HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells

Abstract

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.

Fig. 1.

Biogenesis of lamin A from prelamin A. Prelamin A undergoes four posttranslational processing steps (13). First, the cysteine of the C-terminal CaaX motif is farnesylated by protein FTase. Second, the last three amino acids (-aaX) are clipped off (cleavage P1), a redundant activity of RCE1 and ZMPSTE24 (13). Third, the farnesylcysteine is carboxyl-methylated by ICMT. Fourth, the last 15 amino acids of prelamin A (including the farnesylcysteine methyl ester) are clipped off by ZMPSTE24 (cleavage P2), releasing mature lamin A. Lonafarnib, an FTI, blocks all of the processing steps and leads to the accumulation of nonfarnesylated prelamin A. In this study, we tested the hypothesis that HIV-PIs inhibit ZMPSTE24, causing an accumulation of farnesyl-prelamin A in cells.

Fig. 2.

Accumulation of prelamin A in fibroblasts treated with an FTI and HIV-PIs. (A) Western blots of extracts from WT human fibroblast cell lines (AG08470 and AG07095) that had been treated for 10 days with LPV (20 μM), an FTI (5 μM), or vehicle alone (DMSO). The prelamin A to lamin C ratio, as judged by quantitative image analysis, averaged 0.31 in the LPV-treated cells and 0.39 in the FTI-treated cells; the prelamin A to mature lamin A ratio averaged 0.23 in the LPV-treated cells and 0.96 in the FTI-treated cells. We used an antibody against lamin A/C, two different rabbit antisera against the C terminus of mouse prelamin A (“a” and “b”), and an antibody against actin. Note the greater distance between the prelamin A and lamin A bands in the FTI-treated cells compared with the LPV-treated cells. One of the anti-prelamin A antisera, “a,” bound only nonfarnesylated human prelamin A, whereas the other, “b,” bound to both farnesylated and nonfarnesylated human prelamin A (see Materials and Methods). (B) Western blots of human fibroblasts treated with LPV or an FTI. Again, the prelamin A in LPV-treated cells migrates further than the prelamin A in FTI-treated cells. The lane labeled “RD + WT” represents a mixture of RD (ZMPSTE24-deficient) and WT cell extracts, each treated with DMSO. Note that the prelamin A from the RD extracts migrates at the same position as the prelamin A from the LPV-treated fibroblasts. (C) Western blots of extracts from mouse fibroblasts treated for 48 h with LPV (20 μM), atazanavir (ATV) (20 μM), an FTI (5 μM), or vehicle (DMSO). (D) Western blot analysis of Rce1^−/− and Icmt^−/− mouse fibroblasts that had been treated for 24 h with LPV (20 μM), an FTI (5 μM), or vehicle (DMSO). Icmt deficiency is associated with some prelamin A accumulation (15), which is increased further with LPV. (E) Western blots of extracts from WT mouse fibroblasts treated for 10 days with LPV (20 μM), NVP (200 μM and 400 μM), STV (20 μM and 40 μM), or vehicle (DMSO).

Fig. 3.

HIV-PIs block the processing of an enhanced GFP-prelamin A fusion protein. (A) Schematic representations of the GFP-prelamin A fusion protein and its cleavage by ZMPSTE24. The cDNA sequences encoding amino acids 548–665 from mouse prelamin A (represented by the blue oval) were ligated in-frame to GFP (represented as a green oval). The location of the prelamin A cleavage reaction carried out exclusively by ZMPSTE24 (cleavage P2) is indicated, as are the locations of epitopes for antibodies against GFP, mature lamin A, and prelamin A. (B) Western blot analysis of HEK293 cells transiently transfected with a GFP expression vector (pEGFP-C1) or the GFP-prelamin A fusion construct. Antibody binding was detected with an Odyssey system (Li-Cor Biosciences). (Left) Red signal indicates binding of the anti-GFP antibody, and green signal indicates binding of the anti-mature lamin A antibody. The merged signals result in a yellow/green color. (Right) Red signal indicates binding of the anti-GFP antibody, and green signal indicates binding of the anti-prelamin A antibody. (C) Western blot, using an antibody against GFP, of WT and Zmpste24^−/− fibroblasts transiently transfected with the GFP-prelamin A fusion construct and treated overnight with the vehicle (DMSO), LPV (20 μM), or the FTI (5 μM). (D) Western blot analysis of HeLa cells transiently transfected with the GFP-prelamin A fusion construct and treated overnight with vehicle (DMSO), LPV (20 μM), atazanavir (ATV) (20 μM), STV (20 μM), NVP (20 μM), or an FTI (5 μM). Western blot was performed with antibodies against GFP and prelamin A (Middle and Bottom). Top shows the merged image; the anti-GFP signal is red, and the anti-prelamin A signal is green. (E) Western blots of HEK293 cells transiently transfected with the GFP-prelamin A construct and treated overnight with LPV (20 μM), the FTI (5 μM), or vehicle (DMSO). (F) Western blots of HEK293 cells transiently transfected with the GFP-prelamin A construct and treated overnight with LPV (20 μM), TPV (20–100 μM), or DMSO.

Fig. 4.

Western blots showing that LPV has no effect on protein farnesylation or geranylgeranylation. Wild-type fibroblasts were treated for 24 h with LPV (20 μM), an FTI (5 μM), a geranylgeranyltransferase type I inhibitor (GGTI-298) (15 μM), or vehicle (DMSO). LPV had no effect on the isoprenylation of HDJ-2 or Rap1A.

Fig. 5.

HIV-PIs inhibit ZMPSTE24 but not FTase, ICMT, or RCE1 in vitro. (A) Effect of LPV and ATV on the farnesylation of H-Ras by FTase (mean of triplicate determinations). (B) Effect of LPV on the methylation of N-acetyl-farnesylcysteine by ICMT (mean of three experiments, each point in quadruplicate ± SD). (C) Abilities of LPV and TPV to inhibit the enzymatic activity of mouse ZMPSTE24. Shown are results from a coupled endoproteolysis/methylation assay (17) that tested the ability of membranes from Δste24Δrce1 yeast overexpressing mouse ZMPSTE24 to cleave a yeast a-factor substrate, rendering it susceptible to methylation by Ste14p. Each assay was repeated four to seven times, with each point in duplicate, ± SD. (D) Effect of the HIV-PIs LPV and TPV on the activity of mouse RCE1. This assay tested the ability of membranes from Δste24Δrce1 yeast overexpressing RCE1 to cleave an a-factor substrate and then be methylated by Ste14p. Each assay was performed three times, each point in duplicate, ± SD.

Fig. 6.

Mouse fibroblasts heterozygous for Zmpste24 deficiency (Zmpste24^+/−) exhibit higher levels of prelamin A accumulation with LPV treatment. Primary cultures of Zmpste24^+/+, Zmpste24^+/−, and Zmpste24^−/− fibroblasts, prepared from littermate embryos, were treated for 10 days with LPV (20 μM) or vehicle (DMSO). A and B show two independent experiments with different cell lines. Quantitative PCR studies showed that Zmpste24 mRNA levels in Zmpste24^+/− cells were half-normal (not shown).