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PubMed2004

The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site.

A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.

Amino Acid Sequence↗
PubMed1999

Impact of protease inhibitors on the outcome of human immunodeficiency virus-infected patients with chronic diarrhea.

OBJECTIVE: The effect of protease inhibitors on the outcome of chronic HIV-related diarrhea is unknown. The aim of this study was to compare the response to treatment of chronic HIV-related diarrhea, recurrence of diarrhea, and survival in a large cohort of individuals taking protease inhibitors to the outcome in similar patients not receiving protease inhibitors. METHODS: We reviewed the medical records of all patients referred between October 1993 and October 1996 at Bellevue Hospital for endoscopic evaluation of chronic HIV-related diarrhea after negative stool examination. Only patients presenting after December 1995 received protease inhibitor therapy. Follow-up data were obtained from chart review and direct telephone contact. The success of antidiarrheal therapy was compared between protease inhibitor and nonprotease inhibitor groups for patients receiving pathogen-specific therapy and for those with no pathogens found on endoscopy. RESULTS: Two hundred eighty-two of 307 patients evaluated for chronic diarrhea were followed for a mean of 69.9+/-34.1 weeks. Patients receiving protease inhibitors had a significantly higher rate of successful response to antidiarrheal therapy (62.0% vs 33.5%, p < 0.001). Protease inhibitors were associated with a significant decrease in stool frequency (4.8+/-4.5 vs 3.4+/-4.6 bowel movements per day, p = 0.01), an increase in weight (2.4+/-5.9 vs -1.6+/-6.2 kg, p < 0.001), a decrease in recurrence of diarrhea (34.8% vs 15.3%, p = 0.02), and a longer mean survival (148 vs 118 weeks, p = 0.002). CONCLUSIONS: Protease inhibitors significantly improve the outcome of antidiarrheal therapy and survival in patients with chronic HIV-associated diarrhea.

AIDS-Related Opportunistic Infections↗
PubMed1986

Proteases and their inhibitors in chronic inflammatory periodontal disease.

This article reviews the current knowledge of the sources, function and interactions of proteolytic enzymes and their inhibitors in chronic inflammatory periodontal disease. Proteolytic tissue degradation is a typical phenomenon in chronic inflammatory periodontal disease. The proteolytic enzymes can be both host- and bacteria-derived. The proteases of the inflammatory cells are aimed for digestion of bacteria, enhanced locomotion through connective tissue, demarcation of the site of infection and tissue remodeling. Uncontrolled release of proteases in inflammation causes self-digestion and tissue destruction. The potential of the bacterial proteases in degradation of connective tissue is not yet known. Biochemical and immunologic mediators of inflammation are released by proteolytic reactions. Immunoglobulin-cleaving proteases present a specific mechanism in perturbation of host defenses. The 2 main protease inhibitors in serum, alpha-1-antitrypsin and alpha-2-macroglobulin, are also present in the gingival tissue fluid guarding the function of proteases. It has been suggested, although not confirmed, that deficiency in serum protease inhibiting capacity could be correlated with susceptibility to periodontal disease. Mucous secretions contain local low molecular weight protease inhibitors, but their possible rôle in saliva is not known. Bacteria-derived, antiproteolytic short peptides may prove to be useful in pharmacological control of tissue destruction at inflammatory sites.

Adult↗
PubMed1998

Dual inhibition of human rhinovirus 2A and 3C proteases by homophthalimides.

The 2A and 3C proteases encoded by human rhinoviruses (HRVs) are attractive targets for antiviral drug development due to their important roles in viral replication. Homophthalimides were originally identified as inhibitors of rhinovirus 3C protease through our screening effort. Previous studies have indicated that the antiviral activity of certain homophthalimides exceeded their in vitro inhibitory activity against the viral 3C protease, suggesting that an additional mechanism might be involved. Reported here is the identification of homophthalimides as potent inhibitors for another rhinovirus protease, designated 2A. Several homophthalimides exhibit time-dependent inhibition of the 2A protease in the low-micromolar range, and enzyme-inhibitor complexes were identified by mass spectrometry. Compound LY343814, one of the most potent inhibitors against HRV14 2A protease, had an antiviral 50% inhibitory concentration of 4.2 microM in the cell-based assay. Our data reveal that homophthalimides are not only 3C but also 2A protease inhibitors in vitro, implying that the antiviral activity associated with these compounds might result from inactivation of both 2A and 3C proteases in vivo. Since the processing of the viral polyprotein is hierarchical, dual inhibition of the two enzymes may result in cooperative inhibition of viral replication. On the basis of the current understanding of their enzyme inhibitory mechanism, homophthalimides, as a group of novel nonpeptidic antirhinovirus agents, merit further structure-action relationship studies.

