The regulation of carbamoyl phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) by phosphorylation and protein-protein interactions

Pyrimidines have many important roles in cellular physiology, as they are used in the formation of DNA, RNA, phospholipids, and pyrimidine sugars. The first rate-limiting step in the de novo pyrimidine synthesis pathway is catalyzed by the carbamoyl phosphate synthetase II (CPSase II) part of the multienzymatic complex Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, Dihydroorotase (CAD). CAD gene induction is highly correlated to cell proliferation. Additionally, CAD is allosterically inhibited or activated by uridine triphosphate (UTP) or phosphoribosyl pyrophosphate (PRPP), respectively. The phosphorylation of CAD by PKA and ERK has been reported to modulate the response of CAD to allosteric modulators. While there has been much speculation on the identity of CAD phosphorylation sites, no definitive identification of in vivo CAD phosphorylation sites has been performed. Therefore, we sought to determine the specific CAD residues phosphorylated by ERK and PKA in intact cells. We observed the PKA-induced phosphorylation of Ser1406 and Ser1859 HEK-293 cells. Surprisingly, while ERK phosphorylated CAD on multiple residues in vitro, CAD was not an ERK substrate in HEK-293 cells. We determined the identity of a previously unknown phosphopeptide in CAD isolated from HEK-293 cells. We also observed that the phosphorylation of CAD in HEK-293 cells is important for the maintenance of CPSase II activity. In addition to investigating the regulation of CAD by phosphorylation, we have identified a novel protein-protein interaction between CAD and the human cell cycle checkpoint protein hRad9. The interaction was mapped to the CPSase II portion of CAD, and the binding of hRad9 to CAD induced a significant activation of CPSase II activity. Taken together, these studies demonstrate novel mechanisms of CAD regulation in mammalian cells

URACILATED HIV-1 DNA FOLLOWS DIVERSE FATES DURING INFECTION OF MYELOID LINEAGE CELLS

The uracil nucleobase plays a central role in intrinsic immunity against HIV-1 infection when it is found in DNA rather than RNA. The most well characterized uracil-centric innate immune response involves host DNA cytidine deaminase enzymes (APOBECs), which selectively deaminate cytosine residues during HIV-1 first strand DNA synthesis thereby rendering the viral genome nonfunctional by hypermutation (G→A). Previous work from our group suggested the presence of another uracil-dependent HIV-1 restriction pathway that does not involve APOBEC enzymes. The function of this pathway, which has been historically controversial, involves incorporation of dUTP into viral DNA by reverse transcriptase to produce U/A base pairs (uracilation), which preserve the coding potential of native T/A pairs. Notably, U/A pairs are “invisible” to normal DNA sequencing methods and their presence, persistence and ultimate fate in proviral DNA of HIV-1 infected cells is an important aspect of viral infection that has been largely unexplored. The limiting dNTP pool levels and unique DNA repair capacities of non-dividing cells such as monocytes, macrophages and dendritic cells provides a distinctive and poorly understood environment for HIV replication and infection. This work describes new methods for detecting uracil bases in HIV-1 DNA and provides a detailed examination of their fate over the course of infection in monocyte-derived macrophage (MDM) target cells of HIV-1. We report that a major subpopulation of MDMs has a metabolic phenotype leading to high levels of dUTP incorporation into HIV-1 DNA (U/A pairs) during reverse transcription. Importantly, U/A base pairs are detected in short-lived blood monocytes and alveolar macrophages (but not CD4+ T cells) from antiretroviral therapy (ART)-suppressed HIV-1 infected individuals suggesting that they arise from recent passage of monocytes through a drug resistant viral reservoir. The findings define a restriction/persistence pathway that operates in monocytes, macrophages and perhaps other myeloid lineage cells, but not in CD4+ T cells. This newly defined interplay between host dNTP pools, DNA repair machinery and HIV-1 is important for understanding the potential of tissue macrophages to establish and maintain a long-term HIV reservoir

Proteomic studies on anti-proliferating activities of adenosine and cordycepin in human cancer cell lines.

