Browsing by Subject "Biosynthesis"

Now showing 1 - 5 of 5
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    Mass Spectrometric Approaches to Probing the Redox Function of Ape1
    (2012-07-03) Delaplane, Sarah Ann; Georgiadis, Millie M.; Bosron, William F.; Witzmann, F. A. (Frank A.)
    Human apurinic/apyrimidinic endonuclease 1 (hApe1) is a multi-functional protein having two major functions: apurinic/apyrimidinic endonuclease activity for DNA damage repair and redox activity for gene regulation. Many studies have shown the action of Ape1 in the base excision repair pathway leading to cell survival. It has also been reported that Ape1 reduces a number of important transcription factors that are involved in cancer promotion and progression. Though the repair activity is well understood, the redox mechanism is not yet clear. What is known about Ape1 is its structure and that it contains seven cysteines (C65, C93, C99, C138, C208, C296, and C310), none of which are disulfide bonded. Two of these cysteines, C99 and C138, are solvent-accessible, and C65, C93, and C99 are located in the redox domain. It is believed that one or more cysteines are involved in the redox function and is hypothesized that hApe1 reduces the down-stream transcription factors by a disulfide exchange mechanism. E3330, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquninoyl)]2-nonyl-2-propenoic acid, is a specific inhibitor for the redox function of hApe1. The interaction mechanism is not known. Using N-Ethylmaleimide (NEM) chemical footprinting, combined with Hydrogen/Deuterium Exchange (HDX) data, we propose that a locally unfolded form coexists with the folded form in an equilibrium that is driven by irreversible NEM labeling, and that E3330 interacts with and stabilizes this locally unfolded form. This locally unfolded form is thereby proposed to be the redox-active form. We further support this claim with LC-MS/MS analysis showing an increase of disulfide bonds induced by E3330 among the cysteines in the redox domain, which would be too far apart from each other in the folded form to form a disulfide bond. We also studied three analogs of E3330. The need for an E3330 analog is to develop a more efficient and effective compound that would allow for sub-micromolar levels of activity (E3330 requires a micromolar amount). Study of the analogs will also allow us to gain perspective of the mechanism or mechanisms of E3330’s activity in Ape1’s redox function.
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    Mechanisms of hexosamine-induced cholesterol accumulation and therapeutic actions of chromium
    (2014-01-03) Penque, Brent A.; Elmendorf, Jeffrey S.; Atkinson, Simon; Considine, Robert V.; Evans-Molina, Carmella; Kempson, Stephen A.
    Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.
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    The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation
    (Public Library of Science, 2017-12-29) Long, Quanxin; Yan, Ran; Hu, Jieli; Cai, Dawei; Mitra, Bidisha; Kim, Elena S.; Marchetti, Alexander; Zhang, Hu; Wang, Soujuan; Liu, Yuanjie; Huang, Ailong; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.
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    Starve Cancer Cells of Glutamine: Break the Spell or Make a Hungry Monster?
    (MDPI, 2019-06-11) Jiang, Jie; Srivastava, Sankalp; Zhang, Ji; Biochemistry & Molecular Biology, IU School of Medicine
    Distinct from normal differentiated tissues, cancer cells reprogram nutrient uptake and utilization to accommodate their elevated demands for biosynthesis and energy production. A hallmark of these types of reprogramming is the increased utilization of, and dependency on glutamine, a nonessential amino acid, for cancer cell growth and survival. It is well-accepted that glutamine is a versatile biosynthetic substrate in cancer cells beyond its role as a proteinogenic amino acid. In addition, accumulating evidence suggests that glutamine metabolism is regulated by many factors, including tumor origin, oncogene/tumor suppressor status, epigenetic alternations and tumor microenvironment. However, despite the emerging understanding of why cancer cells depend on glutamine for growth and survival, the contribution of glutamine metabolism to tumor progression under physiological conditions is still under investigation, partially because the level of glutamine in the tumor environment is often found low. Since targeting glutamine acquisition and utilization has been proposed to be a new therapeutic strategy in cancer, it is central to understand how tumor cells respond and adapt to glutamine starvation for optimized therapeutic intervention. In this review, we first summarize the diverse usage of glutamine to support cancer cell growth and survival, and then focus our discussion on the influence of other nutrients on cancer cell adaptation to glutamine starvation as well as its implication in cancer therapy.
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    What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira.
    (PLOS, 2016-02) Fouts, Derrick E.; Matthias, Michael A.; Adhikarla, Haritha; Adler, Ben; Amorim-Santos, Luciane; Berg, Douglas E.; Bulach, Dieter; Buschiazzo, Alejandro; Chang, Yung-Fu; Galloway, Renee L.; Haake, David A.; Haft, Daniel H.; Hartskeerl, Rudy; Ko, Albert I.; Levett, Paul N.; Matsunaga, James; Mechaly, Ariel E.; Monk, Jonathan M.; Nascimento, Ana L. T.; Nelson, Karen E.; Palsson, Bernhard; Peacock, Sharon J.; Picardeau, Mathieu; Ricaldi, Jessica N.; Thaipandungpanit, Janjira; Wunder, Elsio A.; Yang, X. Frank; Zhang, Jun-Jie; Vinetz, Joseph M.; Department of Microbiology & Immunology, IU School of Medicine
    Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.