Colorimetry↗
PubMed1998

Antimalarial synergy of cysteine and aspartic protease inhibitors.

It has been proposed that the Plasmodium falciparum cysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparum parasites, cysteine and aspartic protease inhibitors exhibited synergistic effects in blocking parasite metabolism and development. The inhibitors also demonstrated apparent synergistic inhibition of plasmodial hemoglobin degradation both in culture and in a murine malaria model. When evaluated for the treatment of murine malaria, a combination of cysteine and aspartic protease inhibitors was much more effective than higher concentrations of either compound used alone. These results support a model whereby plasmodial cysteine and aspartic proteases participate in the degradation of hemoglobin, and they suggest that combination antimalarial therapy with inhibitors of the two classes of proteases is worthy of further study.

Animals↗
PubMed2000

Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28.

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M(w)-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N'-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30 degrees C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.

Amylases↗
PubMed2000

Immunoglobulins in nasal secretions of healthy humans: structural integrity of secretory immunoglobulin A1 (IgA1) and occurrence of neutralizing antibodies to IgA1 proteases of nasal bacteria.

Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11 healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and, subsequently, concentrations in undiluted secretions could thereby be calculated. IgA, mainly in the secretory form, was found by enzyme-linked immunosorbent assay to be the dominant isotype in all subjects, and the vast majority of IgA (median, 91%) was of the A1 subclass, corroborating results of previous analyses at the level of immunoglobulin-producing cells. Levels of serum-type immunoglobulins were low, except for four subjects in whom levels of IgG corresponded to 20 to 66% of total IgA. Cumulative levels of IgA, IgG, and IgM in undiluted secretions ranged from 260 to 2,494 (median, 777) microg ml(-1). IgA1 protease-producing bacteria (Haemophilus influenzae, Streptococcus pneumoniae, or Streptococcus mitis biovar 1) were isolated from the nasal cavities of seven subjects at 2.1 x 10(3) to 7.2 x 10(6) CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, alpha-chain fragments characteristic of IgA1 protease activity were not detected in secretions from any subject by immunoblotting. Neutralizing antibodies to IgA1 proteases of autologous isolates were detected in secretions from five of the seven subjects but not in those from two subjects harboring IgA1 protease-producing S. mitis biovar 1. alpha-chain fragments different from Fc(alpha) and Fd(alpha) were detected in some samples, possibly reflecting nonspecific proteolytic activity of microbial or host origin. These results add to previous evidence for a role of secretory immunity in the defense of the nasal mucosa but do not help identify conditions under which bacterial IgA1 proteases may interfere with this defense.

Adult↗
PubMed1984

Purification and characterization of four proteases from a clinical isolate of Serratia marcescens kums 3958.

Four distinct proteases were purified to homogeneity from culture filtrates of Serratia marcescens kums 3958, a fresh isolate from a patient with a severe corneal ulcer. Purification was achieved by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex gel filtration chromatography. The proteases were differentiated from each other by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate and by immunodiffusion in agarose gels. The molecular weights of these purified proteases were estimated to be 56 X 10(3), 60 X 10(3), and 73 X 10(3) (hereafter designated 56K, 60K, and 73K proteases, respectively). The 73K protease was separated into 73Ka and 73Kb upon isoelectricfocusing. The isoelectric points of the 56K (major) and 60K, 73Ka, and 73Kb proteases (minors) were approximately 5.3, 4.4, 5.8, and 7.3, respectively. Both 56K and 60K enzymes were completely inactivated by EDTA at pH 5.0 and were reactivated by zinc ion; thus, they are metalloenzymes, whereas 73K (73Ka and 73Kb) enzymes appear to be thiol proteases. Carbohydrate, cysteine, and cystine were not detected in the 56K and 60K proteases. Amino acid compositions, partial amino acid sequence, and enzymological and immunological properties revealed that these four enzymes are distinct from each other.

