The calcium and integrin binding protein (CIB) family is comprised of four evolutionarily conserved members (CIB1-4) that have diverse roles within cells. Whilst there appears to be some overlapping roles and interactions between the CIB family members, such as the interaction with integrin αIIbβ3, they also perform distinct roles within cells. CIB1 is involved in the regulation of cell survival and proliferation, cell migration, cell adhesion and apoptosis, as well as calcium signalling. Whereas CIB2 predominantly plays a critical role in hearing and mechanoelectrical transduction. CIB family members have also been found to have opposing roles within cells, where CIB1 promotes pro-proliferative and pro-migratory signalling in cancer, whilst CIB2 overexpression hinders neoplastic growth and cell migration. The broad cellular expression and involvement of CIB1 and CIB2 proteins in countless cell signalling pathways makes the discovery of their interaction networks of key interest to inform the dynamic role of this family of proteins within cells.
A novel proximity-dependent labelling system, Antibody and Small-tag Assembly on Proteins for Interaction Detection (ASAP-ID), was applied to CIB1 and CIB2 to allow for the discovery of novel interacting proteins. An APEX2-nanobody fusion was expressed, purified and applied to cells overexpressing either CIB1 or CIB2 to allow for proximity labelling of interacting proteins within fixed cells. Labelling of interacting proteins is validated by immunofluorescence and western blot. Biotinylated target proteins are identified using proteomics (LC/MS/MS). A proximity ligation assay (PLA) was established to demonstrate the detection of CIB1 interactions which will allow for the validation of novel protein partners discovered. In addition, we observe the changes in CIB1 interactions that occur under various stimuli known to alter CIB1 activity.
We aimed to leverage the novel ASAP-ID technique to further map CIB1 and CIB2 interactions beyond what has been achieved by conventional interaction studies. Further characterisation of CIB1 and CIB2 interactions will provide greater insight into their functions and establish unique or overlapping roles between the two proteins. Additionally, further characterising the interactome of CIB1 will provide insight into its role in signalling pathways and allow validation of CIB1-directed therapeutics for cancer.