The neonatal period in mammals is characterized by high growth rates and is dominated by skeletal muscle hypertrophy. Low birth weight (LBWT) neonates experience restricted growth and development of skeletal muscle, leading to metabolic perturbations later in life. The overall hypothesis of this dissertation was that in utero disturbances in glucose metabolism and increased energy requirements predisposes LBWT neonatal pigs to metabolic disturbances after birth. We sought to increase growth of skeletal muscle and improve glucose production through increasing dietary energy and to determine the changes in glucose catabolism and metabolic flexibility in different skeletal muscle fiber types in LBWT neonates. Piglets were considered normal birth weight (NBWT) and LBWT when birth weight was within 0.5 SD and below 2 SD of the litter average, respectively. Increasing dietary energy increased lean deposition in the longissimus dorsi (LD) in both NBWT and LBWT neonates. Although glucose rate of appearance was greater in LBWT compared to their NBWT sibling, glucose concentrations were reduced in LBWT compared to NBWT pigs, regardless of diet fed. Postprandial glucose concentrations were lower in LBWT compared to NBWT pigs, regardless of diet fed, although rate of appearance did not differ between them. This would suggest that glucose is being absorbed in the peripheral tissues to be utilized. However, expression of enzymes related to glycolysis were downregulated in both the soleus and LD of LBWT compared to NBWT neonatal pigs. In addition, expression of enzymes related to the catabolism of glucose in the serine biosynthetic pathway were decreased in both the soleus and LD muscles of LBWT compared to NBWT neonatal pigs. Expression of the pentose phosphate pathway was slightly increased in LBWT compared to NBWT siblings in both muscle types. Increased expression of pyruvate dehydrogenase 4 was exhibited in both the soleus and LD of LBWT pigs compared to NBWT siblings. This would indicate a switch in fuel utilization to more fatty acid oxidation. By contrast, CO2 production from the oxidation of palmitate was reduced in LBWT compared with NBWT pigs along with reduced oxidation of glucose and pyruvate. In conclusion, lipid supplementation increased growth at the expense of fat deposition in the liver of NBWT and LBWT pigs. However, supplementing with fat did not increase glucose production due to the contribution of glycerol remaining constant. Hypoglycemia cannot be attributed to greater catabolism in skeletal muscle due to decreased expression of glycolytic genes and the addition of fatty acids did not spare glucose oxidation in skeletal muscle of LBWT pigs. / PHD / During the neonatal period animals display the fastest growth rates, especially pertaining to muscle growth. Muscle development in low birth weight (LBWT) is restricted, leading not only to impaired postnatal growth but increases the risk for developing metabolic diseases later in life such as obesity and type 2 diabetes. LBWT is also characterized by decreased glucose concentrations and decreased body fat content at birth. In the present studies we sought to increase growth and improve glucose production by supplementing with a high energy diet and to compare the changes in glucose catabolism in different skeletal muscle fiber types along with analyzing the ability to switch fuel substrates in LBWT and NBWT neonatal pigs. Increasing dietary energy increased longissimus dorsi (LD) weight as a percentage of bodyweight, regardless of growth status. In addition, during fasting glucose production was higher in LBWT compared to their NBWT siblings, regardless of diet. However, glucose concentration in LBWT were lower compared to NBWT neonatal pigs. Although glucose concentrations were lower in LBWT compared to NBWT pigs after a meal, glucose production rate was unchanged among LBWT and NBWT siblings fed either a high or low energy diet. This suggests that glucose uptake is increased in peripheral tissues of LBWT pigs. However, enzymes related to glycolysis in the LD and soleus of LBWT pigs had lower expression than their NBWT sibling. In addition, the enzyme responsible for the shift in fuel selection, pyruvate dehydrogenase kinase 4 (PDK4) was highly expressed in LBWT compared to NBWT neonatal pigs in both the LD and soleus. This would suggest a switch in glucose oxidation to fatty acid oxidation in the skeletal muscle of LBWT neonatal pigs. However, oxidation of fatty acids in both the soleus and LD of LBWT was reduced compared to NBWT neonatal pigs. In conclusion, lipid supplementation increased growth at the expense of lipid deposition in the liver and did not increase glucose production. Reduced glucose concentrations are not due to greater catabolism in skeletal muscle due to decreased expression of glycolytic genes and the addition of fatty acids did not spare glucose oxidation in the skeletal muscle of LBWT pigs.

