Browsing by Subject "HEK 293 cells"

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    An angiotensin I-converting enzyme mutation (Y465D) causes a dramatic increase in blood ACE via accelerated ACE shedding
    (Public Library of Science, 2011) Danilov, Sergei M; Gordon, Kerry; Nesterovitch, Andrew B; Lünsdorf, Heinrich; Chen, Zhenlong; Castellon, Maricela; Popova, Isolda A; Kalinin, Sergey; Mendonca, Emma; Petukhov, Pavel A
    BACKGROUND: Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. METHODOLOGY/PRINCIPAL FINDINGS: HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. Conclusions/Significance The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).
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    Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion
    (Public Library of Science, 2013) de Voux, Alex; Chan, Mei-Chi; Folefoc, Asongna T; Madziva, Michael T; Flanagan, Colleen A
    The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion.