Indiana University-Purdue University Indianapolis (IUPUI) / Thiamin diphosphate (ThDP)-dependent enzymes catalyze a wide range of reactions including the oxidative and nonoxidative decarboxylation of 2-keto acids, carboligation reactions, the cleavage of C-C bonds, and the formation of C-S, C-N, and C-O bonds. Surprisingly, given this diversity, all ThDP-dependent enzyme catalyzed reactions proceed through essentially the same intermediate. This suggests that these enzymes share a common ancestry and have evolved to become the diverse group of enzymes seen today. Sequence alignments have revealed that all ThDP-dependent enzymes share two common ThDP binding domains, the PYR domain and the PP domain. In addition to these conserved domains, over time, other domains have been added creating further diversity in this superfamily. For instance, the TH3 domain, found in many ThDP-dependent enzymes, serves the function of binding additional cofactors such as FAD in enzymes like acetohydroxyacid synthase (AHAS) but in others, like pyruvate decarboxylase (PDC), it has lost this function completely. The work presented here focuses on ThDP-dependent decarboxylases. In this thesis, several evolutionary aspects of this group of enzymes will be examined including (i) the characterization of an evolutionary forerunner in the presence of a mechanism-based inhibitor, (ii) the characterization of the minor isozymes of pyruvate decarboxylase from Saccharomyces cerevisiae, and (iii) the development of a selection method to increase the efficiency of the site-saturation mutagenesis used to study ThDP-dependent enzyme evolution.

Indiana University-Purdue University Indianapolis (IUPUI) / The expression of pyruvate dehydrogenase kinase (PDK) 2 and 4 are increased in the fasted state to inactivate the pyruvate dehydrogenase complex (PDC) by phosphorylation to conserve substrates for glucose production. To assess the importance of PDK2 and PDK4 in regulation of the PDC to maintain glucose homeostasis, PDK2 knockout (KO), PDK4 KO, and PDK2/PDK4 double knockout (DKO) mice were generated. PDK2 deficiency caused higher PDC activity and lower blood glucose levels in the fed state while PDK4 deficiency caused similar effects in the fasting state. DKO intensified these effects in both states. PDK2 deficiency had no effect on glucose tolerance, PDK4 deficiency produced a modest effect, but DKO caused a marked improvement, lowered insulin levels, and increased insulin sensitivity. However, the DKO mice were more sensitive than wild-type mice to long term fasting, succumbing to hypoglycemia, ketoacidosis, and hypothermia. Stable isotope flux analysis indicated that hypoglycemia was due to a reduced rate of gluconeogenesis. We hypothesized that hyperglycemia would be prevented in DKO mice fed a high saturated fat diet for 30 weeks. As expected, DKO mice fed a high fat diet had improved glucose tolerance, decreased adiposity, and were euglycemic due to reduction in the rate of gluconeogenesis. Like chow fed DKO mice, high fat fed DKO mice were unusually sensitive to fasting because of ketoacidosis and hypothermia. PDK deficiency resulted in greater PDC activity which limited the availability of pyruvate for oxaloacetate synthesis. Low oxaloacetate resulted in overproduction of ketone bodies by the liver and inhibition of ketone body and fatty acid oxidation by peripheral tissues, culminating in ketoacidosis and hypothermia. Furthermore, when fed a ketogenic diet consisting of low carbohydrate and high fat, DKO mice also exhibited hypothermia, ketoacidosis, and hypoglycemia. The findings establish that PDK2 is more important in the fed state, PDK4 is more important in the fasted state, survival during long term fasting depends upon regulation of the PDC by both PDK2 and PDK4, and that the PDKs are important for the regulation of glucose and ketone body metabolism.

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Dendritic cells (DCs) are the sentinels of the immune system. They mature at the encounter of the appropriate stimuli or danger signals, which induce them to perform pro-inflammatory antigen presentation to naïve and memory T cells, resulting in inflammation. Remaining in an immature state however, DCs acquire a tolerogenic phenotype. When activated by TLR ligands, DCs undergo metabolic re-programming and switch to TBK1/IKKe/AKT-induced glycolysis at the early activation phase, which is sustained due to nitric oxide (NO)-mediated inhibition of mitochondrial metabolism at the later activation phase. Targeting DC activation in the view of promoting less activated or tolerogenic DCs could be an approach to reduce or abrogate inflammation in settings such as solid organ transplant rejection or in autoimmune diseases such as systemic lupus erythematosus (SLE). In this thesis, we present data pertaining to three different approaches for targeting DC activation including 1) the use of ethyl p / Microbiology and Immunology

Mitochondria shape cytosolic Ca2+ (cCa2+) transients. Ca2+ entry into the mitochondria is driven by the highly negative mitochondrial membrane potential and through a highly selective channel, the Mitochondrial Calcium Uniporter (MCU). Mitochondrial Ca2+ (mCa2+) is utilized by the matrix dehydrogenases for maintaining cellular bioenergetics. The TCA cycle-derived NADH and FADH2 are mCa2+ dependent thus, feed into the electron transport chain (ETC) to generate ATP. Either loss of mCa2+ or metabolite uptake by the mitochondria results in a bioenergetic crisis and mitochondrial dysfunction. Reciprocally, sudden elevation of cCa2+ under conditions of stroke or ischemia/reperfusion injury (I/R) drives excessive mCa2+ overload that in turn leads to the opening of a large channel, the mitochondrial permeability transition pore (PTP) that triggers necrotic cell death. Thus, Ca2+ and metabolite equilibrium is essential to maintain a healthy mitochondrial pool. Our laboratory has previously showed that loss of mCa2+ uptake leads to decreased ATP generation and cell survival through autophagy. Although metabolite scarcity also results in similar reduction in ATP generation, the molecular mechanisms by which metabolites control mitochondrial ion homeostasis remain elusive. Deprivation of glucose or supplementation of mitochondrial pyruvate carrier (MPC) transport blocker UK5099 and or carnitine-dependent fatty acid blocker etomoxir triggered an increase in the expression of MICU1, a regulator of the mitochondrial calcium uniporter (MCU) but not the MCU core subunit. Consistently, either RNAi-mediated deletion of MPC isoforms or dominant negative human mutant MPC1 R97W showed significant induction of MICU1 protein abundance and inhibition of MCU-mediated mCa2+ uptake. Moreover, TCA cycle substrate-dependent MICU1 expression is under the control of EGR1 transcriptional regulation. Reciprocally, the MICU1 dependent inhibition of mCa2+ uptake exhibited lower NADH production and oxygen consumption and ATP production. The reduction of mitochondrial pyruvate by MPC knockdown is linked to higher production of mitochondrial ROS and elevated autophagy markers. These studies reveal an unexpected regulation of MCU-mediated mCa2+ flux machinery involving major TCA cycle substrate availability and possibly MICU1 to control cellular switch between glycolysis and oxidative phosphorylation. While mCa2+ is required for energy generation, sustained elevation of mCa2+ results in mitochondrial swelling and necrotic death. Hence, it was thought that preventing mCa2+ overload can be protective under conditions of elevated cCa2+. Contrary to this, mice knocked-out for MCU, that demonstrated no mCa2+ uptake and hence no mitochondrial swelling, however failed protect cells from I/R- mediated cell death. MCU-/- animals showed a similar infarct size comparable to that of control animals, suggesting that prevention of MCU-mediated mCa2+ overload alone is not sufficient to protect cells from Ca2+ -induced necrosis. The absence of mCa2+ entry revealed an elevation in the upstream cCa2+ transients in hepatocytes from MCUDHEP. Ultra-structural analysis of liver sections from MCU-/- (MCUDHEP) and MCUfl/fl animals revealed stark contrast in the shape of mitochondria: MCUfl/fl liver sections showed long and filamentous mitochondria (spaghetti-like) while MCUDHEP mitochondria were short and circular (donut-like). Furthermore, challenging MCUfl/fl and MCUDHEP hepatocytes with ionomycin caused a marked increase in cCa2+ and a simultaneous change in mitochondrial shape (from spaghetti to donut), a phenomenon we termed mitochondrial shape transition (MiST) that was independent of mitochondrial swelling. The cCa2+-mediated MiST is induced by an evolutionarily conserved mitochondrial surface EF-hand domain containing Miro1. Glutamate and Ca2+ -stress driven cCa2+ mobilization cause MiST in neurons that is suppressed by expression of Miro1 EF1 mutants. Miro1-dependent MiST is essential for autophagosome formation that is attenuated in cells harboring Miro1 EF1 mutants. Remarkably, loss of cCa2+ sensitization by Miro1 prevented MiST and mitigated autophagy. These results demonstrate that an interplay of ions and metabolites function in concert to regulate mitochondrial shape that in turn dictates the diverse mitochondrial processes from ATP generation to determining mechanisms of cell death. / Biomedical Sciences

R-phenylacetylcarbinol (PAC) is used as a precursor for production of ephedrine and pseudoephedrine, which are anti-asthmatics and nasal decongestants. PAC is produced from benzaldehyde and pyruvate mediated by pyruvate decarboxylase (PDC). A strain of Rhizopus javanicus was evaluated for its production of PDC. The morphology of R. javanicus was influenced by the degree of aeration/agitation. A relatively high specific PDC activity (328 U decarboxylase g-1 mycelium) was achieved when aeration/agitation were reduced significantly in the latter stages of cultivation. The stability of partially purified PDC and crude extract from R. javanicus were evaluated by examining the enzyme deactivation kinetic in various conditions. R. javanicus PDC was less stable than Candida utilis PDC currently used in our group. A kinetic model for the deactivation of partially purified PDC extracted from C. utilis by benzaldehyde (0?00 mM) in 2.5 M MOPS buffer has been developed. An initial lag period prior to deactivation was found to occur, with first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration. A mathematical model for the enzymatic biotransformation of PAC and its associated by-products has been developed using a schematic method devised by King and Altman (1956) for deriving the rate equations. The rate equations for substrates, product and by-products have been derived from the patterns for yeast PDC and combined with a deactivation model for PDC from C. utilis. Initial rate and biotransformation studies were applied to refine and validate a mathematical model for PAC production. The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U carboligase ml-1. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde on the rate of PAC production followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde and acetoin. Implementation of digital pH control for PAC production in a well-stirred organic-aqueous two-phase biotransformation system with 20 mM MOPS and 2.5 M dipropylene glycol (DPG) in aqueous phase resulted in similar level of PAC production [1.01 M (151 g l-1) in an organic phase and 115 mM (17.2 g l-1) in an aqueous phase after 47 h] to the system with a more expensive 2.5 M MOPS buffer.