Department of Biochemistry and Molecular Biology Works

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    Mutations in the CDP-Choline Pathway for Phospholipid Biosynthesis Bypass the Requirement for an Essential Phospholipid Transfer Protein
    (Cell Press, 1991) Cleves, Ann E.; McGee, Todd P.; Whitters, Eric A.; Champion, Kathleen M.; Aitken, Jacqueline R.; Dowhan, William; Goebl, Mark; Bankaitis, Vytas A.; Biochemistry and Molecular Biology, School of Medicine
    SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.
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    Regulation of neuregulin-mediated acetylcholine receptor synthesis by protein tyrosine phosphatase SHP2
    (Society for Neuroscience, 1999-11-01) Tanowitz, Michael; Si, Jutong; Yu, De-Hua; Feng, Gen-Sheng; Mei, Lin; Biochemistry and Molecular Biology, School of Medicine
    Synapse-specific expression of the nicotinic acetylcholine receptor (AChR) is believed to be mediated by neuregulin, an epidermal growth factor-like trophic factor released by somatic motoneurons at the neuromuscular junction (NMJ). Neuregulin stimulates ErbB2, ErbB3, and ErbB4, members of the ErbB family of receptor tyrosine kinases. SHP2 is a cytoplasmic protein tyrosine phosphatase containing two Src homology 2 domains near its N terminus, and has been shown to be a positive mediator of mitogenic responses to various growth factors. We found that SHP2 interacted with ErbB2 and ErbB3 after neuregulin stimulation of muscle cells. Expression of SHP2 in C2C12 mouse muscle cells attenuated the neuregulin-induced expression of an AChR epsilon-promoter reporter gene, whereas a catalytically inactive SHP2 mutant or a mutant lacking the N-terminal Src homology 2 (SH2) domain enhanced reporter expression, suggesting that SHP2 negatively regulates the neuregulin signaling pathway. In fibroblast cells that express a mutant SHP2 with a targeted deletion of the N-terminal SH2 domain, neuregulin-mediated activation of the Ras/Raf/extracellular signal-regulated kinase cascade was enhanced. Furthermore, we found that SHP2 immunoreactivity colocalized with the staining of alpha-bungarotoxin, a marker of the NMJ. These results demonstrate a negative role of SHP2 in the neuregulin signal that leads to AChR gene expression at the NMJ.
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    Transthyretin: a review from a structural perspective
    (Springer, 2001) Hamilton, J. A.; Benson, M. D.; Biochemistry and Molecular Biology, School of Medicine
    Transthyretin (formerly called prealbumin) plays important physiological roles as a transporter of thyroxine and retinol-binding protein. X-ray structural studies have provided information on the active conformation of the protein and the site of binding of both ligands. Transthyretin is also one of the precursor proteins commonly found in amyloid deposits. Both wild-type and single-amino-acid-substituted variants have been identified in amyloid deposits, the variants being more amyloidogenic. Sequencing of the gene and the resulting production of a transgenic mouse model have resulted in progress toward solving the mechanism of amyloid formation and detecting the variant gene in individuals at risk.
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    The collaborative study on the genetics of alcoholism: an update
    (The National Institute on Alcohol Abuse and Alcoholism, 2002) Edenberg, Howard J.; Biochemistry and Molecular Biology, School of Medicine
    The Collaborative Study on the Genetics of Alcoholism (COGA) is a large-scale family study designed to identify genes that affect the risk for alcoholism (i.e., alcohol dependence) and alcohol-related characteristics and behaviors (i.e., phenotypes1). This collaborative project is funded by the National Institute on Alcohol Abuse and Alcoholism. Data collection, analysis, and/or storage for this study take place at nine sites across the United States. Because alcoholism is a complex genetic disorder, the COGA researchers expected that multiple genes would contribute to the risk. In other words, there will be no single “gene for alcoholism” but rather variations in many different genes that together, interacting with the environment, place some people at significantly higher risk for the disease. This genetic and environmental variability (i.e., heterogeneity) makes the task of identifying individual genes difficult. However, the COGA project was designed with these difficulties in mind and incorporated strategies to meet the challenges. This article briefly reviews these strategies and summarizes some of the results already obtained in the ongoing COGA study.
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    Synergistic activation of p70S6 kinase associated with stem cell factor in MO7e cells
    (Springer Nature, 2003-06) Lee, Younghee; Broxmeyer, Hal E.; Mantel, Charlie; Kwon, Hyung-Joo; Wha Kim, Jae; Sook Kim, Jin; Kwon, Durhan; Seong Chloe, In; Biochemistry and Molecular Biology, School of Medicine
    Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.
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    Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions
    (Elsevier, 2003-11-10) Villanueva, Rodrigo A.; Jonsson, Colleen B.; Jones, Jennifer; Georgiadis, Millie M.; Roth, Monica J.; Biochemistry and Molecular Biology, School of Medicine
    Retroviral integrases (IN) catalyze the integration of the reverse-transcribed viral DNA into the host genome, an essential process leading to virus replication. For Moloney murine leukemia virus (M-MuLV) IN, the limited solubility of the recombinant protein has restricted the development of biophysical and structural analyses. Herein, recombinant M-MuLV IN proteins, either full length or two nonoverlapping domain constructs, were purified under non-denaturing conditions from solubilized bacterial extracts by Ni2+-NTA resins. Additionally, WT IN was further purified by heparin chromatography. All of the purified proteins were shown to be active and stable. WT M-MuLV IN chromatographed with a peak corresponding with a dimer by gel filtration chromatography. In contrast, the single point mutant C209A IN migrated predominantly as a tetramer. For both proteins, fractions in equilibrium between dimers and tetramers were competent to assemble concerted two-end integrations and yielded a unique strand-transfer profile in the presence of a 28-mer U5 oligonucleotide substrate, indicative of a distinct conformation within the synaptic complex. This specific target-site selection was not observed with a shorter 20-mer U5 substrate. These studies provide the foundation for biophysical and structural analysis on M-MuLV IN and the mechanism of retroviral integration.
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    Lack of EGF receptor contributes to drug sensitivity of human germline cells
    (2005-01) Park, S.J.; Armstrong, S.; Kim, C.H.; Yu, M.; Robertson, K.; Kelley, Mark R.; Lee, S.H.
    Germline mutations have been associated with generation of various types of tumour. In this study, we investigated genetic alteration of germline tumours that affect the drug sensitivity of cells. Although all germline tumour cells we tested were hypersensitive to DNA-damaging drugs, no significant alteration was observed in their DNA repair activity or the expression of DNA repair proteins. In contrast, germline tumours expressed very low level of epidermal growth factor receptor (EGFR) compared to drug-resistant ovarian cancer cells. An immunohistochemical analysis indicated that most of the primary germline tumours we tested expressed very low level of EGFR. In accordance with this, overexpression of EGFR in germline tumour cells showed an increase in drug resistance, suggesting that a lack of EGFR, at least in part, contributes to the drug sensitivity of germline tumours.
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    Lack of EGF receptor contributes to drug sensitivity of human germline cells
    (Springer, 2005-01) Park, S.J.; Armstrong, S.; Kim, C.H.; Yu, M.; Robertson, K.; Kelley, Mark R.; Lee, S.H.; Biochemistry and Molecular Biology, School of Medicine
    Germline mutations have been associated with generation of various types of tumour. In this study, we investigated genetic alteration of germline tumours that affect the drug sensitivity of cells. Although all germline tumour cells we tested were hypersensitive to DNA-damaging drugs, no significant alteration was observed in their DNA repair activity or the expression of DNA repair proteins. In contrast, germline tumours expressed very low level of epidermal growth factor receptor (EGFR) compared to drug-resistant ovarian cancer cells. An immunohistochemical analysis indicated that most of the primary germline tumours we tested expressed very low level of EGFR. In accordance with this, overexpression of EGFR in germline tumour cells showed an increase in drug resistance, suggesting that a lack of EGFR, at least in part, contributes to the drug sensitivity of germline tumours.
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    Antidiabetic thiazolidinediones induce ductal differentiation but not apoptosis in pancreatic cancer cells
    (Elsevier, 2005-02-28) Ceni, Elisabetta; Mello, Tommaso; Tarocchi, Mirko; Crabb, David W.; Caldini, Anna; Invernizzi, Pietro; Surrenti, Calogero; Milani, Stefano; Galli, Andrea; Department of Biochemistry and Molecular Biology, IU School of Medicine
    AIM: Thiazolidinediones (TZD) are a new class of oral antidiabetic drugs that have been shown to inhibit growth of same epithelial cancer cells. Although TZD were found to be ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), the mechanism by which TZD exert their anticancer effect is presently unclear. In this study, we analyzed the mechanism by which TZD inhibit growth of human pancreatic carcinoma cell lines in order to evaluate the potential therapeutic use of these drugs in pancreatic adenocarcinoma. METHODS: The effects of TZD in pancreatic cancer cells were assessed in anchorage-independent growth assay. Expression of PPARgamma was measured by reverse-transcription polymerase chain reaction and confirmed by Western blot analysis. PPARgamma activity was evaluated by transient reporter gene assay. Flow cytometry and DNA fragmentation assay were used to determine the effect of TZD on cell cycle progression and apoptosis respectively. The effect of TZD on ductal differentiation markers was performed by Western blot. RESULTS: Exposure to TZD inhibited colony formation in a PPARgamma-dependent manner. Growth inhibition was linked to G1 phase cell cycle arrest through induction of the ductal differentiation program without any increase of the apoptotic rate. CONCLUSION: TZD treatment in pancre
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    Description of the data from the Collaborative Study on the Genetics of Alcoholism (COGA) and single-nucleotide polymorphism genotyping for Genetic Analysis Workshop 14
    (Springer Nature, 2005-12-30) Edenberg, Howard J.; Bierut, Laura J.; Boyce, Paul; Cao, Manqiu; Cawley, Simon; Chiles, Richard; Doheny, Kimberly F.; Hansen, Mark; Hinrichs, Tony; Jones, Kevin; Kelleher, Mark; Kennedy, Giulia C.; Liu, Guoying; Marcus, Gregory; McBride, Celeste; Shaw Murray, Sarah; Oliphant, Arnold; Pettengill, James; Porjesz, Bernice; Pugh, Elizabeth W.; Rice, John P.; Rubano, Todd; Shannon, Stu; Steeke, Rhoberta; Tischfield, Jay A.; Tsai, Ya Yu; Zhang, Chun; Begleiter, Henri; Biochemistry and Molecular Biology, School of Medicine
    The data provided to the Genetic Analysis Workshop 14 (GAW 14) was the result of a collaboration among several different groups, catalyzed by Elizabeth Pugh from The Center for Inherited Disease Research (CIDR) and the organizers of GAW 14, Jean MacCluer and Laura Almasy. The DNA, phenotypic characterization, and microsatellite genomic survey were provided by the Collaborative Study on the Genetics of Alcoholism (COGA), a nine-site national collaboration funded by the National Institute of Alcohol and Alcoholism (NIAAA) and the National Institute of Drug Abuse (NIDA) with the overarching goal of identifying and characterizing genes that affect the susceptibility to develop alcohol dependence and related phenotypes. CIDR, Affymetrix, and Illumina provided single-nucleotide polymorphism genotyping of a large subset of the COGA subjects. This article briefly describes the dataset that was provided.