Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Visvanathan Ramamurthy

Committee Co-Chair

Andrew Dacks

Committee Member

Peter Mathers

Committee Member

Maxim Sokolov

Committee Member

Shuo Wei

Abstract

The efficient folding, assembly and trafficking of phototransduction proteins from site of synthesis in the inner segment (IS) to the site of action in the outer segment (OS) is crucial for photoreceptor cell survival and function. Defects in this process are known to cause blinding diseases in humans including retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA). The lack of a therapeutic approach for treatment of various retinal degenerations is likely due a knowledge gap in the mechanisms underlying the folding, assembly and transport of prenylated phototransduction proteins. Prenylation and " CAAX" processing is thought to be involved not only in membrane anchorage of proteins but also in trafficking and regulating interactions between proteins. However, in-vivo experimental evidence scrutinizing the role of prenylation in retinal neurons is absent. Moreover, there are a growing number of discoveries linking prenylation defects with different retinal disorders, such as RP, LCA, rod and cone dystrophy, achromatopsia. The purpose of my dissertation is to understand the role of prenylation in biosynthesis, transport and function of key players of phototransduction cascade in photoreceptor neuron. To investigate the role of prenylation and methylation in photoreceptor neurons, we created mice models lacking prenyl transferases (chapter 2 and 3) and methyl transferases (chapter 4) in photoreceptor neurons. In Chapter 1 of this dissertation, we discuss the general significance of prenylation in photoreceptor neurons. We focused on the essential role of prenylation in the function and stability of a variety of prenylated proteins involved in phototransduction pathway. In Chapter 2, data is presented from the first animal model we generated that lacks prenylation in cone photoreceptor neurons. In the study, we demonstrated that the geranylgeranyl lipid anchors on cone PDE6 acts as a "molecular grip" to facilitate either the interaction between cone PDE6 and chaperone AIPL1 or assembly, a step needed for synthesis of functional PDE6 in cones. In chapter 3, we investigated defects in retina and in various phototransduction protein due to lack of prenylation in retina. Here we illustrate that lack of farnesylation affects the proper localization of rod transducin and also results in defective translocation kinetics. Our findings from this works also shows that single lipid anchor on PDE6 is sufficient for its assembly. Chapter 4, we discuss the potential role for ICMT mediated methyl esterification in photoreceptor morphogenesis and function. Our study demonstrates the in-vivo requirement of ICMT- mediated methylation of transducin gamma and cone PDE6 for their membrane anchorage highlighting the key role of ICMT in retinal neurons. Finally, in Chapter 5 we discuss the most significant and novel findings from our work and strategies to fill the gaps in knowledge that remain concerning the role of prenylation in photoreceptor cells. Overall, our findings highlight the intricate role of prenylation and methylation in photoreceptor neurons in-vivo.

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