Functional characterisation of the SH3BGRL3 protein
Creators: name: DI PISA, FILIPPO
Publisher: name: Universit� degli studi di Genova
Year: 2020

description: SH3-domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) belongs to the SH3BGR family, it is ubiquitously expressed and encodes for a 93 AA thioredoxin-like evolutionary conserved protein. Its altered expression and tumor promoting role were described in certain malignancies e.g. lung, papillary and urothelial cancer. SH3BGRL3 shows a significant homology with Escherichia coli Glutaredoxin 3 however it is devoid of potential redox enzymatic activity, lacking the enzymatic site. This suggests some possible role as a modulator of GRXs activities or other proteins (e.g. its potential binding to Grb2 adaptor or EGFR proteins was emphasized in certain cell lines). Our previous co-immunoprecipitation experiments demonstrated that SH3BGRL3 (and SH3BGRL as well) is able to interact Myo1c (via IQ domains) motor protein in SKBR3 cell line. Here we investigated the involvement of Ca2+ on the condition of this binding through pull-down experiments in SKBR3 cell lysates, the localization of the SH3BGRL3/Myo1c complex in MDA-MB-231 model and further functional role of SH3BGRL3 protein in correlation with tumor cell growth, survival and metastatic behavior were studied. Co-IP, pulldown, genetic modification (over-expressing and silencing) experiments, cellular localization studies, Western blots analysis, migration and invasion assays, metabolic analysis and tamoxifen sensitivity assays were performed in selected breast cancer cell lines (SKBR3, MDA-MB-231 and MCF-7) in order to investigate the functional role of the SH3BGRL3 protein. In this work, the Ca2+ dependence of SH3BGRL3/Myo1c interaction were highlighted in SKBR3. The SH3BGRL3/Myo1c complex formation was also investigated in the highly invasive MDA-MB-231 cell line. However, such ability to complex formation of SH3BGRL/Myo1c could not be detected in this cell line, differently from what we sound in SKBR3 model. Moreover, our results could not confirm that SH3BGRL3 (or SH3BGRL) interact with ErbB2, EFGR or Grb2 protein adaptor. In addition, the SH3BGRL3/Myo1c/actin complex formation and the role of SH3BGRL3 in cell motility, migration and invasion were shown in the MDA-MB-231 cell line. This motility regulatory function was SH3BGRL3 protein specific in our model, e. g. the other member of the protein family SH3BGR (e.g. SH3BGRL) had no such effect and was not able to co-immunoprecipitate with Myo1c. It was an interesting observation that Myo1c is detectable in exosomes isolated from MDA-MB-231 suggesting this protein as a potentially useful exosomes marker. The specific tumorigenic roles of the SH3BGRL3 protein were further confirmed using SH3BGRL3 negative MCF-7 cell line in our experiments. Decreased ER?, increased motility and metabolic alterations (elevated mitochondria oxidation and glycolytic function) were detected in case of SH3BGRL3 over-expression in genetically modified MCF-7 cells. In parallel with these, 4?OH-TAM sensitivity was reduced in vitro compared to control MCF-7. Based on these findings, SH3BGRL3 protein expression could influence cell motility, cell invasiveness and it could be involved in resistance against the hormone therapy of breast cancer cell lines.