<p>This thesis describes the structural studies of four different proteins from two organisms. Xyloglucan endotransglycosylases, XETs, are involved in plant cell wall expansion and remodeling by splitting and reconnecting xyloglucan-cellulose crosslinks. The first crystal structure of a XET enzyme has been determined to 1.8 Å. The structure provides insights into how XETs are able to bind a heavily branched xyloglucan sugar, as well as hints about the XET-transglycosylation mechanism.</p><p><i>Mycobacterium tuberculosis</i> (Mtb) is the cause of enormous human mortality each year. Despite the sequencing of the complete Mtb-genome, the biological function of a large fraction of the <i>M. tuberculosis </i>proteins is still unknown. We here report the crystal structures of three such proteins, Rv2740, Rv0216 and Rv0130. Rv2740 forms a Cystatin α+b fold with a deep active site pocket similar to a limonene-1,2-epoxide hydrolase from <i>Rhodococcus erythropolis</i>. However, in contrast to the small limonene-based substrate of the <i>Rhodococcus</i> enzyme, Rv2740 is able to degrade large fatty acid and sterol epoxides, giving suggestions for the physiological substrates of this enzyme.</p><p>The structure of <i>M. tuberculosis</i> Rv0216 exhibits a so-called double hotdog fold. Rv0216 shows similarity to a number of enzymes using thiol esters as substrates, including several <i>R</i>-enoyl hydratases and β-hydroxyacyl dehydratases. However, only parts of the hydratase / dehydratase catalytic site are conserved in Rv0216. Rv0130 in contrast, contains a highly conserved <i>R</i>-hydratase motif, housed in a dimer of two single hotdog folded molecules. This active site is situated in a long tunnel, formed by a sharp kink in the Rv0130 central helix. A number of previously predicted single / double hotdog folded proteins from <i>M. tuberculosis</i> seem to feature a similar substrate-binding tunnel, indicating that Rv0130 as well as some of these proteins, might act on long fatty enoyl chains. </p>

This thesis describes the structural studies of four different proteins from two organisms. Xyloglucan endotransglycosylases, XETs, are involved in plant cell wall expansion and remodeling by splitting and reconnecting xyloglucan-cellulose crosslinks. The first crystal structure of a XET enzyme has been determined to 1.8 Å. The structure provides insights into how XETs are able to bind a heavily branched xyloglucan sugar, as well as hints about the XET-transglycosylation mechanism. Mycobacterium tuberculosis (Mtb) is the cause of enormous human mortality each year. Despite the sequencing of the complete Mtb-genome, the biological function of a large fraction of the M. tuberculosis proteins is still unknown. We here report the crystal structures of three such proteins, Rv2740, Rv0216 and Rv0130. Rv2740 forms a Cystatin α+b fold with a deep active site pocket similar to a limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis. However, in contrast to the small limonene-based substrate of the Rhodococcus enzyme, Rv2740 is able to degrade large fatty acid and sterol epoxides, giving suggestions for the physiological substrates of this enzyme. The structure of M. tuberculosis Rv0216 exhibits a so-called double hotdog fold. Rv0216 shows similarity to a number of enzymes using thiol esters as substrates, including several R-enoyl hydratases and β-hydroxyacyl dehydratases. However, only parts of the hydratase / dehydratase catalytic site are conserved in Rv0216. Rv0130 in contrast, contains a highly conserved R-hydratase motif, housed in a dimer of two single hotdog folded molecules. This active site is situated in a long tunnel, formed by a sharp kink in the Rv0130 central helix. A number of previously predicted single / double hotdog folded proteins from M. tuberculosis seem to feature a similar substrate-binding tunnel, indicating that Rv0130 as well as some of these proteins, might act on long fatty enoyl chains.

