Browsing by Subject "Cysteine"

Now showing 1 - 4 of 4

Biosynthesis of sulfur containing heterocycles in natural products
(2016-12) Gengler, Jon Peter; Liu, Hung-wen, 1952-
This thesis is a comprehensive review of the biosynthesis of sulfur containing heterocycles in natural metabolites. The review focuses on sulfur incorporation and cyclization of the moieties, with a lesser examination of the role these heterocycles play in the chemistry of their compound's activity.
Characterization of proteins and peptides via enhanced 266 nm ultraviolet photodissociation mass spectrometry utilizing a selenium based chromophore
(2016-05) Parker, William Ryan; Brodbelt, Jennifer S.; Eberlin, Livia S.
Mass spectrometry and chemical derivatization have been used as tools for the identification of proteins in both top-down and bottom-up studies. Cysteine is the rarest and most nucleophilic amino acid thus making it a popular target for chemical tagging strategies. Ultraviolet photodissociation (UVPD) is a versatile activation technique for fragmentation of peptides and proteins. For successful photodissociation, ions of interest must contain a suitable chromophore that matches the wavelength of irradiation. N-(Phenylseleno)phthalimide (NPSP) is a fast reacting reagent which attaches a selenium based chromophore that absorbs at 266 nm light to free thiols. In the studies presented in this thesis, NPSP was used to derivatize free cysteine residues in both intact proteins and tryptic peptides. Activation with 266 nm photons causes a dominant neutral loss of the benzeneselenol groups on the tagged protein or peptide ions. This diagnostic neutral loss allows the determination of the number of free versus bound cysteine residues in intact proteins. Additionally, tagging peptides with benzeneselenol provides a means to target only the cysteine-containing peptides in bottom-up proteomics experiments. Both of these methods provide a significantly reduced search space for identification of cysteine-containing proteins. Counting the number of cysteine residues also provides an effective way to restrict the number of protein candidates for database searches. Moreover, cysteine peptides are inherently more unique than other peptides created upon enzymatic digestion of proteins due to the low frequency of cysteine in the proteome, thus allowing these peptides to be used as surrogates for protein identification.
Cysteine responsive copper(II) based ¹⁹F MRI probes
(2018-01-26) Enriquez, José Santiago; Que, Emily
Cysteine plays an important role upholding cellular homeostasis by maintaining a proper redox environment. Perturbations in cysteine concentration may lead to cardiovascular and liver disease, and cancer, making cysteine an important amino acid to detect and monitor. In order to detect cysteine in vitro and in vivo it is advantageous to choose an imaging tool with high tissue penetration, low background noise, and less radiation, which led us to ¹⁹F MRI. Herein we report a series of copper complexes with fluorine moieties appended to the macrocycle cyclam for use of ¹⁹F MRI detection of cysteine. In this study, an efficient “turn-on” response was observed upon reduction of the Cu(II) complexes by cysteine. This response was characterized using UV/Vis absorption spectroscopy, NMR, EPR, and ¹⁹F MRI. Early biological results suggest that these probes can detect cysteine in red blood cells, opening the doors for further animal studies.
Engineering and validation of a human cyst(e)ine degrading enzyme as a novel cancer therapeutic
(2017-05) Cramer, Shira Lyla; Georgiou, George; Stone, Everett; Alper, Hal; Maynard, Jennifer; Ehrlich, Lauren; DiGiovanni, John
Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. As a precursor for the biosynthesis of GSH, L-cysteine (L-Cys) availability is critical for maintaining the intracellular thiol redox potential and under conditions of elevated ROS, endogenous L-Cys production is insufficient for GSH synthesis. This necessitates the import of extracellular L-cyst(e)ine (predominantly in its disulfide form, L-cystine (CSSC)) to meet cellular antioxidant requirements. Since L-Cys is a non-essential amino acid in animals, eliminating L-Cys and CSSC uptake should selectively impact tumors that display increased ROS production and thus exhibit a higher demand for antioxidants, without causing an adverse effect on normal physiology. This can be accomplished by eliminating the extracellular pool of L-Cys and CSSC through the action of an enzyme that selectively converts these amino acids into non-toxic products. Unfortunately, no human enzyme displays sufficient catalytic properties towards both L-Cys and CSSC to be relevant for clinical applications. In the chapters that follow, we describe the engineering of a human L-Cys and CSSC degrading enzyme (cyst(e)inase) as a novel and potent therapeutic for tumors displaying elevated levels of ROS. We show that administration of cyst(e)inase mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates at a therapeutically useful rate. In a wide variety of models, treatment with this enzyme suppresses tumor growth in mice, yet results in no apparent toxicities even after months of continuous treatment. Through additional engineering, we describe the isolation of novel enzymes that display the requisites for clinical development; including increases in soluble protein yields and catalytic activity towards both L-Cys and CSSC, which together translates to practical doses for a human therapeutic at reasonable manufacturing costs. Finally, upon further investigation into the therapeutic effect of cyst(e)inase, we show that cyst(e)inase treatment dramatically inhibits metastasis, as well as suggesting an important role of ROS regulation and the immune system in a syngeneic mouse model.