Chromatography, DEAE-Cellulose↗
PubMed1969

Inhibition of protease production of various bacteria by ammonium salts: its effect on toxin production and virulence.

Production of protease by many bacteria was found to be inhibited by ammonium salts, and the enzyme production was more sensitive to the salts than was growth of the organisms. Inhibition of protease production by some pathogenic bacteria may result in the recognition of an exotoxin which otherwise would have been digested by the protease. In the case of Pseudomonas aeruginosa, qualitatively different toxicities could be demonstrated in the culture fluids, depending on the presence or absence of protease in such a fluid. The toxicity of the culture in the presence of a high titer of protease may be due primarily to the protease, whereas the toxicity exhibited in the absence of protease could be due to proteinacious exotoxin. Producers of high titers of protease tended to be less virulent in vivo than producers of low titers of the enzyme, which exert their toxicities by a separate exotoxin.

Aeromonas↗
PubMed1996

Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.

Amino Acid Sequence↗
PubMed1999

Effects of human immunodeficiency virus type 1 resistance to protease inhibitors on reverse transcriptase processing, activity, and drug sensitivity.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors often display a reduced replicative capacity as a result of an impairment of protease function. Such fitness-impaired viruses display Gag precursor maturation defects. Here, we report that some protease inhibitor-resistant viruses also display abnormalities in the processing of reverse transcriptase (RT) by the protease. In three recombinant viruses carrying resistant protease sequences from patient plasma, we observed a marked decrease in the amount of mature RT subunits and of particle-associated RT activity compared to their parental pretherapy counterparts. We investigated the possibility that a decrease in the amount of particle-associated mature RT could affect the sensitivity of the corresponding virus to RT inhibitors. We observed a twofold increase of sensitivity to zidovudine (AZT) when a virus which carried AZT mutations was processed by a resistant protease. Interestingly, the presence of AZT-resistance mutations partially rescued the replication defect associated with the mutated protease. The interplay between resistance to protease inhibitors and to RT inhibitors described here may be relevant to the therapeutic control of HIV-1 infection.

Anti-HIV Agents↗
PubMed2000

Generation and characterization of a hepatitis C virus NS3 protease-dependent bovine viral diarrhea virus.

Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, P(H/B) (wild-type HCV NS3 protease) and P(H/B(S139A)) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the P(H/B) clone yielded viable viruses, whereas the mutant clone, P(H/B(S139A)), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV-Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV-Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.

Amino Acid Sequence↗
PubMed2002

Molecular and enzymatic characterization of the porcine endogenous retrovirus protease.

The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M(r) of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.

Amino Acid Sequence↗
PubMed2004

Regulation of poliovirus 3C protease by the 2C polypeptide.

Poliovirus-encoded nonstructural polypeptide 2C is a multifunctional protein that plays an important role in viral RNA replication. 2C interacts with both intracellular membranes and virus-specific RNAs and has ATPase and GTPase activities. Extensive computer analysis of the 2C sequence revealed that in addition to the known ATPase-, GTPase-, membrane-, and RNA-binding domains it also contains several "serpin" (serine protease inhibitor) motifs. We provide experimental evidence suggesting that 2C is indeed capable of regulating virus-encoded proteases. The purified 2C protein inhibits 3C(pro)-catalyzed cleavage of cellular transcription factors at Q-G sites in vitro. It also inhibits cleavage of a viral precursor by the other viral protease, 2A(pro). However, at least three cellular proteases appear not to be inhibited by 2C in vitro. The 2C-associated protease inhibitory activity can be depleted by anti-2C antibody. A physical interaction between 2C and His-tagged 3C(pro) can be demonstrated in vitro by coimmunoprecipitation of 2C with anti-His antibody. Deletion analysis suggests that the 2C central and C-terminal domains that include several serpin motifs are important for 3C(pro)-inhibitory activity. To examine the 2C protease inhibitory activity in vivo, stable HeLa cell lines were made that express 2C in an inducible fashion. Infection of 2C-expressing cells with poliovirus led to incomplete (or inefficient) processing of viral precursor polypeptides compared to control cell lines containing the vector alone. These results suggest that 2C can negatively regulate the viral protease 3C(pro). The possible role of the 2C protease inhibitory activity in viral RNA replication is discussed.

