Design, synthesis, and applications of probes to monitor post-translational lipid modifications and protease activity in cells

Abstract

Post-translational modifications create a diverse set of proteins with different functions and activity from a single genome. As a result of post-translational modifications protein activity can be tightly regulated and the protein’s cellular location can change. Furthermore, when proteins change location, they can activate or inhibit signaling pathways that are necessary for cell cycle progression. This thesis has four different chapters with the first chapter describing protein prenylation, a post-translational lipid modification. Before prenylation, proteins have a cytosolic localization and after prenylation proteins localize to membranes. Thus, chapters 2 and 3 discuss methods to track the localization of prenylated proteins and peptides in cells using fluorescence microscopy. Finally, chapter 4 discusses the design and synthesis of an activity based probe to measure MMP activity in cells and discusses initial experiments to measure MMP-14 activity in cells. Prenylomics is a type of proteomics that involves adding isoprenoid analogues containing an alkyne to cells, then using click chemistry to identify and quantify the types of prenylated proteins in cells. The results from these experiments can identify novel prenylated proteins or track how the amount and type of prenylated proteins change in different diseases. However, it is important to ensure proteins localize properly when modified with isoprenoid analogues to ensure the results obtained are biologically relevant. Additionally, there are two types of protein prenylation, farnesylation and geranylgeranylation. One isoprenoid analogue, referred to as C15AlkOPP, is a dual substrate for farnesylation and geranylation. Thus, this probe identifies the amount and type of prenylated proteins in cells, but does not indicate if the protein is farnesylated, geranylgeranylated, or both. Thus, the first aim of this work involved treating cells expressing GFP-H-Ras with stain, which depletes endogenous isoprenoids and causes GFP-H-Ras to mislocalize to the cytoplasm. Then, different isoprenoid analogues were added to determine how efficiently the probe is incorporated into farnesylated proteins by measuring the amount of GFP-H-Ras plasma membrane localization. The work in this chapter identified that shorter isoprenoid analogues, C15AlkOPP, are more efficient at restoring farnesylation than longer isoprenoid analogues, such as C15PentOPP. Additionally, this work also determined that the bioactive diphosphate, C15AlkOPP is more efficiently incorporated into farnesylated proteins in cells compared to the alcohol precursor, C15AlkOH. Recently, chaperone proteins, smgGDS-607 and smgGDS-558, have been shown to regulate protein entry into the prenylation pathway, and trafficking of prenylated proteins to their respective cellular membrane, respectively. Several proteins containing a polybasic region can interact with smgGDS-607 and smgGDS-558 in their pre-prenylated and prenylated form, respectively. In chapter 3 a K-Ras peptide was synthesized containing the polybasic region and prenylation motif. The peptide contained a NDBF protected cysteine, which spatiotemporally initiates prenylation by exposing the C-terminal cysteine to Prenyltransferase enzymes. Additionally, a fluorophore was attached to the K-Ras N-terminus and a cell penetrating peptide was attached to the K-Ras peptide using a disulfide bond at the N-terminus of TAT and K-Ras. Initial experiments indicated endosomal entrapment was preventing cytosolic delivery, so a different peptide was synthesized and cytosolic delivery was achieved, but no plasma membrane localization was detected after the NDBF protecting group was removed with UV light which indicates the peptide was not prenylated. Although, the initial goal was not achieved in this work, this work indicates that FTase and GGTase activity may be tightly regulated when apoptosis is initiated, which is why no plasma membrane localization was observed after delivering peptides to the cytoplasm. Two observations that occur in aging cells include lipid accumulation, and accumulation of senescent cells. It is hypothesized that MMP, matrix metalloprotease, activity may be dysregulated in senescent cells which may contribute to chronic disease development during aging. Thus, in chapter 4 an activity based probe was designed and synthesized to measure MMP activity in cells. MMP activity is essential for embryonic development, wound healing, and angiogenesis. However, it’s activity must be tightly regulated because aberrant activity is involved in fibrosis and cancer. In a bulk cell analysis of senescent cells, there was an increased amount of MMP-14 activity measured. However, there are several different subpopulations of senescent cells, so it is essential to have a probe capable of measuring MMP-14 activity at a single cell level. In this chapter, a covalent MMP inhibitor, was synthesized, then modified to contain an alkyne using organic synthesis. Then, an in-gel fluorescence assay was used to identify labeled proteins, which include MMP-14 and other MMP enzymes. The work in this chapter identified a change in protein labeling when treated with statin which may be related to activation of an ERRα transcription factor.