Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2-bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization. / October 2016

No description available.

Triazoles have been widely used as amide bond isosteres in chemical biology as linkers and to enhance proteolytic stability. The use of triazoles has grown exponentially since the discovery of the copper (I) catalysed alkyne azide cycloaddition reaction in 2002 as the reaction is solvent and functional group tolerant, and usually high yielding. The reaction is also orthogonal to reactions used in nature, meaning it has become a powerful coupling tool. In post-translational modification (PTM), proteins are modified by covalent attachment of functional groups to amino acid side chains. These PTM processes are generally thought to be dynamic and highly regulated by cell machinery, controlling protein function in response to stimuli. The ability to control function post protein synthesis allows organisms to have a smaller genome, which is advantageous as it reduces the energy required for DNA replication and repair. Research into the function of PTMs has been limited by the difficulty in generating recombinant proteins that bear a single PTM in a specific location. Although many elegant methods have been proposed that solve this problem, to date cysteine alkylation is one of the most successful techniques. For lysine PTMs, thia-lysine II (sLys) derivatives have been shown to be excellent mimics of lysine, where the only perturbation between the native lysine-containing analogue is the switch of a CH2 for S in the side chain. Biotin is a well-known PTM in biotin dependent carboxylases, where biotin is involved in CO2 transfer. Recently biotinylation has also been shown to be a PTM on many other proteins, however the role of biotinylation is not well understood. Biotin triazole III has been shown to be a good mimic of the biotin amide bond and retains excellent affinity to Avidin (Av). In Chapter 1 the effects of modification to the valeryl side chain, and orientation of the biotin triazole bond affect affinity to Av using ITC are investigated. Compounds III, V and VI are shown to have a KD < 120 pM, but further information on the binding affinity of these compounds could not be assessed by ITC. Biotin triazoles III-VI were also shown to be resistant to hydrolysis in serum, unlike the native biotin amide bond, which is hydrolysed by the enzyme biotinidase (BTD). Generation of amide sLys derivatives has been shown to be synthetically challenging. In Chapter 2, the synthesis and applications of chloromethyl-triazole biotin as a sulfhydryl selective alkylation reagent are investigated. The electron withdrawing nature of the triazole was proposed to give a ‘pseudo-benzylic’ halide α to the triazole, thus increasing reactivity. The controlled alkylation of peptides and proteins has shown that chloromethyl-triazole biotin shows enhanced reactivity over many commercial alkylation reagents and also gives good selectivity for cysteine. Alkylation of histone H4K12C gave the singly alkylated product, accompanied by low amounts of double alkylation. Biotinylation was confirmed by Western blot with anti-biotin. Due to the wide range of readily available functional azides, it was envisaged that halomethyltriazoles could be incorporated into other PTM mimics. In Chapter 3, efforts to expand the range of PTMs accessible using halomethyl-triazoles and further enhance the reactivity of chloromethyl triazoles by preparation of bromo- and iodomethyl triazoles are detailed. Synthesis of reagents to mimic malonylation, succinylation and GlcNAcylation PTMs is described and the reactivity of these halomethyl-triazole reagents is assessed. An alternate approach to the development of PTM mimics through cysteine propargylation and subsequent CuAAC coupling is also described in chapter 3. In conclusion, a series of new reagents have been developed to mimic protein PTMs through alkylation of cysteine. The reagents, which include biotin, GlcNAc, succinyl and malonyl mimics, are based on a halomethyl-triazole scaffold and have been successfully reacted with cysteine containing peptides and proteins.

Lysine methylation is a post-translational modification that regulates a large array of functionally diverse processes that are vital for cellular function. The role of methylation is best characterized on histone proteins due to their high concentration in the cell, but alongside histone modifications, lower abundance non-histone methylation is emerging as a prevalent and functionally diverse regulator of cellular processes. The direct biological impact of non-histone lysine methylation is less well understood because they are difficult to detect. The dynamic concentration range of the proteome masks their signal during proteomic analysis which impedes the detection of these low abundance methylated proteins. Increasing the concentration of proteins bearing methylation is required for improved discovery. This requires enriching the post-translational modification with a capturing reagent prior to analysis. This thesis details an optimized method for using the supramolecular host p-sulfonatocalix[4]arene as a stationary phase methyllysine enrichment reagent for real-life cell-extracted proteins. Prior to the optimizations described in this thesis, cell-derived peptide extracts were not retained within an early generation upper-rim modified calixarene column. But with the new protocols detailed in this thesis, proteins extracted from both cultured prostate cancer cells and industrially sourced brewer’s yeast were successfully retained by a lower-rim modified calixarene column. Thousands of methylated proteins with diverse functions and cellular localization were discovered using this method. Detection of low abundance methylated proteins will aid our discovery of all cellular methylation marks, which in turn, will help delineate their biological functions. / Graduate / 2020-11-30

The molecular target of epigenetic signaling is chromatin. Histones are extensively post-translationally modified (PTM), and many of these individual modifications have been studied in depth. As PTMs occur at multiple positions within histones, the degree to which these modifications might influence each other remains one of the major challenges of chromatin biology. Although major discoveries in understanding the complex repertoire of histone modifications were achieved using reductionist experimental systems with synthetic histone peptides, they do not explain the role of putative PTM cross-talks in a chromatin context. However, generating chromatin substrates of defined modification status has proved to be a technically challenging task. In this thesis, I first demonstrate our work on establishing a novel approach to produce libraries of modified nucleosomes. We employed protein trans-splicing and sortase-mediated ligation strategies to incorporate chemical modifications on histone tails of ‘ligation-ready’ nucleosomes. Subsequently, the ‘designer’ nucleosome libraries were used for testing the binding of heterochromatin protein 1 (HP1) and elucidated the previously uncharacterized crosstalk of H3K9me2 and S28ph marks. Further investigations explained the mechanism of this crosstalk and highlighted the importance of developing chemical biology tools for elucidating complex chromatin signaling. Second, I describe our reconstitution systems for the assembly of semisynthetic recombinant chromatin carrying methylation marks on DNA and distinct modifications on histones, e.g. H3K9me3. I aimed to understand the mechanisms of the interplay between chromatin and one of the DNA maintenance methylation factors, UHRF1. I showed that UHRF1 strongly interacts with nucleosomes containing linker DNA. However, it exerts only residual enzymatic activity in this context. Based on functional H3 ubiquitylation assays in vitro, I found that hemi-methylated nucleosomes stimulate enzymatic activity of UHRF1, suggesting that the protein’s chromatin targeting and activation are a two-step process. The positioning of hemi-methylated CpG on nucleosome regulates UHRF1 target selectivity. Further, mutational analysis revealed that the PHD domain of the factor is indispensable for H3 binding and that its SRA domain is required for catalytic activation. Overall, our work adds a new layer of positional complexity to the me½CpG-dependent regulation of UHRF1 and expands the current model of DNA methylation maintenance.

No description available.

No description available.

No description available.

Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content. / Ph. D.

Yes / Protein O-GlcNAcylation is a reversible post-translational modification of serines/threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. Drosophila melanogaster OGT/supersex combs (sxc) is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved, and the underlying mechanisms linking these phenotypes to embryonic development, are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, amongst others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development. / Wellcome Trust Investigator Award (110061); MRC grant (MC_UU_12016/5); and Royal Society Research Grant.