3C Viral Proteases↗
PubMed2004

Genetic screen for monitoring severe acute respiratory syndrome coronavirus 3C-like protease.

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.

Amino Acid Sequence↗
PubMed1989

Characterization of the proteases in the crude mite extract.

Characterization of the proteases was performed in the crude mite extract fractionated by Sephacryl S-200 gel filtration. Three peaks of protease activities were detected in the fractions. From the results of substrate specificity and susceptibility to the inhibitors, PK.1 protease (about 60 kD) is suggested to be a trypsin-like protease of mites. From the results of susceptibility to various agents, PK.2 (about 30 kD) and PK.3 (about 20 kD) proteases may be cysteine proteases, e.g., papain and cathepsin B. PK.3 protease existed in the precipitate of 60% ammonium sulfate fractionation. The data in the present study suggest the possibility that Dermatophagoides farinae I allergen might be a cysteine protease probably derived from the gastrointestinal tract of the house dust mite.

Allergens↗
PubMed1994

Structure-assisted design of nonpeptide human immunodeficiency virus-1 protease inhibitors.

The discovery of therapeutic agents has, in the past, started primarily with random screening efforts. These screens, although effective, are time-consuming and expensive. Attempts are now being made to design more efficient methods of screening that take into account available information about the three-dimensional structure of a target receptor or enzyme. In the case of acquired immunodeficiency syndrome, the structure of a proteolytic enzyme, the human immunodeficiency virus (HIV) protease, an aspartyl protease that plays a crucial role in the viral life cycle, has been determined and extensively characterized. Using the protease structure and the computer program DOCK, the active site of the protease was mapped and its shape used to screen a subset of the Cambridge Structural Database. Among the molecules whose shape was complementary to the active site was the antipsychotic agent haloperidol. This molecule and several chemically modified derivatives were shown to bind competitively with micromolar affinity to the HIV protease but not to cellular aspartyl proteases. X-ray structures of the HIV protease complexed with haloperidol derivatives show the molecules binding in the predicted position at the active site. In an attempt to overcome the problems associated with low-affinity competitive inhibitors, reactive groups that enable the molecule to serve as a specific irreversible inhibitor of the HIV protease were introduced onto the haloperidol scaffold. These inhibitors demonstrated an ability to block viral polyprotein processing in a tissue culture model of HIV-1 infection, although their cytotoxicity is pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug Design↗
PubMed2003

HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages.

Protease inhibitors decrease the viral load in HIV patients, however the patients develop hypertriglyceridemia, hypercholesterolemia, and atherosclerosis. It has been assumed that protease inhibitor-dependent increases in atherosclerosis are secondary to the dyslipidemia. Incubation of THP-1 cells or human PBMCs with protease inhibitors caused upregulation of CD36 and the accumulation of cholesteryl esters. The use of CD36-blocking antibodies, a CD36 morpholino, and monocytes isolated from CD36 null mice demonstrated that protease inhibitor-induced increases in cholesteryl esters were dependent on CD36 upregulation. These data led to the hypothesis that protease inhibitors induce foam cell formation and consequently atherosclerosis by upregulating CD36 and cholesteryl ester accumulation independent of dyslipidemia. Studies with LDL receptor null mice demonstrated that low doses of protease inhibitors induce an increase in the level of CD36 and cholesteryl ester in peritoneal macrophages and the development of atherosclerosis without altering plasma lipids. Furthermore, the lack of CD36 protected the animals from protease inhibitor-induced atherosclerosis. Finally, ritonavir increased PPAR-gamma and CD36 mRNA levels in a PKC- and PPAR-gamma-dependent manner. We conclude that protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages.

Animals↗