Tam Wai-Kwan Karen.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 109-128).Abstracts in English and Chinese.Thesis committee --- p.iStatement --- p.iiAbstract --- p.iiiAcknowledgements --- p.viAbbreviations --- p.viiTable of Contents --- p.ixList of Tables --- p.xiiList of Figures --- p.xivChapter 1. --- Introduction --- p.1Chapter 2. --- Literature Review --- p.2Chapter 2.1 --- Introduction of Cordyceps --- p.2Chapter 2.2 --- Pharmacological functions of Cordyceps --- p.3Chapter 2.2.1 --- Functions in respiratory system --- p.3Chapter 2.2.2 --- Functions in renal system --- p.7Chapter 2.2.3 --- Functions in hepatic system --- p.8Chapter 2.2.4 --- Functions in cardiovascular system --- p.9Chapter 2.2.5 --- Functions in endocrine and steroid system --- p.10Chapter 2.2.6 --- Functions in the immune system --- p.11Chapter 2.2.7 --- Functions in nervous system --- p.15Chapter 2.2.8 --- Controls in glucose metabolism --- p.15Chapter 2.2.9 --- Anti-oxidation activity --- p.16Chapter 2.2.10 --- Anti-tumor activity --- p.18Chapter 2.3 --- Active ingredients of Cordyceps and their related biological activities --- p.20Chapter 2.3.1 --- Polysaccharides --- p.20Chapter 2.3.2 --- Nucleosides --- p.21Chapter 2.3.2.1 --- Adenosine --- p.21Chapter 2.3.2.2 --- Cordycepin --- p.24Chapter 2.4 --- Proteomic tools in studies of the change in protein expression --- p.25Chapter 2.4.1 --- Two-dimensional electrophoresis --- p.27Chapter 2.4.2 --- Mass Spectrometry --- p.28Chapter 3. --- Methods and Materials --- p.30Chapter 3.1 --- Cell lines and culture conditions --- p.30Chapter 3.2 --- Trypan blue exclusion method --- p.30Chapter 3.3 --- Cell counting --- p.31Chapter 3.4 --- Anti-proliferation assay --- p.31Chapter 3.5 --- Anti-proliferation assay of normal cell line --- p.32Chapter 3.6 --- Determination of ic50 --- p.33Chapter 3.7 --- Sample preparation for proteins studies --- p.33Chapter 3.8 --- Protein quantitation --- p.34Chapter 3.9 --- Gel electrophoresis --- p.36Chapter 3.10 --- Image analysis --- p.37Chapter 3.11 --- In-gel digestion and MALDI-ToF MS --- p.37Chapter 3.12 --- Statistical Analysis --- p.39Chapter 3.13 --- Chemicals --- p.39Chapter 4. --- Results --- p.41Chapter 4.1 --- MTT assay --- p.41Chapter 4.1.1 --- The anti-proliferating activity of adenosine against cancer cell lines (HepG2 and SV7tert) and normal cell line (Hs68) --- p.41Chapter 4.1.2 --- The anti-proliferating activity of cordycepin against cancer cell lines (HepG2 and SV7tert) and normal cell line (Hs68) --- p.42Chapter 4.1.3 --- The anti-proliferation effects of adenosine and cordycepin --- p.42Chapter 4.2 --- Changes in protein expression --- p.50Chapter 4.2.1 --- "Corresponding drug treatment of cell lines (HepG2, SV7tert and Hs68)" --- p.50Chapter 4.2.2 --- "Comparison of protein profiles from cells (HepG2, SV7tert or Hs68) under the normal and drug treated (with either adenosine or cordycepin) conditions" --- p.51Chapter 4.2.2.1 --- HepG2 study --- p.51Chapter 4.2.2.2 --- SV7tert study --- p.52Chapter 4.2.2.3 --- Hs68 study --- p.52Chapter 4.2.3 --- Protein identification --- p.53Chapter 4.2.3.1 --- HepG2 cell line --- p.53Chapter 4.2.3.2 --- HepG2-changes in protein expressions after adenosine treatment --- p.54Chapter 4.2.3.3 --- HepG2-changes in protein expressions after cordycepin treatment --- p.54Chapter 4.2.3.4 --- SV7tert cell line --- p.54Chapter 4.2.3.5 --- SV7tert-changes in protein expressions after cordycepin treatment --- p.55Chapter 4.2.3.6 --- Hs68 cell line --- p.55Chapter 4.2.3.7 --- Hs68-changes in protein expressions after cordycepin treatment --- p.56Chapter 5. --- Discussion --- p.89Chapter 5.1 --- anti-proliferation assays --- p.89Chapter 5.2 --- changes in protein expression: --- p.90Chapter 5.2.1 --- Protein alterations in HepG2 --- p.91Chapter 5.2.1.1 --- Changes in protein expression (membrane protein and transport: Trimethyllysine hydroxylase) --- p.91Chapter 5.2.1.2 --- Changes in protein expression (protein synthesis and folding: carboxypeptidase E) --- p.92Chapter 5.2.1.3 --- Changes in protein expression (membrane proteins and transport: calumenin and electron transfer flavoproteins) --- p.93Chapter 5.2.2 --- Protein alterations in SV7tert --- p.94Chapter 5.2.2.1 --- Changes in protein expression (protein synthesis and folding: BiP(GRP78)) --- p.94Chapter 5.2.2.2 --- Changes in protein expression (cell defense and tolerance: Hsp60 (chaperonin); TANK binding kinase-1) --- p.96Chapter 5.2.2.3 --- Changes in protein expression (metabolism: prolyl 4-hydroxylase; aldolase A; glyceraldehyde-3-phosphate dehydrogenase) --- p.97Chapter 5.2.2.4 --- Changes in protein expression (cell growth and division: βII tubulin; HnRNP Al) --- p.100Chapter 5.2.3 --- Protein alterations in Hs68 --- p.101Chapter 5.2.3.1 --- Changes in protein expression (metabolism: triosephosphate isomerse 1) --- p.101Chapter 6. --- Discussion --- p.103Chapter 6.1 --- The antiproliferating activities of adenosine and cordycepin --- p.103Chapter 6.2 --- "Effects of adenosine and cordycepin on the changes in protein expressions in HepG2, SV7tert and Hs68" --- p.104Chapter 6.3 --- Problems and improvements in two-dimensional gel electrophoresis --- p.105Chapter 7. --- Conclusion and future prospectives --- p.107References --- p.10

Contribution of Glucose Metabolism to the B Lymphocyte Responses

Thesis advisor: Thomas C. ChilesB-lymphocytes respond to environmental cues for their survival, growth, and differentiation through receptor-mediated signaling pathways. Naïve Blymphocytes must acquire and metabolize external glucose in order to support the bioenergetics associated with maintaining cell volume, ion gradients, and basal macromolecular synthesis. The up-regulation of glycolytic enzyme expression and activity via engaged B-cell receptor mediated-events was glucose-dependent. This suggests an essential role for glucose energy metabolism in the promotion of B cell growth, survival, and proliferation in response to extracellular stimuli. In addition, the activity of ATP-citrate lyase (ACL) was determined to be crucial for ex vivo splenic B cell differentiation to antibody-producing cells wherein B cells undergo endomembrane synthesis and expansion. This investigation employed knockout murine models as well as chemical inhibitors to determine the signaling components and enzymes responsible for glucose utilization and incorporation into membrane lipids. These results point to a critical role for phosphatidylinositol 3- kinase (PI3K) in orchestrating cellular glucose energy metabolism and glucosedependent de novo lipogenesis for B lymphocyte responses.Thesis (PhD) — Boston College, 2012.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology