Dissolution Dynamic Nuclear Polarization of Polypeptides

Nuclear Magnetic Resonance (NMR) spectroscopy provides remarkable site resolution, but often requires signal averaging because of low sensitivity. Dissolution dynamic nuclear polarization (DNP), which offers large signal enhancements, has been used to follow reactions involving small molecules that typically have long spin-lattice relaxation times. This thesis presents work in the development of dissolution DNP to directly hyper-polarize and observe polypeptides, which can subsequently be used for the study of a time dependent process, such as folding. Dissolution DNP involves hyperpolarizing samples in the solid state, dissolving the samples with a stream of hot solvent and rapid transfer of the sample into an NMR tube for measurement in the solution state. Since protein samples are prone to foam under conditions for rapid sample injection, solvent systems were optimized. Solvents such as water/acetonitrile and water/methanol mixtures were utilized. An unlabeled peptide, bacitracin, was hyperpolarized on ^(1)H nuclei and enhancements of 30, 45 and 180 were obtained for amide, aliphatic and aromatic protons respectively. Although these enhancements are already significant, loss of hyperpolarization during sample injection was further alleviated by the use of isotopically enriched polypeptides. In [^(13)C, 50% ^(2)H] labeled samples of denatured L23, a 96 amino acid long ribosomal protein, signal enhancements of more than 500 times were obtained on the ^(13)C nuclei. This signal enhancement was then exploited to follow the protein folding process, using L23 as a model. Time resolved spectra of hyperpolarized L23 were measured after a pH jump and protein folding was monitored by observing changes in the carbonyl region of the spectra, which are indicative of the formation of secondary structures. Despite signal overlap in the protein spectra, using the statistical distribution of ^(13)C chemical shifts, the fractions of secondary structure elements were estimated for each scan of the DNP-NMR experiment. Additionally, individual resonances for methyl groups upfield of other protein resonances became resolved in the later transients. An option for the improvement of such site resolution by NMR experiments using coherence selection is discussed. While DNP-NMR offers the capability to observe transient species, identification of such species is difficult in cases where not all chemical shifts are known. Here, a new strategy for the analysis of DNP-NMR data is proposed based on non-negative matrix factorization (NNMF). NNMF enables identification of various sources that contribute to an observed signal. This capability is demonstrated using a series of spectra measured from an enzymatic conversion reaction of oxaloacetic acid to malic acid. Simulations were carried out to evaluate the performance of NNMF under different experimental constraints, and the strengths and limitations of the method are discussed based on the simulations

In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase

Hyperpolarized Dihydroxyacetone Is a Sensitive Probe of Hepatic Gluconeogenic State

Type II diabetes and pre-diabetes are widely prevalent among adults. Elevated serum glucose levels are commonly treated by targeting hepatic gluconeogenesis for downregulation. However, direct measurement of hepatic gluconeogenic capacity is accomplished only via tracer metabolism approaches that rely on multiple assumptions, and are clinically intractable due to expense and time needed for the studies. We previously introduced hyperpolarized (HP) [2-13C]dihydroxyacetone (DHA) as a sensitive detector of gluconeogenic potential, and showed that feeding and fasting produced robust changes in the ratio of detected hexoses (6C) to trioses (3C) in the perfused liver. To confirm that this ratio is robust in the setting of treatment and hormonal control, we used ex vivo perfused mouse livers from BLKS mice (glucagon treated and metformin treated), and db/db mice. We confirm that the ratio of signal intensities of 6C to 3C in 13C nuclear magnetic resonance spectra post HP DHA administration is sensitive to hepatic gluconeogenic state. This method is directly applicable in vivo and can be implemented with existing technologies without the need for substantial modifications

Application of Carbon-13 Isotopomer Analysis to Assess Perinatal Myocardial Glucose Metabolism in Sheep

Ovine models of pregnancy have been used extensively to study maternal–fetal interactions and have provided considerable insight into nutrient transfer to the fetus. Ovine models have also been utilized to study congenital heart diseases. In this work, we demonstrate a comprehensive assessment of heart function and metabolism using a perinatal model of heart function with the addition of a [U-13C]glucose as tracer to study central energy metabolism. Using nuclear magnetic resonance spectroscopy, and metabolic modelling, we estimate myocardial citric acid cycle turnover (normalized for oxygen consumption), substrate selection, and anaplerotic fluxes. This methodology can be applied to studying acute and chronic effects of hormonal signaling in future studies

In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma.

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase