How an Inhibitor of the HIV-I Protease Modulates Proteasome Activity

Abstract

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 M). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor 125 I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 M) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 M) protein degradation was affected, and the cell cycle was arrested in the G 1 /S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response. The human immunodeficiency virus, type I (HIV-I)1 encodes an aspartic endoprotease that is required for cleavage of the viral gag-pol polyprotein. Inhibition of the HIV-I protease leads to the release of noninfectious virus particles and has thus been the aim of drug development in AIDS therapy (1). Several cleavages performed by the HIV-I protease lie between the amino acids phenylalanine (or tyrosine) and proline. Endoproteolytic cleavages N-terminal of proline residues have not been frequently observed in mammalian proteases, which was the premise for the design of transition state mimetics of the PhePro bond as potential inhibitors of the HIV-I protease (2). Interestingly, an exception to this premise arose from the recent analysis of the cleavage specificity of the 20 S proteasome (3) that quite frequently cleaved polypeptide substrates Nterminal of proline residues (4 -8).A number of HIV-I protease inhibitors have been successfully used in clinical therapy of HIV-I infection (9). Patients treated with highly active antiretroviral therapy consisting of HIV-I protease inhibitors and nucleoside analogues showed a marked decrease in viremia with a simultaneous increase in CD4 ϩ helper T cells after the initiation of treatment. Remarkably, even in patients that (because of resistance of the virus) remained viremic upon highly active antiretroviral therapy, the number of CD4 ϩ helper T cells in peripheral blood increased suggesting that there was a direct effect of highly active antiretroviral thera...

The Selective Proteasome Inhibitors Lactacystin and Epoxomicin Can Be Used to Either Up- or Down-Regulate Antigen Presentation at Nontoxic Doses

“…This immunomodulatory effect was neither due to the inhibition of viral replication nor to a direct effect on CTL activation, but was attributed to a reduction in the presentation of immunodominant LCMV epitopes. Interestingly, Ritonavir was found to inhibit the chymotrypsin-like activity of the 20S proteasome in vitro, while the trypsin-like activity was enhanced (41,42). We reasoned that the modulation of proteasome activity would account for the in vivo reduction of Ag presentation, but we could not exclude that this effect was, at least in part, due to the inhibition of other proteases in APC.…”

The Ubiquitin-like Protein FAT10 Forms Covalent Conjugates and Induces Apoptosis

“…This investigation was warranted, particularly because Liu et al (10) had reported an association of FAT10 with MAD2, which is a protein involved in mitotic arrest. We stained nuclei of viable TB1N cells with propidium iodide 1 or 2 days after FAT10 induction, and the DNA content was assessed by flow cytometry as described previously (18). In this analysis, no change in the distribution of DNA content in TB1N cells could be observed when cells grown in the absence or presence of tet were compared (data not shown).…”

“…For cell cycle analysis, nuclei were prepared from TB1N cells 0, 24, and 48 h after the induction of FAT10 expression, and nuclei were stained with propidium iodide exactly as described previously (18). Fluorescence of cells or nuclei was analyzed using a FACScan flow cytometer and LYSIS II software (Becton Dickinson).…”

Role of endoplasmic reticulum stress in drug‐induced toxicity

“…Proteasome inhibition has been shown for atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, although the potency of this inhibitory effect seemed variable between these drugs (Schmidtke et al. 1999; Pajonk et al. 2002; Piccinini et al.…”