ABSTRACT
We reported that several HIV protease inhibitors (HIV-PIs) interfere with the endoproteolytic processing of two farnesylated proteins, yeast a-factor and mammalian prelamin A. We proposed that these drugs interfere with prelamin A processing by blocking ZMPSTE24, an integral membrane zinc metalloproteinase known to play a critical role in its processing. However, because all of the drug inhibition studies were performed with cultured fibroblasts or crude membrane fractions rather than on purified enzyme preparations, no definitive conclusions could be drawn. Here, we purified Ste24p, the yeast ortholog of ZMPSTE24, and showed that its enzymatic activity was blocked by three HIV-PIs (lopinavir, ritonavir, and tipranavir). A newer HIV-PI, darunavir, had little effect on Ste24p activity. None of the HIV-PIs had dramatic effects on the enzymatic activity of purified Ste14p, the prenylprotein methyltransferase. These studies strongly support our hypothesis that HIV-PIs block prelamin A processing by directly affecting the enzymatic activity of ZMPSTE24, and in this way they may contribute to lipodystrophy in individuals undergoing HIV-PI treatment.
Subject(s)
HIV Protease Inhibitors/pharmacology , Membrane Proteins/antagonists & inhibitors , Metalloendopeptidases/antagonists & inhibitors , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Catalysis/drug effects , Humans , Lamin Type A , Lopinavir , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Metalloendopeptidases/genetics , Metalloendopeptidases/isolation & purification , Protein Methyltransferases/antagonists & inhibitors , Protein Methyltransferases/genetics , Protein Methyltransferases/isolation & purification , Pyridines/pharmacology , Pyrimidinones/pharmacology , Pyrones/pharmacology , Ritonavir/pharmacology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , SulfonamidesABSTRACT
HIV protease inhibitors (HIV-PIs) are key components of highly active antiretroviral therapy, but they have been associated with adverse side effects, including partial lipodystrophy and metabolic syndrome. We recently demonstrated that a commonly used HIV-PI, lopinavir, inhibits ZMPSTE24, thereby blocking lamin A biogenesis and leading to an accumulation of prelamin A. ZMPSTE24 deficiency in humans causes an accumulation of prelamin A and leads to lipodystrophy and other disease phenotypes. Thus, an accumulation of prelamin A in the setting of HIV-PIs represents a plausible mechanism for some drug side effects. Here we show, with metabolic labeling studies, that lopinavir leads to the accumulation of the farnesylated form of prelamin A. We also tested whether a new and chemically distinct HIV-PI, darunavir, inhibits ZMPSTE24. We found that darunavir does not inhibit the biochemical activity of ZMPSTE24, nor does it lead to an accumulation of farnesyl-prelamin A in cells. This property of darunavir is potentially attractive. However, all HIV-PIs, including darunavir, are generally administered with ritonavir, an HIV-PI that is used to block the metabolism of other HIV-PIs. Ritonavir, like lopinavir, inhibits ZMPSTE24 and leads to an accumulation of prelamin A.
Subject(s)
HIV Infections/diet therapy , HIV Protease Inhibitors/pharmacology , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Protein Processing, Post-Translational/drug effects , Sulfonamides/pharmacology , Animals , Darunavir , HIV Infections/enzymology , HIV Infections/genetics , HIV Protease Inhibitors/adverse effects , HIV Protease Inhibitors/therapeutic use , Humans , Lamin Type A , Lipodystrophy/chemically induced , Lipodystrophy/enzymology , Lipodystrophy/genetics , Lopinavir , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Metabolic Syndrome/chemically induced , Metabolic Syndrome/enzymology , Metabolic Syndrome/genetics , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/genetics , Mice , Mice, Knockout , Pyrimidinones/adverse effects , Pyrimidinones/pharmacology , Pyrimidinones/therapeutic use , Ritonavir/adverse effects , Ritonavir/pharmacology , Ritonavir/therapeutic use , Sulfonamides/adverse effects , Sulfonamides/therapeutic useABSTRACT
HIV protease inhibitors (HIV-PIs) target the HIV aspartyl protease, which cleaves the HIV gag-pol polyprotein into shorter proteins required for the production of new virions. HIV-PIs are a cornerstone of treatment for HIV but have been associated with lipodystrophy and other side effects. In both human and mouse fibroblasts, we show that HIV-PIs caused an accumulation of prelamin A. The prelamin A in HIV-PI-treated fibroblasts migrated more rapidly than nonfarnesylated prelamin A, comigrating with the farnesylated form of prelamin A that accumulates in ZMPSTE24-deficient fibroblasts. The accumulation of farnesyl-prelamin A in response to HIV-PI treatment was exaggerated in fibroblasts heterozygous for Zmpste24 deficiency. HIV-PIs inhibited the endoproteolytic processing of a GFP-prelamin A fusion protein. The HIV-PIs did not affect the farnesylation of HDJ-2, nor did they inhibit protein farnesyltransferase in vitro. HIV-PIs also did not inhibit the activities of the isoprenyl-cysteine carboxyl methyltransferase ICMT or the prenylprotein endoprotease RCE1 in vitro, but they did inhibit ZMPSTE24 (IC(50): lopinavir, 18.4 +/- 4.6 microM; tipranavir, 1.2 +/- 0.4 microM). We conclude that the HIV-PIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. The inhibition of ZMPSTE24 by HIV-PIs could play a role in the side effects of these drugs.