No description available.

The normal adult heart prefers fatty acids as an energy substrate. In the case of heart failure, the heart switches its preference from fatty acids to glucose, adopting a pattern similar to fetal metabolism. PGC-1α is heavily involved in the shift towards glucose oxidation. p65, which belongs to the NF-κB transcription factor family is another crucial molecule involved in maintaining cardiac homeostasis. There is a substantial amount of evidence suggesting that PGC-1α and NF-κB directly interact, thereby connecting metabolic and inflammatory processes. Dysregulation of either PGC-1α or NF-κB signalling correlates to many diseases including heart disease. In this study, we provide further evidence that the NF-κB family has the ability to repress PGC-1α. We also show that the PGC-1α promoter contains a p65 binding site through which p65 imparts control on the PGC-1α gene. Metabolic homeostasis and inflammation pathways are closely linked and play crucial roles in heart dysfunction.

This thesis presents the cloning, purification, crystallization, and structural studies of two unknown proteins from Mycobacterium tuberculosis, and of an aminotransferase from Mycobacterium smegmatis. Structural knowledge of these proteins is of highest interest for structure-based drug design, which is one of the approaches that can be used in order to fight tuberculosis (TB). The structure of the conserved hypothetical protein Rv0216 was refined to a resolution of 1.9 Å. The structure exhibits a so-called double hotdog-fold, similar to known hydratases. However, only parts of the hydratase active site are conserved in Rv0216, and no function could be assigned to the protein. Several Rv0216-like protein sequences were found in a variety of actino- and proteobacteria, suggesting that these proteins form a new protein family. Furthermore, other hotdog-folded proteins in M. tuberculosis were identified, of which a few are likely to be hydratases or dehydratases involved in the fatty acid metabolism. The structure of Rv0130 exhibits a single hotdog-fold and contains a highly conserved R-hydratase motif. Rv0130 was shown to hydrate fatty acid coenzyme A derivatives with a length of six to eight carbons. The Rv0130 active site is situated in a long tunnel, formed by a kink in the central hotdog-helix, which indicate that it can utilize long fatty acid chains as well. A number of previously predicted hotdog-folded proteins also feature a similar tunnel. The structure of branched chain aminotransferase (BCAT) of M. smegmatis was determined in the apo-form and in complex with an aminooxy inhibitor. Mycobacterial BCAT is very similar to the human BCAT, apart for one important difference in the active site. Gly243 is a threonine in the human BCAT, a difference that offers specificity in inhibition and substrate recognition of these proteins. The aminooxy compound and MES were found to inhibit the mycobacterial BCAT activities. The aminooxy compound inhibits by blocking the substrate-pocket. A second inhibitor-binding site was identified through the binding of a MES molecule. Therefore, both the MES-binding site and the substrate-pocket of M. smegmatis BCAT are suggested to be potential sites for the development of new inhibitors against tuberculosis.

Protein palmitoylation refers to the process of adding a 16-carbon saturated fatty acid to the cysteine of a substrate protein, and this can in turn affect the substrate’s localization, stability, folding, and several other processes. This process is catalyzed by a family of 23 mammalian protein acyltransferases (PATs), a family of transmembrane enzymes that modify an estimated 10% of the proteome. At this point in time, no structure of a protein in this family has been solved, and therefore there is poor understanding about the regulation of the enzymes and their substrates. Most proteins, including palmitoylation enzymes and substrates, have some level of intrinsic disorder, and this flexibility can be important for signaling processes such as protein- protein interactions and post-translational modifications. Therefore, we assumed that examining intrinsic disorder in palmitoylation enzymes and substrates would yield insight into their regulatory mechanisms. First, we found that among other factors, utilizing intrinsic disorder predictions led to a palmitoylation predictor that significantly outperformed existing predictors. Next, we discovered a conserved region of predicted disorder-to-order transition in the disordered C-termini of the PAT family. In Erf2, the yeast Ras PAT, we developed a model where this region reversibly interacts with membranes, and we found that this region mediates interaction with Acc1, an enzyme involved in fatty acid metabolism processes. Finally, we found that an XLID-associated nonsense mutation in zDHHC9, the mammalian Ras PAT, removed a disordered region that was critical for enzyme localization. Future studies of palmitoylation utilizing the framework of intrinsic disorder may lead to additional insights about this important regulatory process.

Die nicht-invasive bildgebende Diagnostik hat bei koronarer Herzkrankheit einen wichtigen Stellenwert für die Diagnosestellung und Therapieplanung. In diesem Zusammenhang liefern nuklearmedizinische Verfahren wichtige Daten zur Gewebsperfusion und erlauben die bildliche Darstellung und Quantifizierung relevanter Details des kardiomyozytären Stoffwechsels. Die quantitativ bedeutendste Methode der nuklearmedizinischen Herzdiagnostik ist die Perfusionsszintigraphie mit Tl-201 oder einem der Tc-99m-markierten Tracer (Sestamibi, Tetrofosmin). Die Perfusionsszintigraphie gibt eine Darstellung der Perfusionsverhältnisse auf zellulärem Niveau während Belastung und Ruhe und erlaubt zusätzlich die Einschätzung der myokardialen Vitalität. Neben der Darstellung der Myokard-Perfusion ist die Untersuchung des myokardialen Energiestoffwechsels von besonderem Interesse, da jegliche Kontraktion auf der Bereitstellung einer ausreichenden Menge energiereicher Phosphate beruht und Störungen des Energiestoffwechsels zu unmittelbaren Konsequenzen für die Kontraktion führen. In der klinischen Diagnostik hat sich die Darstellung des myokardialen Glukosestoffwechsels mit F-18-Fluorodesoxyglukose (FDG) durchsetzen können und gilt als Goldstandard für den Vitalitätsnachweis. Jede Perfusionsstörung wirkt sich unmittelbar auf den myokardialen Energiehaushalt aus. Als Indikator für die Störung des Energiehaushalts kann die veränderte Nutzung unterschiedlicher energieliefernder Substrate dienen. Die Folgen von Koronarstenosen für den kardiomyozytären Energiehaushalt lassen sich demnach durch die veränderte Nutzung radioaktiv markierter Substrate erfassen und bildlich mittels nuklearmedizinischer Methoden darstellen. Während die Darstellung des myokardialen Glukosestoffwechsels bereits Eingang in die klinische Diagnostik gefunden hat, ist die Analyse des kardialen Lipidmetabolismus aufgrund der Komplexität der möglichen Stoffwechselwege schwieriger und bislang ohne klinische Bedeutung. Lipide sind aber - zumindest theoretisch - von besonderem Interesse, da ihre Oxidation einerseits für den Hauptteil der ATP-Produktion verantwortlich ist und andererseits Abbauprodukte aus dem Lipidstoffwechsel zu einer Schädigung des Herzens beitragen können. Das Ziel dieser Arbeit war dementsprechend die Bestimmung der Extraktion langkettiger Fettsäuren in (chronisch) ischämischem (hibernierendem) und in reperfundiertem ("stunned myocardium") Myokard. Als Fettsäure wurde I-123-Iodophenyl-Pentadekansäure verwendet, die vergleichbar mit Palmitinsäure in die Zellen aufgenommen und dann entweder der beta-Oxidation zugeführt oder in intrazelluläre Lipidpools integriert wird. Im ersten Teil der Arbeit erfolgten Experimente mit isolierten Rattenherzen, die flußkonstant nach Langendorff perfundiert wurden. Der Fettsäuremetabolismus wurde mittels Indikator-Verdünnungsmethode untersucht, wobei Tc-99m-Albumin als intravaskulärer Referenztracer diente. Es erfolgten Experimente zur Charakterisierung der Fettsäure-Extraktion während unterschiedlicher Flußraten und während 90minütiger Reperfusion nach 20minütiger Ischämie (Flußreduktion auf 25% des Kontrollwertes). Als Perfusat diente eine modifizierte Krebs-Henseleit-Lösung, der entweder 10 mmol/l Glukose oder 10 mmol/l Glukose + 5 I.E./l Alt-Insulin zugesetzt wurden. Die Ergebnisse der Untersuchungen am isolierten Rattenherz zeigen, daß die Extraktion von Fettsäuren auch in (akut) minderperfundiertem Myokard erhalten ist. Die I-123-IPPA-Extraktion stieg bei Reduktion des Blutflusses zunächst exponentiell und bei Reduktion unter 25% des Kontrollflusses mehr als exponentiell an. Bei Zusatz von Insulin zum Perfusat war die Beziehung zwischen Fluß und Fettsäure-Extraktion qualitativ nicht verändert. Quantitativ ergaben sich Differenzen, die insbesondere das Ausmaß der Nettoextraktion bei hohen Flußraten betrafen (höher bei Zusatz von Insulin). Während Reperfusion hing die Fettsäureextraktion von der Zusammensetzung des Perfusats ab. Bei Insulinzusatz (gute intrazelluläre Glukoseverfügbarkeit) war die Fettsäureextraktion reduziert, ohne Insulinzusatz diskret gesteigert. Die Erholung der Wandbewegung ging der Normalisierung der Fettsäureextraktion zeitlich voraus. Das kapilläre Permeabilitäts-Oberflächen-Produkt (PS-Produkt) für I-123-IPPA war in beiden Perfusatgruppen während Reperfusion deutlich auf 20-30% des Kontrollwerts vermindert. Der Abfall des PS-Produkts ist primär Ausdruck einer Ischämie- (oder Reperfusions-) assoziierten Endothelschädigung mit verminderter Permeabilität für I-123-IPPA. Im zweiten Teil der Arbeit wurden Patienten mit koronarer Herzkrankheit untersucht. Dabei handelte es sich einerseits um Patienten nach akutem Myokardinfarkt mit effektiver Reperfusion ("stunning") und andererseits um Patienten mit chronischer KHK und eingeschränkter linksventrikulärer Funktion ("hibernation"). Die Ergebnisse der Patientenuntersuchungen zeigen (ähnlich wie die Daten der Experimente am isolierten Herzen), daß sich myokardiales stunning und hibernation durch unterschiedliche Muster der Fettsäureverwertung unterscheiden. Chronisch minderperfundiertes Myokard zeigte eine erhaltene Fettsäureextraktion, während die Fettsäureaufnahme in reperfundiertem Myokard vermindert war. In reperfundiertem Myokard überdauerte die Reduktion der Fettsäureextraktion die Störung der regionalen Wandbewegung. Insgesamt zeigen die Ergebnisse der Experimente am isolierten Herzen wie auch der Patientenstudien, daß durch Szintigraphie mit radioaktiv markierten Fettsäuren unterschiedliche ischämische Syndrome (stunning, hibernation) differenziert werden können. Allerdings ist die Störung der Fettsäureextraktion unspezifisches Zeichen einer Myokardschädigung und auch nach Beseitigung des schädigenden Einflußes noch über einen relativ langen Zeitraum nachweisbar. Der potentielle klinische Nutzen der Fettsäureszintigraphie wird erheblich davon abhängen, inwieweit es gelingt, unterschiedliche Fettsäure-Verwertungsmuster mit der individuellen Prognose eines Patienten zu korrelieren. / The most important single procedure in nuclear cardiology is myocardial perfusion imaging with Tl-201 or one of the Tc-99m labeled tracers (sestamibi, tetrofosmin). Perfusion scintigraphy allows the assessment of perfusion on a cellular level during stress and at rest, and of myocardial viability. Besides the assessment of myocardial perfusion, the examination of myocardial energy metabolism is of special interest, because every contraction relies on a sufficient amount of high-energy phosphates and every disturbance of energy metabolism is directly followed by a disturbance of myocardial contraction. In clinical cardiology, imaging of myocardial glucose