Browsing by Author "Doraisamy, Sri Suhashini"

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved, secreted protein. Structurally, MANF has a saposin-like and SAP-like domain that is commonly found in proteins in the lysosome and nucleus, respectively. In addition to it, it has a RTDL motif which is a KDEL-like sequence representing the ER retention signal. Previous studies have revealed that MANF has cytoprotective effects in several disease models, making it a putative therapeutic molecule. However, its exact function and molecular mechanism are yet to be elucidated. Therefore, knowing the exact function of MANF is crucial for its effective use as a therapeutic. Subcellular localisation is an effective way to determine a protein’s exact function as it provides an enclosed environment and surface for possible chemical interaction in which a protein can execute its specialised function(s). Due to the lack of studies on the subcellular localisation of endogenous MANF at a basal state and preliminary unpublished results from the group on MANF’s ability to regulate catalase mRNA expression, the aim of this study was to investigate MANF’s subcellular localisation through the development and validation of biochemical methods. Methods such as immunofluorescence, immunoelectron microscopy and biochemical fractionation were explored and validated for studying subcellular localisation. Here, the limitations of immunoelectron microscopy and biochemical fractionation are described, as the protocols used need further optimisation. Using immunofluorescence microscopy, MANF was detected particularly in the perinuclear region and ER. The findings are suggestive of MANF localisation but not conclusive due to the inadequate physiological condition of the cells; indicating the need for further optimisation of the methodology to study MANF’s localisation.