (Lv.TGFBR2-SE/Fc). Additionally, we identified the interactome of purified TGFBR2-SE/Fc by screening of the Huprot v3.0 Human Proteome array. This assay indicated that TGFBR2-SE is able to bind to a panel of 155 proteins. This set of proteins was compared with exosomal Vesiclepedia data using FunRich, indicating that TGFBR2?SE interacts with 79 proteins present in exosomes, of which 67 are found either only in the cytoplasm (22), only in the nucleus (11) or both (34). Finally, we confirm exosomal secretion of TGFBR2-SE and the Fc-tag protein by Western blot of 293T cell microvesicles with mAbs (-TGFBR2-SE and -CD63). These results show for the first time important TGFBR2-SE characteristics contributing to unravel the function of the newly described isoform.