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Title: Intracellular trafficking and regulation of variant B cystatin C in retinal pigment epithelial cells
Author: Riccio, A.
ISNI:       0000 0004 6422 576X
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Cystatin C is an important inhibitor of lysosomal and extracellular cysteine proteases. It is synthesized as a precursor containing a signal sequence that targets the protein to the secretory pathway. In retinal pigment epithelial (RPE) cells, cystatin C is abundantly expressed and secreted, suggesting a critical role in the regulation of the proteolytic activity in the extracellular space, which, if altered, may contribute to diseases such as Age-related Macular Degeneration (AMD). An amino acid substitution in the signal sequence of the precursor cystatin C generates the variant B, which has been linked to an increased risk of developing AMD and Alzheimer's disease (AD). This mutation has been reported to affect protein trafficking, resulting in decreased secretion and mitochondrial mislocalization. Recently, however, both its disease association as well as the effect on the protein trafficking have been questioned. The main aim of my study was to further investigate the trafficking and secretion of the mutated cystatin C. Recombinant wild-type and variant B cystatin C constructs, untagged or linked to different tags (EGFP, FLAG, HA), were transfected into RPE cell lines. Intracellular localization was investigated by imaging and subcellular fractionation. Protein trafficking and secretion were examined by analysing protein release into the culture media and the use of inhibitors of the secretory pathway. In addition, glycosylation sites were engineered to monitor transition through the Endoplasmic Reticulum (ER) and Golgi apparatus. Constructs encoding the signal sequence linked to EGFP were also used to evaluate the targeting properties of the variant B signal sequence. In the second part of my study, the efficiency of signal sequence cleavage was investigated by analysing the levels and localization of the precursor fraction by immunoprecipitation and imaging. The presence of alternatively cleaved forms and glycosylation modifications was also analysed, using immunodetection, mass spectrometry and deglycosylation treatments. In the last part, mechanisms of protein regulation, including degradation, internalization, dimerization were investigated to identify possible differences between the wild-type and variant B proteins. Interacting proteins were identified by mass spectrometry. My results showed no major differences in trafficking and regulation between wild-type and variant B proteins. Only the EGFP-tagged constructs resulted in mistrafficking to mitochondria, and this was the case for both cystatin C forms, suggesting that EGFP is not a suitable tag to study cystatin C and excluding mitochondrial mislocalization as a factor contributing to disease association. There were also no differences in secretion, regulation and interaction partners between wild-type and variant B. However, precursor processing for the variant B was slightly less efficient, resulting in a slightly higher fraction of non-processed precursor protein and an alternatively cleaved, possibly O-glycosylated form. The mutation in the signal sequence therefore appears to affect the precursor processing rather than targeting. These forms may accumulate over-time generating proteolytic imbalance and protein aggregates, which ultimately may lead to cellular dysfunction and degeneration associated with AMD and AD. Interestingly, inhibition of the ubiquitin-proteasome system or the autophagy-lysosome pathway, which become functionally impaired during aging and disease, led to a decrease or increase of cystatin C protein levels, respectively, suggesting a possible involvement in the altered levels of cystatin C in the structural and functional changes seen in age-related conditions.
Supervisor: Tew, S. T. ; Jackson, M. J. ; Hunziker, W. H. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral