Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Western diet changes cardiac acyl-CoA composition in obese rats: a potential role for hepatic lipogenesis.

The "lipotoxic footprint" of cardiac maladaptation in diet-induced obesity is poorly defined. We investigated how manipulation of dietary lipid and carbohydrate influenced potential lipotoxic species in the failing heart. In Wistar rats, contractile dysfunction develops at 48 weeks on a high-fat/high-carbohydrate "Western" diet, but not on low-fat/high-carbohydrate or high-fat diets. Cardiac content of the lipotoxic candidates--diacylglycerol, ceramide, lipid peroxide, and long-chain acyl-CoA species--was measured at different time points by high-performance liquid chromatography and biochemical assays, as was lipogenic capacity in the heart and liver by qRT-PCR and radiometric assays. Changes in membranes fluidity were also monitored using fluorescence polarization. We report that Western feeding induced a 40% decrease in myocardial palmitoleoyl-CoA content and a similar decrease in the unsaturated-to-saturated fatty acid ratio. These changes were associated with impaired cardiac mitochondrial membrane fluidity. At the same time, hepatic lipogenic capacity was increased in animals fed Western diet (+270% fatty acid elongase activity compared with high-fat diet), while fatty acid desaturase activity decreased over time. Our findings suggest that dysregulation of lipogenesis is a significant component of heart failure in diet-induced obesity.

Regulation of Set1-mediated methylation of Dam1

Eukaryotic genomes exist within a dynamic structure named chromatin in which DNA is wrapped around an octamer of histones forming the nucleosome. Histones are modified by a range of posttranslational modifications including methylation, phosphorylation, and ubiquitination, which are integral to a range of DNA-templated processes including transcriptional regulation. A hallmark for transcriptional activity is methylation of histone H3 on lysine (K) 4 within active gene promoters. In S. cerevisiae, H3K4 methylation is mediated by Set1 within the COMPASS complex. Methylation requires prior ubiquitination of histone H2BK123 by the E2-E3 ligases Rad6 and Bre1, as well as the Paf1 transcriptional elongation complex. This regulatory pathway exemplifies cross-talk in trans between posttranslational modifications on distinct histone molecules. Set1 has an additional substrate in the kinetochore protein Dam1, which is methylated on K233. This methylation antagonizes phosphorylation of adjacent serines by the Ipl1 Aurora kinase. The discovery of a second Set1 substrate raised the question of how Set1 function is regulated at the kinetochore. I hypothesized that transcriptional regulatory factors essential for H3K4 methylation at gene promoters might also regulate Set1-mediated methylation of Dam1K233. Here I show that the regulatory factors essential for COMPASS activity at gene promoters is also indispensable for the methylation of Dam1K233. Deletion of members of the COMPASS complex leads to loss of Dam1K233 methylation. In addition, deletion of Rad6, Bre1, or members of the Paf1 complex abolishes Dam1 methylation. The role of Rad6 and Bre1 in Dam1 methylation is dependent on H2BK123 ubiquitination, as mutation of K123 within H2B results in complete loss of Dam1 methylation. Importantly, methylation of Dam1K233 is independent of transcription and occurs at the kinetochore. My results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription at the kinetochore. My data provide the first example of cross-talk in trans between modifications on a histone and a non-histone protein. Additionally, my results indicate that several factors previously thought to be required for Set1 function at gene promoters are more generally required for the catalytic activity of the COMPASS complex regardless of substrate or cellular process.

Metabolism and action of 9-$\beta$-D-arabinofuranosyl-2-fluoroadenine

9-$\beta$-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and 2$\sp\prime$-deoxyadenosine with potent antitumor activity both in vitro and in vivo. The mechanism of action of F-ara-A was evaluated both in whole cells and in experimental systems with purified enzymes. F-ara-A was converted to its 5$\sp\prime$-triphosphate F-ara-ATP in cells and then incorporated into DNA in a self-limiting manner. About 98% of the incorporated F-ara-AMP residues were located at the 3$\sp\prime$-termini of DNA strands, suggesting a chain termination property of this compound. DNA synthesis in CEM cells was inhibited by F-ara-A treatment with an IC$\sb{50}$ value of 1 $\mu$M. Cells were not able to restore the normal level of DNA synthesis even after being cultured in drug-free medium for 40 h. A DNA primer extension assay with M13mp18(+) single-stranded DNA template using purified human DNA polymerases $\alpha$ and further revealed that F-ara-ATP competed with dATP for incorporation into the A sites of the elongating DNA strands. The incorporation of F-ara-AMP into DNA resulted in a termination of DNA synthesis at the incorporated A sites. Pol $\alpha$ and $\delta$ were not able to efficiently extend the DNA primer with F-ara-AMP at its 3$\sp\prime$-end. Furthermore, the presence of F-ara-AMP at the 3$\sp\prime$-end of an oligodeoxyribonucleotide impaired its ligation with an adjacent DNA fragment by human and T4 ligases. Human DNA polymerase $\alpha$ incorporated more F-ara-AMP into DNA than polymerase $\delta$ and was more sensitive to the inhibition by F-ara-ATP, suggesting that polymerase $\alpha$ may be a preferred target for this analogue. On the other hand, DNA-dependent nucleotide turnover experiments and sequencing gel analysis demonstrated that DNA polymerase $\delta$ was able to remove the incorporated F-ara-AMP residue from the 3$\sp\prime$-end of the DNA strand with its 3$\sp\prime$-5$\sp\prime$ exonuclease activity in vitro, subsequently permitting further elongation of the DNA strand.^ The incorporation of F-ara-AMP into DNA was linearly correlated both with the inhibition of DNA synthesis and with the loss of clonogenicity. Termination of DNA synthesis and deletion of genetic material resulted from F-ara-AMP incorporation may be the mechanism responsible for cytotoxicity of F-ara-A. (Abstract shortened with permission of author.) ^

Citric acid cycle flux and pressure-volume work in the isolated working rat heart

It has been demonstrated previously that the mammalian heart cannot sustain physiologic levels of pressure-volume work if ketone bodies are the only substrates for respiration. In order to determine the metabolic derangement responsible for contractile failure in hearts utilizing ketone bodies, rat hearts were prefused at a near-physiologic workload in a working heart apparatus with acetoacetate and competing or alternate substrates including glucose, lactate, pyruvate, propionate, leucine, isoleucine, valine and acetate. While the pressure-volume work for hearts utilizing glucose was stable for 60 minutes of perfusion, performance fell by 30 minutes for hearts oxidizing acetoacetate as the sole substrate. The tissue content of 2-oxoglutarate and its transamination product, glutamate, were elevated in hearts utilizing acetoacetate while succinyl-CoA was decreased suggesting impaired flux through the citric acid cycle at the level of 2-oxoglutarate dehydrogenase. Further studies indicated that the inhibition of 2-oxoglutarate dehydrogenase developed prior to the onset of contractile failure and that the inhibition of the enzyme may be related to sequestration of the required cofactor, coenzyme A, as the thioesters acetoacetyl-CoA and acetyl-CoA. The contractile failure was not observed when glucose, lactate, pyruvate, propionate, valine or isoleucine were present together with acetoacetate, but the addition of acetate or leucine to acetoacetate did not improve performance indicating that improved performance is not mediated through the provision of additional acetyl-CoA. Furthermore, addition of competing substrates that improved function did not relieve the inhibition of 2-oxoglutarate dehydrogenase and actually resulted in the further accumulation of citric acid cycle intermediates "upstream" of 2-oxoglutarate dehydrogenase (2-oxoglutarate, glutamate, citrate and malate). Studies with (1-$\sp{14}$C) pyruvate indicate that the utilization of ketone bodies is associated with activation of NADP$\sp+$dependent malic enzyme and enrichment of the C4 pool of the citric acid cycle. The results suggest that contractile failure induced by ketone bodies in rat heart results from inhibition of 2-oxoglutarate dehydrogenase and that reversal of contractile failure is dissociated from relief of the inhibition, but rather is due to the entry of carbon units into the citric acid cycle as compounds other than acetyl-CoA. This mechanism of enrichment (anaplerosis) provides oxaloacetate for condensation with acetyl-CoA derived from ketone bodies allowing continued energy production by sustaining flux through a span of the citric acid cycle up to the point of inhibition at 2-oxoglutarate dehydrogenase for energy production thereby producing the reducing equivalents necessary to sustain oxidative phosphorylation. ^

