The HIV Protease Inhibitors Nelfinavir and Saquinavir, but Not a Variety Of

Antiviral Therapy 10:215–223 The HIV protease inhibitors nelfinavir and saquinavir, but not a variety of HIV reverse transcriptase inhibitors, adversely affect human proteasome function Marco Piccinini1, Maria T Rinaudo1*, Annalisa Anselmino1, Barbara Buccinnà1, Cristina Ramondetti1, Antonio Dematteis1, Emanuela Ricotti 2, Lucia Palmisano3, Michael Mostert2 and Pier-Angelo Tovo2 1Department of Medicine and Experimental Oncology and 2Department of Paediatric Sciences, University of Turin, Turin, Italy 3Istituto Superiore di Sanità, Rome, Italy *Corresponding author: Tel: +39 011 670 5303 Fax: +39 011 670 5311; E-mail: [email protected] Background: In HIV-infected patients some clinical and drugs at different concentrations. Intracellular protea- immunological benefits of antiretroviral therapy, which some proteolytic activity was evaluated by searching for frequently include a combination of HIV protease inhibitors ubiquitin-tagged proteins in HL60 cells incubated with (PIs) and reverse transcriptase inhibitors (RTIs), cannot be and without the drugs. solely explained by the drugs’ action on viral enzymes. Results: At therapeutic dosages, nelfinavir and saquinavir Proteasomes constitute the central protease of the ubiq- inhibited proteasome peptidase activity and caused uitin ATP-dependent pathway involved in many cellular intracellular accumulation of polyubiquitinated proteins, processes, as well as in HIV maturation and aggressiveness. a hallmark of proteasome proteolytic inhibition in vivo; Objective: To explore whether the PIs nelfinavir and the RTIs failed to evoke either effect. saquinavir and the RTIs abacavir, nevirapine, delavirdine, Conclusion: Proteasomes are targeted by the two PIs but stavudine and didanosine affect proteasome function in not the RTIs. Therefore, in HIV-infected patients the vitro and in vivo. beneficial effect of a therapy including one of the two Methods: Peptidase activity of purified human 26S and PIs should partly rely on inhibition of host proteasome 20S proteasomes was assayed with and without the function. Introduction HIV-related morbidity and mortality have dramatically degradation of foreign or damaged proteins, as well as decreased since the introduction of highly active anti- of short half-life proteins playing a key role in a retroviral therapy (HAART), commonly based on the variety of primary cell functions. These include cell combined administration of specific HIV protease cycle progression, processing of transcriptional factors inhibitors (PIs) and reverse transcriptase inhibitors such as the nuclear factor κB (NF-κB) and its inhibitor (RTIs). HAART usually leads to suppression of viral IκB, production of pro-inflammatory cytokines, replication, reversal of HIV-driven immune alterations expression of surface protein receptors, apoptosis, and slower disease progression. Failure of antiretro- T-cell reactivity and degradation of intracellular antiviral therapy is ascribed to the development of resistant gens and their presentation onto MHC class I viral mutants. However, about 40% of patients treated molecules [15–18]. In addition, this proteolytic for more than 1 year exhibit a steady increase in CD4+ pathway regulates the maturation and release of infec- cell counts and do well clinically, despite a plasma viral tious HIV particles [19]. RNA level rebound [1,2]. The reason for this discrep- Overall, the ubiquitin ATP-dependent pathway ancy remains unclear. involves two discrete steps: covalent, ATP-dependent An increasing body of evidence highlights direct attachment of oligomers of ubiquitin molecules to the immunomodulatory effects of antiretroviral agents protein targeted for proteolysis and degradation of [3–11], and a few of these agents have been shown to the marked protein to small peptides by the 26S affect proteasome function [8,12–14]. Proteasomes proteasome [20]. constitute the central protease of the ubiquitin ATP- The 26S proteasome is a large multi-subunit particle dependent proteolytic pathway involved in the comprised of a catalytic core, the 20S proteasome, © 2005 International Medical Press 1359-6535 215 M Piccinini et al. which is placed between two caps formed by the 19S UK); antibodies to ubiquitin and actin were from Santa regulatory component. The 20S proteasome, which Cruz Biotechnology (Heidelberg, Germany) and also exists as a free particle, has a hollow cylindrical Sigma-Aldrich (Milan, Italy), respectively. The other shape; in mammals it consists of 14 pairs of α and β products were from Sigma-Aldrich and Roche- subunits staked in four rings, the external ones Boehringer (Monza, Italy). Red blood cell units and composed of α subunits and the internal ones of β fresh blood samples were from Associazione Volontari