metabolism with F-18 fluoro-deoxyglucose (FDG) is the accepted gold standard for myocardial viability. Every disturbance of perfusion exerts direct influence on energy metabolism. The altered use of the different energy-yielding substrates can be regarded as an indicator of the degree of metabolic disturbance. Therefore, the consequences of coronary stenoses on energy metabolism can be assessed with radioactive substrates and imaged with nuclear medicine methods. Whereas imaging of glucose metabolism is part of today's clinical cardiology, the analysis of lipid metabolism is - due to the complexity of possible metabolic pathways - more difficult and so far without clinical relevance. However, lipids are of special importance, because (1) their oxidation is responsible for the major part of ATP production and (2) degradation products from the lipid metabolism contribute to cardiac damage. The aims of these studies were to examine the extraction of long-chain fatty acids in chronically ischemic ("hibernating") and in reperfused ("stunned") myocardium. In the experiments, I-123 iodophenylpentadecanoic acid (IPPA) served as labeled long-chain fatty acid. Cellular uptake of IPPA is comparable to palmitic acid and - after uptake - IPPA undergoes either beta-oxidation or is integrated into intracellular lipid pools. The first part of these studies deals with experiments in isolated rat hearts which were subjected to retrograde perfusion according to Langendorff. Fatty acid metabolism was analyzed in these hearts using the indicator-dilution technique with Tc-99m albumin as intravascular reference. Fatty acid extraction was assessed at different flow rates and during reperfusion after flow reduction to 25% of control for 20 min. All hearts were perfused with a modified Krebs-Henseleit solution with the addition of either 10 mmol/l glucose or 10 mmol/l glucose + 5 IU insulin. The results of the isolated rat heart experiments show that the extraction of long-chain fatty acids is preserved in myocardium subjected to (acute) low flow ischemia. IPPA extraction increased exponentially with reduction of blood flow and even showed a more than exponential increase at flow rates below 25% of control. After adding insulin to the perfusate the relation between flow and fatty acid extraction was not altered qualitatively. Quantitatively, differences were detected which primarily refer to a higher net extraction at high flow rates. Fatty acid extraction during reperfusion depended on the composition of the perfusate. After addition of insulin (high intracellular glucose availability) fatty acid extraction was reduced, whereas it was increased without insulin. Recovery of wall motion preceded the normalization of fatty acid extraction. The capillary permeability-surface product (PS product) for IPPA was reduced in both perfusate groups to 20-30% of control. The decrease of the PS product can be primarily attributed to ischemia- and/or reperfusion-associated endothelial damage. The second part of these studies deals with the examination of (1) patients after acute myocardial infarction with effective reperfusion (clinical model of "stunning") and (2) patients with chronic coronary artery disease and reduced left ventricular function ("hibernation"). The results in these patient groups show (comparable to the data of the isolated heart experiments) that myocardial stunning and hibernation can be distinguished by different patterns of fatty acid utilization. Fatty acid extraction was preserved in chronically hypoperfused myocardium, whereas it was reduced in reperfused segments. In reperfused myocardium, the reduction of fatty acid extraction outlasted the wall motion abnormality. In conclusion, the results show that scintigraphy with radio-labeled long chain fatty acids can distinguish between different ischemic syndromes (stunning, hibernation). However, the alteration of fatty acid extraction is an unspecific sign of cardiac damage which persists after removal of the harmful stimulus. The potential clinical benefit of fatty acid scintigraphy will largely depend on the definition of different fatty acid utilization patterns and the correlation of these utilization patterns with the prognosis of individual patients.