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

EGFR and Notch signaling pathways are regulated by distinct isoforms of Drosophila cbl

Activation of cell surface receptors transduces extracellular signals into cellular responses such as proliferation, differentiation and survival. However, the appropriate spatial and temporal down-regulation of signaling receptors is essential for normal development and homeostasis. The Cbl family of E3-ubiquitin ligases plays a major role for the ligand-dependent inactivation of growth factor receptors through ubiquitin-mediated endocytosis and lysosomal degradation. Here, we report the D-cbl mutant phenotypes in the Drosophila eye. D-cbl mutants display overgrowth, inhibition of apoptosis, differentiation defects and increased ommatidial spacing. Many of these phenotypes are caused by lack of down-regulation of the Drosophila EGFR signaling. However, not all D-cbl phenotypes can be explained by inappropriate EGFR activity. We found that D-Cbl also negatively regulates Notch activity during eye and wing development. D-cbl produces two isoforms by alternative splicing. Strikingly, the long isoform, D-CblL, preferentially regulates the EGFR, whereas the short isoform, D-CblS, preferentially regulates Notch. Taken together, these data suggest that D-Cbl controls at least two signaling pathways, EGFR and Notch, through production of two alternatively spliced isoforms during development in Drosophila.^

Analysis of the E1-ubiquitin activating enzyme, Uba1, in cell death and tissue growth in Drosophila

Ubiquitination is an essential process involved in basic biological processes such as the cell cycle and cell death. Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Subsequently, ubiquitin is transferred to target proteins via ubiquitin ligases (E3). Defects in ubiquitin conjugation have been implicated in several forms of malignancy, the pathogenesis of several genetic diseases, immune surveillance/viral pathogenesis, and the pathology of muscle wasting. However, the consequences of partial or complete loss of ubiquitin conjugation in multi-cellular organisms are not well understood. Here, we report the characterization of nba1, the sole E1 in Drosophila. We have determined that weak and strong nba1 alleluias behave genetically different and sometimes in opposing phenotypes. For example, weak uba1 alleluias protect cells from cell death whereas cells containing strong loss-of-function alleluias are highly apoptotic. These opposing phenotypes are due to differing sensitivities of cell death pathway components to ubiquitination level alterations. In addition, strong uba1 alleluias induce cell cycle arrest due to defects in the protein degradation of Cyclins. Surprisingly, clones of strong uba1 mutant alleluias stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner resulting in severe overgrowth phenotypes in the mosaic fly. I have determined that the observed overgrowth phenotypes were due to a failure to downregulate the Notch signaling pathway in nba1 mutant cells. Aberrant Notch signaling results in the secretion of a local cytokine and activation of JAK/STAT pathway in neighboring cells. In addition, we elucidated a model describing the regulation of the caspase Dronc in surviving cells. Binding of Dronc by its inhibitor Diap1 is necessary but not sufficient to inhibit Dronc function. Ubiquitin conjugation and Uba1 function is necessary for the negative regulation of Dronc. ^

The combined effect of pravastatin and aspirin on clinical cardiovascular events

The 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, can achieve significant reductions in plasma low-density lipoprotein (LDL)-cholesterol levels. Experimental and clinical evidence now shows that some statins interfere with formation of atherosclerotic lesions independent of their hypolipidemic properties. Vulnerable plaque rupture can result in thrombus formation and artery occlusion; this plaque deterioration is responsible for most acute coronary syndromes, including myocardial infarction (MI), unstable angina, and coronary death, as well as coronary heart diseaseequivalent non-hemorrhagic stroke. Inhibition of HMG-CoA reductase has potential pleiotropic effects other than lipid-lowering, as statins block mevalonic acid production, a precursor to cholesterol and numerous other metabolites. Statins' beneficial effects on clinical events may also thus involve nonlipid-related mechanisms that modify endothelial function, inflammatory responses, plaque stability, and thrombus formation. Aspirin, routinely prescribed to post-MI patients as adjunct therapy, may potentiate statins beneficial effects, as aspirin does not compete metabolically with statins but acts similarly on atherosclerotic lesions. Common functions of both medications include inhibition of platelet activity and aggregation, reduction in atherosclerotic plaque macrophage cell count, and prevention of atherosclerotic vessel endothelial dysfunction. The Cholesterol and Recurrent Events (CARE) trial provides an ideal population in which to examine the combined effects of pravastatin and aspirin. Lipid levels, intermediate outcomes, are examined by pravastatin and aspirin status, and differences between the two pravastatin groups are found. A modified Cox proportional-hazards model with aspirin as a time-dependent covariate was used to determine the effect of aspirin and pravastatin on the clinical cardiovascular composite endpoint of coronary heart disease death, recurrent MI or stroke. Among those assigned to pravastatin, use of aspirin reduced the composite primary endpoint by 35%; this result was similar by gender, race, and diabetic status. Older patients demonstrated a nonsignificant 21% reduction in the primary outcome, whereas the younger had a significant reduction of 43% in the composite primary outcome. Secondary outcomes examined include coronary artery bypass graft (38% reduction), nonsurgical bypass, peripheral vascular disease, and unstable angina. Pravastatin and aspirin in a post-MI population was found to be a beneficial combination that seems to work through lipid and nonlipid, anti-inflammatory mechanisms. ^

Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase $\alpha$ are the molecular targets for two metal ions, Zn$\sp{2+}$ and Cd$\sp{2+},$ and an anticancer drug, F-ara-ATP.^ Human DNA ligases were purified to homogeneity and their AMP binding domains were mapped. Although their AMP-binding domains are similar, there could be difference between the two ligases in their DNA binding domains.^ The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP.^ A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex.^ F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3$\sp\prime$-terminus of DNA nick by DNA polymerase $\alpha.$^ All steps of the DNA ligation reaction were inhibited by Zn$\sp{2+}$ and Cd$\sp{2+}$ in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn$\sp{2+}$ and Cd$\sp{2+}$ showed their contradictory effects on the fidelity of the reaction by human DNA polymerase $\alpha.$ Zn$\sp{2+}$ decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd$\sp{2+}$ increased the frequencies of both misinsertion and mispair extension at very low concentration. Our data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported. ^

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

The Role of K63-linked Ubiquitination Cycles in Akt Kinase Activation

Akt (also known as protein kinase B) serves a central regulator in PI3K/Akt signaling pathways to regulate numerous physiological functions including cell proliferation, survival and metabolism. Akt activation requires the binding of Akt to phospholipid PIP3 on the plasma membrane and subsequent phosphorylation of Akt by its kinases. Growth factor-mediated membrane recruitment of Akt is a crucial step for Akt activation. However, the mechanism of Akt membrane translocation is unclear. Protein ubiquitination is a significant posttranslational modification that controls many biological functions such as protein trafficking and signaling activation. Therefore, we hypothesize that ubiquitination may be involved in Akt signaling activation. We have demonstrated that Akt could be conjugated with non-proteolytic K63-linked ubiquitination by TRAF6 ubiquitin E3 ligase. This modification on Akt was required for membrane recruitment, phosphorylation and activation of Akt in response to growth factor stimulation. The human cancer-associated Akt E17K mutant exhibited an increase in K63-linked ubiquitination, which contributes to the enrichment of membrane recruitment and phosphorylation of Akt. Thus, we conclude that K63-linked ubiquitination is a critical step for oncogenic Akt activation and also involved in human cancer development. Notably, the process of protein ubiquitination can be reversed by deubiquitinating enzymes (DUBs), which play a critical role to terminate signaling activation induced by ubiquitination. To further investigate how ubiquitination cycles regulate Akt activation, we have identified that CYLD as a DUB for Akt, and CYLD inhibited growth factor-induced ubiquitination and activation of Akt. Under serum-depletion condition, CYLD interacts with Akt and keep Akt under inactive state by directly removing K63-linked ubiquitination of Akt. CYLD disassociates with Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. We also demonstrated that CYLD deficiency promoted cancer cell proliferation, survival, glucose metabolism and human prostate cancer development. Therefore, we conclude that CYLD plays a critical role for negatively regulating Akt signaling activation through deubiquitination of Akt. In summary, this study delineated the important mechanism of cycles of ubiquitination and deubiquitination of Akt in regulating membrane translocation and activation of Akt, and TRAF6 and CYLD as central switches for these processes.

Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^