HIV protease inhibitors (HIV-PIs

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 ZMP-STE24, 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 ZMP-STE24 and leads to an accumulation of prelamin A.HIV protease inhibitors (HIV-PIs) 3 are designed to inhibit the HIV aspartyl protease, which is required for generating viral core proteins (1). HIV-PIs have become essential elements of modern antiretroviral regimens, but they have been associated with significant side effects, including partial lipodystrophy and metabolic syndrome (2-4). Similar disease phenotypes have been observed in association with missense mutations in LMNA (the gene for lamins A and C) (5, 6) and with genetic defects associated with defective conversion of prelamin A to mature lamin A (7-9).In 2003, Caron et al. (10) reported that a pair of HIV-PIs, indinavir and nelfinavir, appeared to lead to the accumulation of small amounts of prelamin A in a preadipocyte cell line. This finding was intriguing, but the biochemical mechanism was obscure. Potentially, this finding could have been due to the inhibition of any of four different enzymatic steps in prelamin A metabolism. The biogenesis of mature lamin A from prelamin A involves 1) farnesylation of a C-terminal cysteine by protein farnesyltransferase; 2) the removal of the last three amino acids of prelamin A (a redundant enzymatic activity of Ras converting enzyme 1 (RCE1) and ZMPSTE24); 3) the methylation of a newly exposed farnesylcysteine by isoprenylcysteine carboxyl methyltransferase (ICMT); and 4) the removal of the last 15 residues of the protein, including the farnesylcysteine methyl ester, by ZMPSTE24 (11). Steps 2-4 are utterly dependent on the first post-translational processing step, protein farnesylation.Recently, our laboratories showed that several HIV-PIs, but notably lopinavir, lead to substantial prelamin A accumulation in cultured cells at therapeutically relevant concentrations, and we went on to identify the ...

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 HIVPIs 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. KeywordsZMPSTE24; Ste24p; HIV-PI; lamins; Ste14p; lipodystrophy; protease; methyltransferase Many eukaryotic proteins, such as the mating pheromone a-factor in Saccharomyces cerevisiae, the Ras proteins in both yeast and mammals, and the nuclear lamins in mammals, terminate with a CaaX motif (in which the "C" is cysteine, "a" is often an aliphatic amino acid, and "X" is one of several different amino acids) [1]. The CaaX motif triggers a series of posttranslational modifications. First, the cysteine is farnesylated or geranylgeranylated by cytosolic protein prenyltransferases. Second, the last three amino acids of the protein (i.e., the "aaX") are clipped off and released. This reaction is generally carried out by RCE1, a membrane endoprotease of the endoplasmic reticulum (ER); however, in the case of a-factor in yeast and 5Address correspondence to: Department of Chemistry, Purdue University, 560 Oval Dr., West Lafayette, IN 47907-2084. Tel.: 765-494-7322; Fax: 765-494-0239; E-mail: hrycyna@purdue.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript prelamin A in mammals, this step can be carried out by both RCE1 and a membrane zinc metalloproteinase of the ER, designated Ste24p in yeast and ZMPSTE24 in mammals [2][3][4]. Finally, the newly exposed isoprenylcysteine is methylated by an integral membrane methyltransferase, designated ...

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 an important component of HIV treatment regimens but have been associated with lipodystrophy and other side effects. In both human and mouse fibroblasts, we showed 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 isoprenylcysteine carboxyl methyltransferase ICMT or the prenylprotein endoprotease RCE1 in vitro, but they did inhibit ZMPSTE24 (IC(50): lopinavir, 18.4 +/− 4.6 uM; tipranavir, 1.2 +/− 0.4 uM). We conclude that HIV‐PIs inhibit ZMPSTE24, leading to an accumulation of farnesyl‐prelamin A within cells. The inhibition of ZMPSTE24 by HIV‐PIs could play a role in the side effects of these drugs.