Evidence for the existence of a non-catalytic modifier site of peptide hydrolysis by the 20 S proteasome

The 20 S proteasome is an endoprotease complex that preferentially cleaves peptides C-terminal of hydrophobic, basic, and acidic residues. Recently, we showed that these specific activities, classified as chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing (PGPH) activity, are differently affected by Ritonavir, an inhibitor of human immunodeficiency virus-1 protease. Ritonavir competitively inhibited the chymotrypsin-like activity, whereas the trypsin-like activity was enhanced. Here we demonstrate that the Ritonavir-mediated up-regulation of the trypsin-like activity is not affected by specific active site inhibitors of the chymo-trypsin-like and PGPH activity. Moreover, we show that the mutual regulation of chymotrypsin-like and PGPH activities by their substrates as described previously by a "cyclical bite-chew" model is not affected by selective inhibitors of the respective active sites. These data challenge the bite-chew model and suggest that effectors of proteasome activity can act by binding to non-catalytic sites. Accordingly, we propose a kinetic "two-site modifier" model that assumes that the substrate (or effector) may bind to an active site as well as to a second non-catalytic modifier site. This model appears to be valid as it describes the complex kinetic effects of Ritonavir very well. Since Ritonavir partially inhibits major histocompatibility complex class I restricted antigen presentation, the postulated modifier site may be required to coordinate the active centers of the proteasome for the production of class I peptide ligands.     

Involvement of tryptase-related cellular protease(s) in human immunodeficiency virus type 1 infection

Trypstatin, a new cellular Kunitz-type protease inhibitor purified from rat mast cells, inhibited syncytium formation in human immunodeficiency virus type 1 (HIV-1)-infected CCRF-CEM and uninfected Molt-4 clone 8 at a concentration of 1 microM. Anti-rat tongue mast cell tryptase antibodies reacted with Molt-4 clone 8 cells, as determined by Western blot and by immunofluorescence. In addition, the antibody inhibited syncytium formation. These findings along with homologous sequences with trypstatin and a neutralizing epitope of gp120 of HIV-1 suggest that a tryptase-like cellular enzyme(s) is involved in HIV-1 infection.     

The effect of human immunodeficiency virus-1 protease inhibitors on the toxicity of a variety of cells

Protease inhibitors in combination with other antiretroviral drugs have been shown to be efficacious in treating human immunodeficiency virus-1 (HIV-1) infection. The side effects of such a treatment usually involve perturbations of fat metabolism and insulin responsiveness. This has led to a number of studies on the adverse effects of these drugs in vitro. The concentrations of various protease inhibitors used in many of these studies were >20 microM. Although some investigators did address the toxicity of protease inhibitors, no overall effort was made to examine this effect during differentiation of fat or muscle. In this study, we assessed the toxicity of HIV-1 protease inhibitors over a range of concentrations (i.e., 0 to 100 microM) in nondifferentiating (e.g., human fibroblasts, 3T3-L1 preadipocytes, and L6 myoblasts) and differentiated cells (e.g., L6 myotubes). The most toxic protease inhibitor in all cell types was Saquinavir (sqv), whereas the least toxic protease inhibitor was Indinavir (idv). Ritonavir (rtv) and Amprenavir (apv) were more toxic than idv but not quite as toxic as sqv. In 3T3-L1 preadipocytes, treatment with sqv, rtv, and apv resulted in toxicity, whereas idv was not toxic even at the highest concentration used. Indinavir was not toxic to L6 myoblasts or L6 myotubes; however, sqv, rtv, and apv caused toxicity in L6 myoblasts. Saquinavir decreased L6 myotube viability in a dose-dependent manner. Human immunodeficiency virus-1 protease inhibitors were shown to be toxic in a variety of cell types. These effects on human fibroblasts and muscle cells have not been reported previously.     

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.     

An ultrasensitive human immunodeficiency virus type 1 protease radioimmuno rate assay with a potential for monitoring blood levels of protease inhibitors in acquired immunodeficiency disease syndrome patients.

The angiotensin I-based peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser yields angiotensin I (Ang I) and Leu-Glu-Glu-Ser upon hydrolysis by the human immunodeficiency virus type 1 (HIV-1) protease, but not by human renin. N-terminal sequencing of the reaction products showed that the HIV-1 protease cleaved exclusively at the Leu-Leu bond. The rate of Ang I formation can be measured by a radioimmunoassay, since the parent peptide has minimal cross reactivity in this assay. The rate of enzymatic hydrolysis is maximal at pH 4.5-5.0 and at an ionic strength of 1 M. At 37 degrees C, 0.1 M Na acetate buffer, pH 5.0, 1 M NaCl, 10% glycerol, 5% ethylene glycol, 1 mg/ml bovine serum albumin, and 3 mM EDTA, the reaction obeys Michaelis-Menten type kinetics with Km = 17.2 +/- 3.5 microM and kcat = 2.30 +/- 0.33 min-1. The activity assay readily quantitates as little as 0.25 nM of HIV-1 protease. The production of Ang I by the HIV-1 protease is inhibited in the presence of a HIV-1 protease inhibitor. The newly discovered substrate is relatively insensitive to human or monkey serum. Therefore, the effect of sera from 20 patients with advanced acquired immunodeficiency disease syndrome (AIDS) on Ang I production in the above assay system was examined. Results of this study indicate that it may be possible to adapt the above Ang I-based system to determine blood levels of HIV-1 protease inhibitors in AIDS patients during clinical trials.     

Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome

Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the "acidic" and "neutral" chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting that either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) human platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.4; (b) neither the chymotrypsin-like activity of the proteasome, nor proteasome antigens are detected in the cathepsin A preparation; (c) purified proteasome does not exhibit Cbz-Phe-Ala-hydrolyzing activity; (d) Z-lle-Glu-(Ot-Bu)Ala-leucinal (PSI), a compound that selectively inhibits the chymotrypsin-like activity of the proteasome at a concentration of 10 microM has no inhibitory effect on the carboxypeptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, is completely inhibited by micromolar concentrations of lactacystin/beta-lactone. It is therefore concluded that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.     

HIV protease inhibitor induces fatty acid and sterol biosynthesis in liver and adipose tissues due to the accumulation of activated sterol regulatory element-binding proteins in the nucleus

The mechanism by which human immunodeficiency virus (HIV) protease inhibitor therapy adversely induces lipodystrophy and hyperlipidemia has not been defined. This study explored the mechanism associated with the adverse effects of the prototype protease inhibitor ritonavir in mice. Ritonavir treatment increased plasma triglyceride and cholesterol levels through increased fatty acid and cholesterol biosynthesis in adipose and liver. Ritonavir treatment also resulted in hepatic steatosis and hepatomegaly. These abnormalities, which were especially pronounced after feeding a Western type high fat diet, were due to ritonavir-induced accumulation of the activated forms of sterol regulatory binding protein (SREBP)-1 and -2 in the nucleus of liver and adipose, resulting in elevated expression of lipid metabolism genes. Interestingly, protease inhibitor treatment did not alter SREBP mRNA levels in these tissues. Thus, the adverse lipid abnormalities associated with protease inhibitor therapy are caused by the constitutive induction of lipid biosynthesis in liver and adipose tissues due to the accumulation of activated SREBP in the nucleus.     

The selective proteasome inhibitors lactacystin and epoxomicin can be used to either up- or down-regulate antigen presentation at nontoxic doses

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.     

Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.     

Human immunodeficiency virus protease. Bacterial expression and characterization of the purified aspartic protease

The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 128-135. The enzyme expressed as a nonfusion protein exhibits proteolytic activity with a pH optimum of 5.5 and is inhibited by the aspartic protease inhibitor pepstatin with a Ki of 1.1 microM. Replacement of the conserved residue Asp-25 with an Asn residue eliminates proteolytic activity. Analysis of the minimal peptide substrate size indicates that 7 amino acids are required for efficient peptide cleavage. Size exclusion chromatography is consistent with a dimeric enzyme and circular dichroism spectra of the purified enzyme are consistent with a proposed structure of the protease (Pearl, L.H., and Taylor, W.R. (1987) Nature 329, 351-354). These data support the classification of the human immunodeficiency virus protease as an aspartic protease, likely to be structurally homologous with the well characterized family that includes pepsin and renin.     

A structural basis for the acute effects of HIV protease inhibitors on GLUT4 intrinsic activity

Human immunodeficiency virus (HIV) protease inhibitors (PIs) act as reversible noncompetitive inhibitors of GLUT4 with binding affinities in the low micromolar range and are known to contribute to alterations in glucose homeostasis during treatment of HIV infection. As aspartyl protease inhibitors, these compounds all possess a core peptidomimetic structure together with flanking hydrophobic moieties. To determine the molecular basis for GLUT4 inhibition, a family of related oligopeptides containing structural elements found in PIs was screened for their ability to inhibit 2-deoxyglucose transport in primary rat adipocytes. The peptide oxybenzylcarbonyl-His-Phe-Phe-O-ethyl ester (zHFFe) was identified as a potent inhibitor of zero-trans glucose flux with a K(i) of 26 mum. Similar to PIs, transport inhibition by this peptide was acute, noncompetitive, and reversible. Within a Xenopus oocyte expression system, zHFFe acutely and reversibly inhibited GLUT4-mediated glucose uptake, whereas GLUT1 activity was unaffected at concentrations as high as 1 mm. The related photoactivatable peptide zHFF-p-benzoylphenylalanine-[(125)I]Tyr-O-ethyl ester selectively labeled GLUT4 in rat adipocytes and indinavir effectively protected against photolabeling. Furthermore, GLUT4 bound to a peptide affinity column containing the zHFF sequence and was eluted by indinavir. These data establish a structural basis for PI effects on GLUT4 activity and support the direct binding of PIs to the transport protein as the mechanism for acute inhibition of insulin-stimulated glucose uptake.     

Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.     

Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Conditional human immunodeficiency virus type 1 protease mutants show no role for the viral protease early in virus replication.

The human immunodeficiency virus type 1 protease plays a critical role in the proteolytic processing of precursor polyproteins during virion maturation. Contradictory evidence has been obtained for a possible role for the protease early after infection, i.e., during DNA synthesis and/or integration. We have reexamined this question by using conditional mutants of the protease. In one set of experiments, protease mutants that confer a temperature-sensitive phenotype for processing were used to assess the need for protease activity early after infection. No significant difference from results with wild-type virus was seen when infections were carried out at either 35 or 40 degrees C. In a separate set of experiments, infections were carried out in the presence of a protease inhibitor. In this case, both wild-type virus and a drug-resistant variant were used, the latter as a control to ensure a specific effect of the inhibitor. Infection with either virus was not inhibited at drug concentrations that were up to 10-fold higher than those needed to inhibit intracellular processing by the viral protease. The results obtained by both of these experimental protocols provide evidence that the human immunodeficiency virus type 1 protease does not play a role early after infection.     

2'Deoxycoformycin and deoxyadenosine affect IL 2 production and IL 2 receptor expression of human T cells

Congenital deficiency of the enzyme adenosine deaminase (ADA) leads to severe combined immunodeficiency. 2'Deoxycoformycin (dCF), a tightly binding inhibitor of ADA, can induce the metabolic state of ADA deficiency. In vivo, the drug causes specific impairment of lymphocyte function and shows strong immunosuppressive properties. However, to decide whether inhibition of the enzyme ADA offers an attractive approach for immunosuppressive therapy, more information is needed about the immunologic mechanisms affected. In human T cells, we investigated the effect of dCF and deoxyadenosine (AdR) on cell activation, interleukin 2 (IL 2) production, and IL 2 receptor induction after allogeneic and lectin-induced stimulation. After allogeneic stimulation, dCF and AdR affected several events in T cellular immune response. Early events in T cell activation showed to be most sensitive to the drugs. Primary MLC was completely inhibited by concentrations as low as 1 microM dCF and 1 microM AdR. The addition of human recombinant IL 2 (rIL 2) could not abrogate the inhibitory effect of the drugs. Apart from activation of T cells, the drugs interfered with proliferation of activated T cells. Two events in activated T cells were affected: IL 2 production and IL 2 receptor expression. In secondary MLC, IL 2 production was markedly reduced in the presence of 9 microM dCF and 60 microM AdR. These concentrations appeared also to affect IL 2 receptor expression in 12-day primary MLC cells stimulated with rIL 2. Lectin stimulation was also affected by the drugs. In phytohemagglutinin (PHA)-stimulated cultures, 9 microM dCF and 60 microM AdR resulted in inhibition of proliferation and IL 2 receptor expression, whereas IL 2 production was normal. It is concluded that dCF and AdR interfere with several events in T cellular immune response such as cell activation, IL 2 production, and IL 2 receptor expression. According to these results, inhibition of the enzyme ADA seems an attractive approach to immunosuppressive therapy.     

Selective restriction of Nef-defective human immunodeficiency virus type 1 by a proteasome-dependent mechanism

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) infectivity by facilitating an early postentry step in the virus life cycle. We report here that the addition of MG132 or lactacystin, each a specific inhibitor of cellular proteasome activity, preferentially enhances cellular permissiveness to infection by Nef-defective versus wild-type HIV-1. Pseudotyping by the glycoprotein of vesicular stomatitis virus rendered Nef-defective HIV-1 particles minimally responsive to the enhancing effects of proteasome inhibitors. These results suggest that Nef enhances the infectivity of HIV-1 particles by reducing their susceptibility to proteasomal degradation in target cells.     

Inhibitors of human immunodeficiency virus-1 protease.

We have shown that the interaction of pepstatin A with human immunodeficiency virus-1 protease (HIV-1 protease) can be characterized by a high-affinity mode (Ki = 478 +/- 27 nM), resulting in pure competitive inhibition of the hydrolytic activity of HIV-1 protease toward the fluorogenic substrate. Binding of pepstatin in this mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 10 microM pepstatin. Haloperidol, in contrast, interacts with HIV-1 protease with weaker affinity (Ki = 19 +/- 1 microM) in a mode which results in pure noncompetitive inhibition of the hydrolytic activity of HIV-1 protease. Binding of haloperidol in this mode induces a red shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 200 microM haloperidol. Addition of both pepstatin and haloperidol at concentrations in the range of their Ki values results in additive inhibition of the hydrolytic activity of HIV-1 protease, as well as an additive effect on the tryptophan fluorescence of protease. However, at saturating concentrations of pepstatin and haloperidol, the effect of haloperidol was predominant, as measured by the changes in the intrinsic fluorescence of HIV-1 protease.