Intracellular protein sorting plays a key role in the regulation of cellular metabolism, gene expression, signal transduction and a number of other cellular processes. Proteins targeted to specific cellular compartments contain organelle-specific targeting sequences which interact with various components of the import machinery that are often evolutionarily conserved. For example, proteins targeted to peroxisomes interact with specific receptor proteins through unique peroxisomal targeting signals (PTS) which results in their import into peroxisomal matrix or insertion into peroxisomal membrane. Peroxisomal protein import has been studied in a number of species and several conserved PTS and receptor proteins have been identified. In our study, we report the unexpected finding that cytochrome c (cyt c), which lacks a canonical PTS, is targeted to peroxisomes of the methylotrophic yeast, Pichia pastoris. This is a unique feature of P. pastoris and is not observed in other yeast species such as the conventional yeast, Saccharomyces cerevisiae or other methylotrophic yeasts such as Hansenula polymorpha. Using S. cerevisiae cyc1 null mutant strain as a surrogate model, we demonstrate that P. pastoris cytochrome c (PpCyt c) is targeted to S. cerevisiae peroxisomes indicating that peroxisomal targeting is a unique and inherent property of PpCyt c and the machinery required for this is conserved in S. cerevisiae as well. We further demonstrate that Ppcyt c targeted to the fatty acid-induced peroxisomes of S. cerevisiae is a hemoprotein with covalently attached heme suggesting that PpCyt c synthesized in cytosol is first targeted to mitochondria where heme is added to the apoprotein by cytochrome c heme lyase and the holoprotein is then re-targeted to peroxisomes through an unknown mechanism. Proteins imported into peroxisomes carry specific peroxisomal targeting signals (PTS) known as PTS1 and PTS2. PTS1 is a tripeptide sequence (SKL) at the carboxy terminus of peroxisomal matrix proteins. To investigate whether the carboxy terminus of PpCyt c contain PTS1 or PTS1-like sequences, we made GFP fusion proteins with PpCyt c carboxy terminal amino acids (GFP-ATK, GFP-LAKATK) and examined their ability to localize to peroxisomes. Neither of these two proteins is targeted to peroxisomes indicating that PTS1-like sequences are not involved in peroxisomal targeting of Ppcyt c. Two receptors known as Pex5 and Pex7 are known to be involved in peroxisomal protein import and we therefore examined PpCyt c import into peroxisomes of P. pastoris strains lacking pex5 and pex7. Peroxisomal import of PpCyt c is abolished in pex5 but not pex7 mutant strain indicating that PpCyt c is imported into peroxisomes by a pex5-dependent but PTS1independent pathway. Since we observed significant amino acid differences between PpCyt c and S. cerevisiae cytochrome c (ScCyt c) in their carboxy-and amino-termini, we interchanged these amino acids between PpCyt c and ScCyt c and examined their subcellular localization. Such studies revealed that swapping the N-terminal or C-terminal amino acids of PpCyt c with those of S. cerevisaie cytochrome c (ScCyt c) abolishes peroxisomal localization of PpCyt c. Thus, both N-and C-terminal amino acids of PpCyt c are essential for its import into peroxisomes. Interestingly, in a number of fungal species, the N-and C-terminal amino acid sequences of cytochrome c are identical to those of PpCyt c indicating that peroxisomal targeting of cytochrome c may be observed in other yeast species as well. S. cerevisiae cells expressing PpCyt c exhibit several unique biochemical properties. S. cerevisiae cells expressing PpCyt c grow more rapidly than those expressing ScCyt c when cultured on media containing oleic acid as the sole carbon source and uptake of C-oleic acid from the medium as well as its assimilation into neutral lipids is quantitatively higher in the former. Surprisingly, the phenotype of S. cerevisiae cells expressing PpCyt c is dramatically altered such that the kinetics of growth on fatty acid containing media as well as lipid profile appear to be identical to those of P. pastoris rather than S. cerevisiae. Thus peroxisomal targeting of cytochrome c dramatically alters the kinetics of growth of S. cerevisiae cells in fatty acid containing media as well as the lipid metabolism raising several interesting questions on the molecular mechanisms involved in the alteration of phenotype of S. cerevisiae. It is likely that peroxisomal targeting of cytochrome c results in quantitative as well as qualitative changes in fatty acid metabolism and this opens up new vistas for the bioconversion of fatty acids into value-added lipid products by metabolic engineering. Based on these studies, we propose a new role for cytochrome c in peroxisomal fatty acid metabolism. Our study demonstrates that evolutionarily conserved proteins such as cytochrome c can acquire unique, species-specific functions that may be of great physiological significance to that organism.

