Year: 2022
Type: dissertation
Abstract: The molecular targets for vitreoretinopathy and uveitis (intraocular inflammation) are poorly understood. Mutations in the calcium-activated cysteine protease, Calpain-5 (CAPN5), were discovered as the cause of Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV). CAPN5 is the first non-syndromic uveitis gene, and its discovery provides an unprecedented starting point for investigating the substantial gap in our understanding of the molecular basis of ocular inflammation. CAPN5 is a calcium-activated, intracellular protease expressed in many tissues, including photoreceptors and their nuclei. It is one of 15 calpain proteases, which cleave subdomains from target proteins to irreversibly change their function. ADNIV mutations are predicted to hyperactivate the CAPN5 protease, leading to aberrant cleavage of proteolytic targets and contributing to the retinal disease phenotype. The calpains are a relatively large, well-studied family, but little is known regarding the structure and mechanism of action of CAPN5, a non-classical calpain. Furthermore, little is known about CAPN5’s targets or the downstream sequelae of aberrant calpain activity in the retina and vitreous. In this thesis, I discuss the structure of the CAPN5 protease core domain (CAPN5-PC) and its structural and biochemical features that make it unique to its classical calpain counterparts. I also established the kinetic parameters of CAPN5 enzymatic activity in vitro and demonstrated that ADNIV-causing mutations are indeed hyperactive (Chapter 2). Insight gained from these studies may eventually be used to design future therapies for ADNIV and other calpain-associated pathologies. To help treat ADNIV patients in the near term, our team utilized a personalized proteomics approach to characterize the ADNIV vitreous proteome. In so doing, we identified immune mediators that could be targeted by available drugs and successfully repurposed these drugs for ADNIV therapy (Chapter 3). Utilizing a mass spectrometry-based approach, we further characterized ADNIV vitreous and identified differentially expressed proteins associated with the ADNIV clinical phenotype (Chapter 4). We further applied proteomics to identify the retinal substrates of CAPN5, and the preliminary data hints to sequential determinants of CAPN5 specificity (Appendix).
DOI: 10.17077/etd.006352
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4290643164