PubMed · 7856111
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I Saito. 1994. [Adenovirus vector].. https://doi.org/10.2222/jsv.44.100
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A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.
A recombinant adenovirus coding for rat aquaporin-5 was constructed and plaque purified. The recombinant adenovirus (AdrAQP5) mediated the expression of aquaporin-5 in rat and human salivary cell lines and in dog kidney cells in vitro as demonstrated by Northern blot and Western blot analyses, and by confocal microscopy after immunofluorescent labeling. In kidney cells, expression of the transgene was optimal if cells were infected at their basolateral surface, a phenomenon associated with the distribution of integrin receptors on these cells. The expressed aquaporin-5 protein was functionally active because viral-mediated gene transfer resulted in a significant increase in the osmotically directed net fluid secretion rate across monolayers of kidney cells. AdrAQP5 should provide an efficient and useful means to impart facilitated water permeability to cells lacking such a pathway.
The first step of glucose metabolism is the phosphorylation of glucose, catalyzed by the hexokinase family of enzymes. To address the metabolic impact of increasing glucose phosphorylation capacity in liver, rat primary hepatocytes were treated with recombinant adenoviruses containing the cDNAs encoding either rat liver glucokinase (AdCMV-GKL) or rat hexokinase I (AdCMV-HKI). Maximal glucose phosphorylation in AdCMV-GKL- and AdCMV-HKI-treated hepatocytes was increased 7.1 +/- 1.2- and 6.3 +/- 0.8-fold, respectively, over hepatocytes treated with an adenovirus expressing beta-galactosidase. Glucose usage (measured with 3 and 20 m 2-[3H]glucose and 5-[3H]glucose) was significantly increased in AdCMV-GKL-treated cells preincubated in 1 or 25 mM glucose. Treatment of hepatocytes with AdCMV-HKI also caused enhanced glucose utilization, but the increases were smaller and were less apparent in cells preincubated in high (25 mM) glucose. AdCMV-GKL-treated hepatocytes incubated for 48 h in the presence of variable glucose concentrations had glycogen levels that were maximally 15.0 +/- 0. 6-fold greater than levels in corresponding control cells. AdCMV-HKI-treated hepatocytes incubated under similar conditions had unchanged glycogen levels relative to controls. In AdCMV-GKL-treated cells, lactate output was increased to a maximum of 3.0 +/- 0.4-fold (at 25 mM glucose), glucose oxidation was increased 3.5 +/- 0.3-fold, and triglyceride production was unchanged relative to untreated cells. Among these three parameters, only lactate production was increased in AdCMV-HKI-treated cells, and then only at low glucose concentrations. We conclude that overexpression of glucokinase has potent effects on glucose storage and utilization in hepatocytes and that these effects are not matched by overexpression of hexokinase I.