This thesis examined the translocation of fatty acid (FA) transport proteins to the plasma membrane (PM) in human and rat skeletal muscle during moderate intensity exercise. The responses to the post-exercise period and to acute moderate intensity exercise after 6 weeks of high intensity interval training (HIIT) were also examined in humans. The overall hypotheses were that 1) FAT/CD36 and FABPpm would translocate to the PM in human skeletal muscle during 120 min of moderate intensity exercise, 2) FAT/CD36 and FABPpm would translocate to the PM in rat skeletal muscle during 120 min of moderate intensity exercise and this would correlate to an increase in palmitate uptake, 3) FAT/CD36 and FABPpm would translocate to the PM during 120 min of moderate intensity exercise, but return to basal levels by 45 min post-exercise, 4) six weeks of HIIT would increase PM content of FABPpm but not FAT/CD36 in resting skeletal muscle, 5) six weeks of HIIT would cause a further increase in the translocation of FAT/CD36 and FABPpm to the PM during moderate intensity exercise and this would correspond to an increase in whole body fat oxidation compared to exercise pre-training, and 6) six weeks of HIIT would increase whole muscle content of FATP1 and FATP4. In human skeletal muscle, PM FAT/CD36 and FABPpm increased 75% and 20% respectively after 120 min of cycling at ~60% VO2 peak which corresponded to a 110% increase in whole body fat oxidation. In rat skeletal muscle, PM FAT/CD36 and FABPpm increased 20% and 30% respectively, which correlated to a 30% increase in palmitate uptake following 120 min of treadmill running at ~65% VO2 peak. The PM content of FAT/CD36 increased further to 120% of resting values by 45 min of post-exercise following 120 min of cycling at ~60% VO2peak, which correlated with a heavy reliance on fat as a fuel during the post-exercise period. FABPpm returned to resting levels of PM content by 15 min post-exercise. After 6 wk of HIIT, whole muscle FAT/CD36 (50%), FABPpm (21%) and FATP4 (25%) were increased in human skeletal muscle, while FATP1 remained unchanged. There were no changes in PM content of FAT/CD36 or FABPpm at rest following training. FAT/CD36 and FABPpm were also measured before and after 120 min of cycling at ~60% of pre-trainingVO2 peak following training, but no differences in the magnitude of the PM content increases were seen compared to pre-training, despite a 27% increase in fat oxidation. These studies demonstrate that FA transport proteins translocate to the PM during moderate intensity exercise, which correlates with increased FA uptake and whole body fat oxidation. This relationship does not appear to hold during the post-exercise period, as further increases in the PM content of FAT/CD36 does not correspond with the decrease in fat oxidation. The PM content of FAT/CD36 and FABPpm were not increased at rest following training, and there was no effect of training on the translocation of FAT/CD36 or FABPpm to the PM during moderate intensity exercise at the same absolute power output, however there may be a further increase at a relative power output. / Natural Sciences and Engineering Research Council, Canadian Institute of Health Research, Ontario Graduate Scholarship