Browsing by Subject "Oncogene Proteins"
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Item Behavioral, Neurochemical, and Histological Characterization of Mice Deficient for Parkin, DJ-1, and Antioxidant Proteins(2011-08-10) Seamans, Katherine Webster; Goldberg, Matthew S.Parkinson’s disease is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra. The cause of Parkinson’s disease remains uncertain, however, evidence implicates mitochondrial dysfunction and oxidative stress with selective vulnerability of dopaminergic neurons. Although most cases of Parkinson’s disease are sporadic, 5-10% of cases are caused by mutations in a single gene. Loss-of-function mutations in parkin and DJ-1 were the first to be linked to recessively inherited parkinsonism. Surprisingly, mice bearing similar loss-of-function mutations in parkin and DJ-1 do not show age-dependent loss of nigral dopaminergic neurons or depletion of dopamine in the striatum. Although the normal cellular functions of Parkin and DJ-1 remain unclear, we hypothesized that Parkin and DJ-1 protect cells from oxidative stress and that loss-of-function mutations in these genes cause neurodegeneration in Parkinson’s disease by rendering cells more sensitive to mitochondrial dysfunction and oxidative stress. We crossed mice deficient for Parkin and DJ-1 with mice deficient for the major mitochondrial antioxidant protein Mn-superoxide dismutase or Cu/Zn-superoxide dismutase. Previous studies have shown that mice with reduced levels of Cu/Zn-superoxide dismutase or Mn-superoxide dismutase are more sensitive to dopaminergic neurotoxins whereas mice with increased levels of superoxide dismutase are more resistant to dopaminergic neurotoxins. We predicted that reducing levels of antioxidant proteins in parkin-/-DJ-1-/- mice would result in age-dependent nigral cell loss, striatal dopamine depletion or behavioral abnormalities. Characterization of these mice for behavioral abnormalities, neurotransmitter defects and neuropathology, revealed significant behavioral abnormalities in the mutant mice even in the absence of significant changes to dopamine levels in the striatum, dopamine receptor density, or dopaminergic neuron numbers. Aged parkin-/-DJ-1-/- and Mn-superoxide dismutase triple deficient mice have a surprising enhanced rotorod performance without the presence of an anxiety phenotype or hyperactivity. Cu/Zn-superoxide dismutase and Mn-superoxide dismutase triple deficient mice have elevated levels of dopamine in the striatum, however none of the mice present with nigral cell loss. Levels of D1-like and D2-like dopamine receptors in the striatum were unchanged. It is evident from our studies that on a parkin/DJ-1 null background, additional loss of major antioxidant proteins does not lead to a progressive loss of dopaminergic neurons in mice.Item The Endoplasmic Reticulum Udpase ENTPD5 Promotes Cancer Cell Growth and Survival in the PI3K/PTEN(2011-02-01) Shen, Zhirong; Wang, XiaodongPI3 Kinase and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinase that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. Activation of AKT and downstream PI3 Kinase signaling promotes protein translation, resulting in increased protein flux into ER; this will lead to decreased efficiency of protein folding and accumulation of unfolded proteins in the ER and finally lead to ER stress. How does cancer cell solve this problem of increased folding during rapid growth to avoid ER stress? We discovered that ENTPD5, an endoplasmic reticulum (ER) enzyme, is up-regulated in cell lines and primary human tumor samples with active AKT. AKT upregulates ENTPD5 by relieving transcriptional inhibition by FoxO transcription factors. ENTPD5 hydrolyzes UDP to UMP to promote protein N-glycosylation and folding in ER. Knockdown of ENTPD5 in PTEN-null cells causes ER stress and loss of receptor tyrosine kinases through ER-associated degradation pathway under stress conditions. Consequently, the growth of PTEN-null cells is inhibited both in vitro and in mouse xenograft tumor models. ENTPD5 is therefore an essential component for PI3K/AKT active cancer cells and a potential drug target for anti-cancer therapy.Item Small Molecule Regulator of ENTPD5, and ER Enzyme in the PTEN/AKT Pathway(2011-02-01) Huang, Song; Wang, XiaodongPI3K signaling plays a crucial role in effecting alterations in a broad range of cellular functions in response to diverse extracellular stimuli (insulin, growth factors, integrins and GPCRs etc.). A key downstream effector of PI3K is the serine-threonine kinase Akt, which in response to PI3K activation, phosphorylates and regulates the activity of a number of cellular targets, through which it modulates a variety of cellular functions, including glucose metabolism, protein synthesis, cell proliferation and survival. Tumor supressor gene PTEN encodes a lipid phosphatase that antagonizes PI3K function and consequently inhibits downstream signaling through Akt. Dysregulation of this pathway has been found in a variety of human cancer, mainly by loss of function of PTEN, or amplification and activating mutations of PI3K and/or Akt. We were interested in the anti-apoptotic mechanism of PI3K/Akt signaling. We observed a defect in apoptosome formation in PTEN-null (PTEN -/-) MEF cell lysate. This defect is due to rapid depletion of ATP by a strong ATP hydrolysis activity in PTEN-null MEF lysate, which is absent in PTEN heterozygous (PTEN +/-) lysate. Following this activity, we purified three enzymes, namely ENTPD5, CMPK1 and AK1, that together forms a coupled enzymatic cycle, hydrolyzing ATP to AMP. In the cell, ENTPD5 is an ER localized UDPase that hydrolyzes UDP, the by-product of glycosyl-transferase, into UMP. Only in its monophosphate form can Uridine nucleotide exits ER through an antiporter by exchanging a molecule of UDP-sugar from cytosol. Up-regulation of ENTPD5 in PTEN-null MEF cells accelerates glycosylation substrate replenishment, therefore promotes N-glycosylation and increases ER protein folding capacity to accommendate the increase of protein synthesis resulted from active PI3K/Akt signaling. Knockdown of ENTPD5 in PTEN-null cells suppresses global N-glycosylation, resulting in ER stress and degradation of several growth factor receptors. As a consequence, the growth of PTEN-null cells is inhibited both in vitro and in mouse xenograft tumor models. Given the essential role of ENTPD5 in PI3K/Akt pathway, we performed biochemical high-throughput screen for ENTPD5 inhibitors. The newly identified inhibitors recapitulate the phenotype of ENTPD5 knockdown in vitro. Interestingly, PTEN-null MEF cells are more susceptible to these inhibitors than PTEN heterozygous MEF cells, in terms of the intensity of induced ER stress and cell death. Inhibition of ENTPD5 produces synthetic lethality with PTEN loss or PI3K/Akt hyperactivation, therefore provides a potential therapy for the cancers harboring these lesions.