Advancements in prenylomic analysis: development of new probes, applications to Alzheimer’s disease, and extension to other lipidation types

Abstract

Protein prenylation, a modification involving the addition of an isoprenoid group to proteins near their C-terminus, occurs in approximately 2% of the proteome. The three types of prenylation are farnesylation, type I geranylgeranylation, and type II geranylgeranylation and are linked to the pathogenesis of diseases such as malaria, cancer, Alzheimer's disease, aging, and progeria. These enzymes modify proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP), and due to variance in isoprenoid selectivity analogues with bioorthogonal functionality have been developed. A wide range of bioorthogonal probes, that utilize a wide variety of click chemistry, have been developed to study prenylation. The ability to track many prenylated proteins at once is paramount to determine the role(s) prenylation has in different diseases. In pursuit of this mammalian cells are treated with one of bioorthogonal analogue, which will be transferred to prenylated proteins using the endogenous prenylation enzymes. This metabolic incorporation of the analogue labels the target proteins with chemical handles that can be used to isolate the prenylated proteins for quantitative bottom-up proteomic analysis. Described here is the development of bioorthogonal analogues for the study of prenylation and expansion of the prenylomic method into AD mouse model systems. Additionally, this methodology was adapted for the identification of novel palmitoyl transferase substrates using an orthogonal enzyme substrate strategy. Chapter one introduces the history and current scope of prenylomic analysis using metabolic labeling experiments. Chapter two presents a novel norbornene-containing analogue, which undergoes an inverse electron demand Diels-Alder reaction, providing an alternative to traditional alkyne-based probes. This new approach identified a different subset of prenylated proteins in HeLa cells. Chapter three focuses on the development of a set of analogues that are longer than FPP and more similar to GGPP. Modulation of prenylation enzyme selectivity through probe length, which though prenylomic analysis, demonstrated that long probes selectively identified geranylgeranylated proteins compared to farnesylated proteins. Chapter four expands prenylomic analysis into AD mouse models which found 15 consistently over-prenylated proteins in the brain. This is the first prenylomic analysis in a live animal and it enabled identification of prenylated proteins related to AD pathogenesis. In chapter five the adaptation of the prenylomic methodology to identify novel palmitoylation, another type of lipidation, targets. In this work, two engineered palmitoyl transferase enzymes paired with a bulky palmitoyl analogue were used to overcome the complex substrate- enzyme matrix that has plagued previous proteomic investigations. Chapter six is a summary of work and proposed adaptations of the prenylomic workflow to increase reproducibility and robustness. Overall, this work is a collection of experiments that expand the prenylomic methodology into new systems or using new analogues.