The Functional Analysis of Granulins

UoM administered thesis: Master of Philosophy

  • Authors:
  • Kathryn Young

Abstract

.AbstractProgranulin (PGRN) is a 593 amino acid secreted glycoprotein which is encoded for by the GRN gene. It is ubiquitously expressed in a number of tissues and is associated with numerous cellular functions including angiogenesis, metabolism and acting as a neurotrophic growth factor. The full-length protein comprises 7.5 tandem granulin repeats containing highly conserved cysteine repeats. The full length PGRN can be cleaved by neutrophil elastase and other metalloproteases to produce a series of 6kDa peptides which, in some instances have been shown to have opposing effects on cell proliferation and inflammation to the parent molecule. Levels of PGRN have been shown to be raised in some cancers, inflammation, autoimmune disease and wound healing however it is PGRN haploinsufficiency caused by mutations in the GRN gene that has been implicated in Frontotemporal Lobar Degeneration (FTLD).Although PGRN and the granulins obviously play a significant role in disease processes, it has not been possible to identify a specific measurable function or activity regulated by these proteins. For this reason we decided to investigate the differential gene expression produced by treating differentiated neural cells with exogenous PGRN and granulin peptides in order to try to elucidate specific biological processes which could lead to the identification of disease markers or therapeutic targets.Pure PGRN and GRN peptides were produced by synthesising sequence-specific DNA, cloning into a Halo®-tagged expression vector and expressing in HEK293 cells. The resultant proteins were purified by HPLC and used to treat fully differentiated SH-SY5Y cells. Post-treatment cells were lysed and used to produce cDNA for RNA Seq analysis and real time PCR or protein for validation experiments.The RNA Seq data produced identified a number of genes up and down-regulated in all treatments. PGRN treatment resulted in the down-regulation of several genes associated with both the proteosome and the spliceosome pathways. The fact that, in addition, there was also a trend towards significance in the down-regulation of the spliceosome by granulin treatments suggests that this may be a common reaction of both the parent molecule and its breakdown components. In addition the down-regulation of CHMP1A and CHMP5 may suggest a role for the ESCRTlll pathway in PGRN mediated FTLD.The identification of these pathways may provide evidence for the selection of potential candidates for putative treatment options although further evaluation and validation of the data will be required.

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Original languageEnglish
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Award date1 Aug 2015