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Numerous studies have investigated how bulk lipid metabolism is influenced in obesity and in particular how the composition of triglycerides found in the cytosol change with increased adipocyte expansion. However, in part reflecting the analytical challenge the composition of cell membranes, and in particular glycerophospholipids, an important membrane component, have been seldom investigated. Cell membrane components contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for numerous proteins and providing important pools for metabolites, such as choline for one-carbon metabolism and S-adenosylmethionine for DNA methylation. Here, I have conducted a comprehensive lipidomic and transcriptomic study of white adipose tissue in mice that become obese either through genetic modification (ob/ob genotype), diet (high-fat diet) or a combination of the two across the life course. Specifically, I demonstrated that the changes in triglyceride metabolism that dominate the overall lipid composition of white adipose tissue were distinct from the compositional changes of glycerophospholipids. These latter lipids became more unsaturated to maintain the fluidity and normal function of the membrane in the initiation of obesity but then turned saturated after long-term administration of HFD and aging. This suggests that while triglycerides within the adipose tissue may be a relatively inert store of lipids, the compositional changes occur in cell membranes with more far-reaching functional consequences in both obesity and aging. The two-phase change of phospholipids can be correlated well with transcriptional and one-carbon metabolic changes within the adipocytes. The transcriptomic study demonstrated that the lipid metabolic pathways regulated by the peroxisome, AMPK, insulin and PPARγ signaling were activated in the initiation of obesity but inhibited in the adipose tissue of old ob/ob mice along with up-regulated inflammation pathways. The brown and white adipose tissue of PPARα-knock-out mice were also studied by lipidomic tools to get a deeper understanding of the effect of the peroxisome and PPAR system on adipose tissue and lipid metabolism during obesity. Most of the lipids were increased and became more saturated and shorter in adipose tissues of PPARα null mice, which is in good accordance with the results of the former animal study. In conclusion, my work using different rodent models and multi-omics techniques demonstrated a protective metabolic mechanism activated in the initiation but impaired at the end of the processes of obesity and aging, which could be an explanation of the similarity of obesity and aging in terms of high incidence of the metabolic syndrome and related diseases.

Cells must regulate their metabolism in order to grow, adapt to changes in nutrient availability and maintain homeostasis. Flux, or the turnover of metabolites, through the metabolic network can be regulated at the allosteric and transcriptional levels. While study of allosteric regulation is limited to biochemical examination of individual proteins, transcriptional control of metabolism can be explored at a systems level. We endeavored to elucidate transcriptional mechanisms of metabolic flux regulation in the model organism Caenorhabditis elegans (C. elegans). We also worked to create a visual tool to explore metabolic pathways that will support future efforts in the research of metabolic gene regulation. C. elegans is a small, free-living nematode that feeds on bacteria and experiences a high level of diversity in nutrient level and composition. Previously, we identified a mechanism by which the essential cofactor, vitamin B12, regulates the expression of genes involved in the degradation of propionate, referred to as B12‑mechanism‑I. This mechanism functions to prevent the toxic accumulation of propionate and requires the TFs NHR-10 and NHR-68. Using genetic screens as well as transcriptomic and metabolomic approaches, we discover a second mechanism by which vitamin B12 regulates metabolic gene expression: B12-mechanism-II. Unlike B12-mechanism-I, B12-mechanism-II is independent of propionate, requires the transcription factor NHR-114 and functions to maintain the metabolic activity of the Methionine/S-adenosylmethionine cycle in a tightly regulated regime. We also present WormPaths, an online resource that allows visualization of C. elegans metabolic pathways and enables metabolic pathway enrichment of user-uploaded transcriptomic data.

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No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.

Strikt anaerobe Bakterien wie Clostridium difficile und C. scatologenes verwenden GRE, um die chemisch ungünstige Decarboxylierung von 4-Hydroxyphenylacetat zu p-Cresol zu katalysieren. Das Enzymsystem besteht aus einer Decarboxylase und dem zugehörigen Aktivierungsenzym. Die 4-Hydroxyphenylacetat-Decarboxylase (4Hpad) besitzt zusätzlich zum Protein-basierten Glycinradikal eine weitere Untereinheit mit bis zu zwei [4Fe-4S] Clustern und repräsentiert hierdurch eine neue Klasse von Fe/S-Cluster-haltigen GREs, die aromatische Verbindungen umsetzen. Das Aktivierungsenzym (4Hpad-AE) weicht vom Standardtypus ab, indem es zusätzlich zum S-Adenosylmethionin(SAM)-bindenden [4Fe-4S]-Cluster (RS-Cluster) mindestens einen weiteren [4Fe-4S]-Cluster bindet. In dieser Studie wurden heterologe Expressions- und Reinigungsprotokolle für 4Hpad und 4Hpad-AE entwickelt. Kristallstrukturen von 4Hpad cokristallisiert mit den Substraten (4-Hydroxyphenylacetat, 3,4-Dihydroxyphenylacetat) und dem Inhibitor (4-Hydroxyphenylacetamid) zeigten geringe strukturelle Änderungen im aktiven Zentrum des Proteins. Die Radikalbildung am 4Hpad-AE wurde durch die Überprüfung einer klassischen reduktiven Spaltung von SAM zu den Reaktionsprodukten 5’-Deoxyadenosin und Methionin bestätigt. EPR- und Mössbauer-Spektroskopische Analysen zeigten, dass 4Hpad-AE mindestens einen zusätzlichen [4Fe-4S] Cluster neben dem einzelnen RS-Cluster enthält. Die katalytische Notwendigkeit eines zusätzlichen Clusters wurde durch eine Mutationsanalyse untersucht, wobei eine verkürzte Version des Enzyms ohne die zusätzliche Cystein-reiche Insertion konstruiert wurde. Das verkürzte Mutante ohne die Bindungsmotive für die zusätzlichen Cluster gekennzeichnet, die Konfiguration, Stöchiometrie und die Funktion der zusätzlichen Cluster diagnostizieren. / 4-hydroxyphenylacetate decarboxylase (4Hpad) is a two [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in Clostridium difficile and C. scatologenes by a Kolbe-type decarboxylation. The decarboxylation product p-cresol is a virulence factor of the human pathogen C. difficile. The small subunit of 4Hpad may have a regulatory function with the Fe/S clusters involved in complex formation and radical dissipation in the absence of substrate. The respective activating enzyme (4Hpad-AE) has one or two [4Fe-4S] cluster(s) in addition to the SAM-binding [4Fe-4S] cluster (RS cluster). The role of these auxiliary clusters is still under debate with proposed functions including structural integrity and conduit for electron transfer to the RS cluster. This study shows the optimized expression and purification protocols for the decarboxylase and the co-crystallization experiments and binding studies with 4-hydroxy-phenylacetate and 3,4-dihydroxyphenylacetate and with the inhibitor 4-hydroxy-phenylacetamide. The purification and characterization of active site mutants of decarboxylase are also done. Concerning 4-HPAD-AE, we report on the purification of code-optimized variants, and on spectroscopic and kinetic studies to characterize the respective i) SAM binding enthalpies, ii) rates for reductive cleavage of SAM and iii) putative functions of the additional Fe/S clusters. The truncated mutant lacking the binding motifs for the auxiliary clusters is characterized to diagnose the configuration, stoichiometry and function of the auxiliary clusters.