subunits. Only three subunits (β1, β2 and β5) are Italiani del Sangue (AVIS) (Turin, Italy). catalytically active and are responsible for splitting peptide bonds after acidic, basic and hydrophobic Proteasome purification amino acid residues. These activities are referred to as The 26S and 20S proteasomes were purified from peptidyl glutamyl peptide hydrolytic (PGPH) activity, human red blood cell units as previously reported trypsin-like activity and chymotrypsin-like activity, [13,24] and stored at –20ºC. respectively, and are considered to be the major peptidase activities of the proteasome [21,22]. Substrates Proteasome peptidase activity assay enter the cylinder through the gated channel defined by Chymotrypsin-like, trypsin-like and PGPH activities of the α subunits. In the free 20S proteasome, the channel purified 26S and 20S proteasomes were evaluated by a is too narrow to be crossed by folded proteins, a spectrofluorometric assay in which the fluorogenic feature that explains why this proteasome cannot peptides Suc-LLVY-AMC, Z-LLR-AMC and Z-LLE- degrade native or ubiquitinated proteins [21]. βNA were used as substrates [24,25]. Peptides However, when the 20S proteasome binds with the 19S (130 µM) in dimethylsulphoxide (DMSO) were incu- regulator, its subunits undergo structural modifications bated in a final volume of 100 µl with: (i) 50 µM ATP, µ inducing in the newly formed particle – the 26S protea- 5 mM MgCl2 and 0.125 or 0.250 g of 26S proteasome – widening of the channel, acquisition of some for chymotrypsin-like and trypsin-like activities proteolytic activity on ubiquitinated proteins, enhance- or PGPH activity, respectively; the reaction lasted ment of peptidase activity and ATP dependence of 30 min for the former two activities and 120 min for µ catalytic properties [21–23]. the latter activity; (ii) 5 mM MgCl2 and 2 or 4 g of Here, we explore whether the PIs nelfinavir and 20S proteasome for chymotrypsin-like and trypsin-like saquinavir, and the RTIs abacavir, nevirapine, delavir- activities or PGPH activity, respectively; the reaction dine, stavudine and didanosine, at clinically relevant lasted 30 min for the former two activities and dosages, affect proteasome function in vitro and in 120 min for the latter activity. vivo. In this respect, peptidase activity of purified human 20S and 26S proteasomes, as well as the levels Chymotrypsin-like activity of the 20S proteasome in of ubiquitin-tagged proteins in cultured cell lines, were lysates from human red blood cells evaluated in the presence or absence of the drugs. In order to assess proteasome peptidase activity in a Intracellular accumulation of ubiquitin-tagged proteins context close to the intracellular milieu, red blood cells is considered to be a hallmark of proteasome inhibition from healthy donors were pelleted at 600 ×g at 15ºC, in vivo [8]. washed twice with PBS, and then lysed by hypotonic shock at 7ºC in 20 mM Tris-HCl pH 7.3, supple- Materials and methods mented with 10 mM NaCl, 1 mM dithiothreitol (DTT) and 1 mM EDTA. After 45 min, lysates were added Reagents with 20% glycerol and centrifuged at 4ºC for 60 min The fluorogenic peptides, Suc-LLVY-AMC, MeOSuc- at 30 000 ×g. The clear supernatant was aliquoted and GLF-AMC, Z-LLR-AMC and Z-LLE-βNA (in which stored at –20ºC. Thawed lysate aliquots (6 µg of single-letter amino acid codes are used and AMC is 7- protein) were incubated at 37ºC for 3 h in a final amido-4-methylcoumarin, βNA is β-naphthylamide, Suc volume of 100 µl with 50 mM HEPES pH 7.8, 65 µM is succinyl, MeOSuc is 3-methoxysuccinyl and Z is Suc-LLVY-AMC and 5 mM MgCl2, with and without benzyloxycarbonyl) were purchased from Bachem 50 µM ATP. The activity tested in these conditions was Feinchemikalien AG (Bubendorf, Switzerland). almost completely aspecific because it was 99% Chromatographic supports, ECL Plus and Hyperfilm unaffected by 10 µM lactacystin or epoxomicin, which autoradiography films were from Amersham are irreversible and selective proteasome peptidase and Pharmacia Biotech (Milan, Italy), and Centricon YM- proteolytic activity inhibitors [27]. To potentiate 30 (30 000 MW cut-off) from Amicon-Millipore Corp proteasome-specific activity, the reaction mixture (Milan, Italy). Antibodies to human 20S proteasome α4 described above was supplemented with 0.05% and 19S complex Rpn8 (S12) subunits, as well as lacta- sodium dodecyl sulphate (SDS), which strongly cystin and epoxomicin, were from Affiniti (Exeter, enhances 20S proteasome chymotrypsin-like activity 216 © 2005 International Medical Press Antiretroviral agents and proteasome function [21]. The activity was then evaluated in the presence of immunoblotting with antibody to ubiquitin. Detection either Suc-LLVY-AMC or MeOSuc-GLF-AMC [28], of immunodecorated protein

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