Abstract
Accelerated apoptosis is one mechanism proposed for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type 1 (HIV-1) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of gp160 in Jurkat T-cells results in increased sensitivity to FAS- and ceramide-mediated apoptosis. The pro-apoptotic effect of gp160 expression is blocked by two calmodulin antagonists, tamoxifen and trifluoperazine. This enhanced apoptosis in response to FAS antibody or C(2)-ceramide is associated with activation of caspase 3, a critical mediator of apoptosis. A point mutation in the C-terminal calmodulin-binding domain of gp160 (alanine 835 to tryptophan, A835W) eliminates gp160-dependent enhanced FAS-mediated apoptosis in transiently transfected cells, as well as in vitro calmodulin binding to a peptide corresponding to the C-terminal calmodulin-binding domain of gp160. Stable Tet-off Jurkat cell lines were developed that inducibly express wild type gp160 or gp160A835W. Increasing expression of wild type gp160, but not gp160A835W, correlates with increased calmodulin levels, increased apoptosis, and caspase 3 activation in response to anti-FAS treatment. The data indicate that gp160-enhanced apoptosis is dependent upon calmodulin up-regulation, involves the activation of caspase 3, and requires calmodulin binding to the C-terminal binding domain of gp160.
Highlights
Hallmark of disease progression, understanding the relationship between these phenomena is critical in understanding human immunodeficiency virus type 1 (HIV-1) pathogenesis
The importance of apoptosis in AIDS is controversial there is abundant in vitro evidence supporting a role for apoptosis in the pathogenesis of HIV-1 [12], and mechanisms regulating this apoptosis in vivo are not clear, current evidence implicates the FAS pathway
Effect of Calmodulin Antagonists on gp160-enhanced Ceramide- and FAS-mediated Apoptosis—Jurkat cells were transfected with gp160 as described above and pretreated with 10 M tamoxifen (TMX) or trifluoperazine (TFP) for 30 min prior to addition of FAS antibody (Fig. 2)
Summary
1233–1240, 2000 Printed in U.S.A. Requirement of Calmodulin Binding by HIV-1 gp160 for Enhanced FAS-mediated Apoptosis*. The pro-apoptotic effect of gp160 expression is blocked by two calmodulin antagonists, tamoxifen and trifluoperazine This enhanced apoptosis in response to FAS antibody or C2-ceramide is associated with activation of caspase 3, a critical mediator of apoptosis. Upon binding FAS ligand, FAS recruits signaling molecules to the death-inducing signaling complex (6 – 8) Induction of this pathway leads to activation of caspase 3, subsequent activation of caspase 6, and eventually activation of DNA fragmentation factor and a Ca2ϩ/Mg2ϩ-dependent endonuclease responsible for cleavage of DNA resulting in the typical laddering pattern seen in most forms of apoptosis (9 –11). We demonstrate here that gp160 expression enhances FASmediated apoptosis in Jurkat cells by increasing calmodulin expression and accelerating caspase 3 activation and that these effects that require calmodulin binding to gp160 are blocked by a single point mutation in the C-terminal calmodulin-binding domain
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