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Inhibitors of protein geranylgeranyltransferase-I lead to prelamin A accumulation in cells by inhibiting ZMPSTE24

2012; Elsevier BV; Volume: 53; Issue: 6 Linguagem: Inglês

10.1194/jlr.m026161

ISSN

1539-7262

Autores

Sandy Y. Chang, Sarah E. Hudon-Miller, Shao H. Yang, Hea-Jin Jung, John M. Lee, Emily Farber, Thangaiah Subramanian, Douglas Andres, H. Peter Spielmann, Christine A. Hrycyna, Stephen G. Young, Loren G. Fong,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

Protein farnesyltransferase (FTase) inhibitors, generally called "FTIs," block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis—GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl–prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl–prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing. Protein farnesyltransferase (FTase) inhibitors, generally called "FTIs," block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis—GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl–prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl–prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing. Prelamin A and other proteins containing a carboxyl-terminal CaaX motif undergo a series of posttranslational modifications, beginning with protein prenylation (1.Zhang F.L. Casey P.J. Protein prenylation: molecular mechanisms and functional consequences.Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1733) Google Scholar–4.Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues.Annu. Rev. Biochem. 1992; 61: 355-386Crossref PubMed Scopus (792) Google Scholar). First, a 15-carbon farnesyl or a 20-carbon geranylgeranyl lipid is added to the thiol group of the cysteine (the "C" of the CaaX motif) by a pair of protein prenyltransferases, protein farnesyltransferase (FTase) or protein geranylgeranyltransferase I (GGTase-I) (1.Zhang F.L. Casey P.J. Protein prenylation: molecular mechanisms and functional consequences.Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1733) Google Scholar, 2.Casey P.J. Seabra M.C. Protein prenyltransferases.J. Biol. Chem. 1996; 271: 5289-5292Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 3.Schafer W.R. Rine J. Protein prenylation: genes, enzymes, targets, and functions.Annu. Rev. Genet. 1992; 26: 209-237Crossref PubMed Scopus (344) Google Scholar). Generally, the cysteine is geranylgeranylated if the "X" is a leucine or phenylalanine; otherwise, it is farnesylated (1.Zhang F.L. Casey P.J. 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Cell Sci. 1994; 107: 61-67Crossref PubMed Google Scholar). Next, the last three amino acids of the protein (i.e., the "aaX" of the CaaX motif) are clipped off by a prenylprotein-specific endoprotease (8.Young S.G. Ambroziak P. Kim E. Clarke S. Postisoprenylation protein processing: CXXX (CaaX) endoproteases and isoprenylcysteine carboxyl methyltransferase.in: Tamanoi F. Sigman D.S. The Enzymes. Academic Press, San Diego2000: 155-213Google Scholar). For most proteins, this step is carried out by RCE1 (8.Young S.G. Ambroziak P. Kim E. Clarke S. Postisoprenylation protein processing: CXXX (CaaX) endoproteases and isoprenylcysteine carboxyl methyltransferase.in: Tamanoi F. Sigman D.S. The Enzymes. Academic Press, San Diego2000: 155-213Google Scholar), but in the case of prelamin A, this step can be carried out by both zinc metalloprotease Ste24p ortholog (ZMPSTE24) and RCE1 (9.Bergo M.O. Gavino B. Ross J. Schmidt W.K. Hong C. Kendall L.V. Mohr A. Meta M. Genant H. Jiang Y. et al.Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.Proc. Natl. Acad. Sci. USA. 2002; 99: 13049-13054Crossref PubMed Scopus (347) Google Scholar, 10.Bergo M.O. Young S.G. Zmpste24 (mammalian farnesylated protein-converting enzyme 1).in: Barrett J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2nd. Academic Press, London2004: 677-682Crossref Scopus (0) Google Scholar). Finally, the newly exposed isoprenylcysteine is methylated by ICMT (11.Clarke S. Vogel J.P. Deschenes R.J. Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.Proc. Natl. Acad. Sci. USA. 1988; 85: 4643-4647Crossref PubMed Scopus (294) Google Scholar–13.Xie H. Yamane H.K. Stephenson R.C. Ong O.C. Fung B.K.-K. Clarke S. Analysis of prenylated carboxyl-terminal cysteine methyl esters in proteins.Methods. 1990; 1: 276-282Crossref Scopus (17) Google Scholar). After these CaaX modifications are complete, prelamin A undergoes a second endoproteolytic processing step (14.Kilic F. Dalton M.B. Burrell S.K. Mayer J.P. Patterson S.D. Sinensky M. In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.J. Biol. Chem. 1997; 272: 5298-5304Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar); the last 15 amino acids of the protein, including the farnesylcysteine methyl ester, are clipped off by ZMPSTE24, releasing mature lamin A (9.Bergo M.O. Gavino B. Ross J. Schmidt W.K. Hong C. Kendall L.V. Mohr A. Meta M. Genant H. Jiang Y. et al.Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.Proc. Natl. Acad. Sci. USA. 2002; 99: 13049-13054Crossref PubMed Scopus (347) Google Scholar, 15.Corrigan D.E. 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Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum.J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 19.Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and Ras proteins.EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar, 20.Hrycyna C.A. Clarke S. Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae.Mol. Cell. Biol. 1990; 10: 5071-5076Crossref PubMed Scopus (94) Google Scholar, 21.Otto J.C. Kim E. Young S.G. Casey P.J. Cloning and characterization of a mammalian prenyl protein-specific protease.J. Biol. Chem. 1999; 274: 8379-8382Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar–22.Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Thus, when protein farnesylation is inhibited with a protein farnesyltransferase inhibitor (FTI), lamin A biogenesis is blocked, leading to an accumulation of nonfarnesylated prelamin A (5.Dalton M.B. Fantle K.S. Bechtold H.A. DeMaio L. Evans R.M. Krystosek A. Sinensky M. The farnesyl protein transferase inhibitor BZA-5B blocks farnesylation of nuclear lamins and p21ras but does not affect their function or localization.Cancer Res. 1995; 55: 3295-3304PubMed Google Scholar). The production of mature lamin A also can be blocked by lopinavir, an HIV protease inhibitor that inhibits ZMPSTE24 (9.Bergo M.O. Gavino B. Ross J. Schmidt W.K. Hong C. Kendall L.V. Mohr A. Meta M. Genant H. Jiang Y. et al.Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.Proc. Natl. Acad. Sci. USA. 2002; 99: 13049-13054Crossref PubMed Scopus (347) Google Scholar, 17.Pendás A.M. Zhou Z. Cadiñanos J. Freije J.M.P. Wang J. Hultenby K. Astudillo A. Wernerson A. Rodríguez F. Tryggvason K. et al.Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice.Nat. Genet. 2002; 31: 94-99Crossref PubMed Scopus (422) Google Scholar). In the presence of lopinavir, the farnesylated form of prelamin A accumulates within cells (23.Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24.Coffinier C. Hudon S.E. Farber E.A. Chang S.Y. Hrycyna C.A. Young S.G. Fong L.G. HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 13432-13437Crossref PubMed Scopus (108) Google Scholar). The recognition that a carboxyl-terminal CaaX motif triggers the posttranslational modification of proteins with a lipid (11.Clarke S. Vogel J.P. Deschenes R.J. Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.Proc. Natl. Acad. Sci. USA. 1988; 85: 4643-4647Crossref PubMed Scopus (294) Google Scholar) was a significant development for the field. Soon thereafter, Chen et al. (25.Chen W-J. Andres D.A. Goldstein J.L. Russell D.W. Brown M.S. cDNA cloning and expression of the peptide-binding b subunit of rat p21ras farnesyltransferase, the counterpart of yeast DPR1/RAM1.Cell. 1991; 66: 327-334Abstract Full Text PDF PubMed Scopus (153) Google Scholar) used "CaaX peptide chromatography" to purify and clone the mammalian protein prenyltransferases. Also, CaaX box peptidomimetic inhibitors of the protein prenyltransferases were generated (26.James G.L. Goldstein J.L. Brown M.S. Rawson T.E. Somers T.C. McDowell R.S. Crowley C.W. Lucas B.K. Levinson A.D. Marsters Jr, J.C. Benzodiazepine peptidomimetics: potent inhibitors of Ras farnesylation in animal cells.Science. 1993; 260: 1937-1942Crossref PubMed Scopus (607) Google Scholar, 27.Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides.Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (702) Google Scholar). Peptidomimetic inhibitors of FTase block the prenylation of CaaX proteins that are normally farnesylated, for example, the RAS proteins, HDJ-2, and prelamin A (5.Dalton M.B. Fantle K.S. Bechtold H.A. DeMaio L. Evans R.M. Krystosek A. Sinensky M. The farnesyl protein transferase inhibitor BZA-5B blocks farnesylation of nuclear lamins and p21ras but does not affect their function or localization.Cancer Res. 1995; 55: 3295-3304PubMed Google Scholar, 26.James G.L. Goldstein J.L. Brown M.S. Rawson T.E. Somers T.C. McDowell R.S. Crowley C.W. Lucas B.K. Levinson A.D. Marsters Jr, J.C. Benzodiazepine peptidomimetics: potent inhibitors of Ras farnesylation in animal cells.Science. 1993; 260: 1937-1942Crossref PubMed Scopus (607) Google Scholar); peptidomimetic inhibitors of GGTase-I (GGTIs) block the prenylation of proteins that are normally geranylgeranylated, for example, Rap1a (28.Sun J. Ohkanda J. Coppola D. Yin H. Kothare M. Busciglio B. Hamilton A.D. Sebti S.M. Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice.Cancer Res. 2003; 63: 8922-8929PubMed Google Scholar). The pharmaceutical industry has been keenly interested in both FTase and GGTase-I inhibitors as anticancer agents (29.Basso A.D. Kirschmeier P. Bishop W.R. Thematic review series: lipid posttranslational modifications. Farnesyl transferase inhibitors.J. Lipid Res. 2006; 47: 15-31Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 30.Berndt N. Hamilton A.D. Sebti S.M. Targeting protein prenylation for cancer therapy.Nat. Rev. Cancer. 2011; 11: 775-791Crossref PubMed Scopus (443) Google Scholar). Some farnesylated CaaX proteins, notably KRAS, can be alternately prenylated by GGTase-I when FTase is blocked with an FTI (31.Whyte D.B. Kirschmeier P. Hockenberry T.N. Nunez-Oliva I. James L. Catino J.J. Bishop W.R. Pai J-K. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.J. Biol. Chem. 1997; 272: 14459-14464Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar). More recently, Varela et al. (32.Varela I. Pereira S. Ugalde A.P. Navarro C.L. Suarez M.F. Cau P. Cadinanos J. Osorio F.G. Foray N. Cobo J. et al.Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging.Nat. Med. 2008; 14: 767-772Crossref PubMed Scopus (306) Google Scholar) suggested that alternate prenylation might also apply to prelamin A. In their experiments, they did not observe prelamin A in cells incubated with an FTI, but they did find substantial prelamin A accumulation when cells were treated with both an FTI and a GGTI. They interpreted these findings as suggesting that prelamin A is alternately prenylated by GGTase-I in the presence of an FTI. They concluded that the GGTI/FTI combination was particularly effective in inhibiting the prenylation of prelamin A, accounting for prelamin A accumulation in cells (32.Varela I. Pereira S. Ugalde A.P. Navarro C.L. Suarez M.F. Cau P. Cadinanos J. Osorio F.G. Foray N. Cobo J. et al.Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging.Nat. Med. 2008; 14: 767-772Crossref PubMed Scopus (306) Google Scholar). However, the possibility remained that GGTIs actually lead to prelamin A accumulation by inhibiting other enzymes in the lamin A biogenesis pathway. The notion that a GGTI might inhibit the maturation of prelamin A to lamin A by inhibiting other enzymes in the pathway is not farfetched. Most GGTase-I inhibitors are peptidomimetic compounds that structurally resemble a CaaX motif, and it would not be particularly surprising if these compounds were to affect other enzymes in the pathway that are specific for prenylated CaaX proteins. Our suspicions that ZMPSTE24 might be affected by peptidomimetic GGTIs were heightened by the fact that the substrate specificity of Ste24p, the yeast ortholog of ZMPSTE24, against prenylated peptides is affected by the sequence of the CaaX motif (33.Boyartchuk V.L. Ashby M.N. Rine J. Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.Science. 1997; 275: 1796-1800Crossref PubMed Scopus (305) Google Scholar, 34.Trueblood C.E. Boyartchuk V.L. Picologlou E.A. Rozema D. Poulter C.D. Rine J. The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.Mol. Cell. Biol. 2000; 20: 4381-4392Crossref PubMed Scopus (81) Google Scholar). Mammalian ZMPSTE24 shares the same CaaX motif specificities (18.Leung G.K. Schmidt W.K. Bergo M.O. Gavino B. Wong D.H. Tam A. Ashby M.N. Michaelis S. Young S.G. Biochemical studies of Zmpste24-deficient mice.J. Biol. Chem. 2001; 276: 29051-29058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Given that ZMPSTE24 activity is influenced by the sequence of the substrate's CaaX motif, it is reasonable to hypothesize that some CaaX peptidomimetics (i.e., inhibitors of GGTase-I) might affect the activity of ZMPSTE24. In the current study, we tested the hypothesis that peptidomimetic GGTIs lead to increased amounts of prelamin A in cells by blocking ZMPSTE24. Human fibroblasts were obtained from ATCC. In some experiments, we used ZMPSTE24-deficient human fibroblasts from a patient with restrictive dermopathy (35.Toth J.I. Yang S.H. Qiao X. Beigneux A.P. Gelb M.H. Moulson C.L. Miner J.H. Young S.G. Fong L.G. Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.Proc. Natl. Acad. Sci. USA. 2005; 102: 12873-12878Crossref PubMed Scopus (227) Google Scholar). Two selective FTIs, ABT-100 and PB-43, were used at concentrations of 2–5 μM. PB-43 was obtained from Dr. Michael Gelb (University of Washington), and ABT-100 was obtained from Dr. David Frost (Abbott Laboratories). Three peptidomimetic inhibitors of GGTase-I (GGTI-298, GGTI-2147, and GGTI-2133) were obtained from Sigma-Aldrich or Calbiochem. Urea-soluble extracts of fibroblasts were prepared as previously described (36.Fong L.G. Ng J.K. Meta M. Cote N. Yang S.H. Stewart C.L. Sullivan T. Burghardt A. Majumdar S. Reue K. et al.Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.Proc. Natl. Acad. Sci. USA. 2004; 101: 18111-18116Crossref PubMed Scopus (163) Google Scholar). Extracts were size-fractionated on 4–12% gradient polyacrylamide Bis-Tris gels (Invitrogen), and the proteins transferred to nitrocellulose for Western blotting. Antibody dilutions were 1:400 for anti-lamin A/C goat IgG (sc-6215, Santa Cruz Biotechnology); 1:800 for goat anti-prelamin A (sc-6214, Santa Cruz Biotechnology); 1:1000 for anti-actin goat IgG (sc-1616, Santa Cruz Biotechnology); and 1:5000 for IRDye 800 anti-goat IgG (Rockland) or horseradish peroxidase (HRP)–conjugated anti-goat IgG (Santa Cruz Biotechnology). The IRdye-labeled antibodies were detected with an Odyssey infrared imaging scanner (LI-COR Biosciences), and the HRP-labeled antibodies were detected by Enhanced Chemiluminescence methods and exposure to X-ray film. Primary hepatocytes from mice that do not express GGTase-I in the liver (Pggt1bfl/flAlbCre+) or FTase in the liver (Fntbfl/flAlbCre+) were isolated by collagenase digestion and differential centrifugation (37.Yang S.H. Chang S.Y. Tu Y. Lawson G.W. Bergo M.O. Fong L.G. Young S.G. Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases.J. Lipid Res. 2012; 53: 77-86Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The cells were seeded in collagen-coated plates and allowed to adhere for 4 h before preparing cell extracts. Quantitative RT-PCR studies (37.Yang S.H. Chang S.Y. Tu Y. Lawson G.W. Bergo M.O. Fong L.G. Young S.G. Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases.J. Lipid Res. 2012; 53: 77-86Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) showed that that Pggt1bfl/flAlbCre+ and Fntbfl/flAlbCre+ hepatocytes had a near-complete absence of Pggt1b and Fntb transcripts, respectively. To assess protein farnesylation, fibroblasts were metabolically labeled for 48 h with an analog of farnesol, 8-anilinogeraniol (AG) (50 μM in DMSO) (38.Troutman J.M. Roberts M.J. Andres D.A. Spielmann H.P. Tools to analyze protein farnesylation in cells.Bioconjug. Chem. 2005; 16: 1209-1217Crossref PubMed Scopus (43) Google Scholar). AG is converted to anilinogeranyl diphosphate (AGPP) and used as a substrate by FTase. The incorporation of AG into prelamin A can be detected by Western blotting with an AG-specific monoclonal antibody (1:5000) (38.Troutman J.M. Roberts M.J. Andres D.A. Spielmann H.P. Tools to analyze protein farnesylation in cells.Bioconjug. Chem. 2005; 16: 1209-1217Crossref PubMed Scopus (43) Google Scholar) and an IRDye 680–labeled anti-mouse IgG (1:2500) (LI-COR) (23.Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 37.Yang S.H. Chang S.Y. Tu Y. Lawson G.W. Bergo M.O. Fong L.G. Young S.G. Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases.J. Lipid Res. 2012; 53: 77-86Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 39.Davies B.S. Barnes 2nd, R.H. Tu Y. Ren S. Andres D.A. Spielmann H.P. Lammerding J. Wang Y. Young S.G. Fong L.G. An accumulation of non-farnesylated prelamin A causes cardiomyopathy but not progeria.Hum. Mol. Genet. 2010; 19: 2682-2694Crossref PubMed Scopus (74) Google Scholar). Human fibroblasts were grown on coverslips and grown in the presence of an FTI (ABT-100) or a GGTI (GGTI-298). Cells were incubated with a rat monoclonal antibody against prelamin A (7G11; 1:50 dilution) or a mouse monoclonal antibody against Lap2β (BD Biosciences; 1:400 dilution) for 2 h. After washing, cells were incubated with a 1:200 dilution of Alexa Fluor 568–conjugated anti-rat antibody or Alexa Fluor 488–conjugated anti-mouse antibody (Invitrogen). DNA was stained with DAPI to visualize nuclei. Images were obtained with a Leica SP2 1P-FCS confocal microscope with a 63× objective. Endoprotease-coupled methylation assays of ZMPSTE24 activity (23.Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24.Coffinier C. Hudon S.E. Farber E.A. Chang S.Y. Hrycyna C.A. Young S.G. Fong L.G. HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 13432-13437Crossref PubMed Scopus (108) Google Scholar, 40.Hudon S.E. Coffinier C. Michaelis S. Fong L.G. Young S.G. Hrycyna C.A. HIV-protease inhibitors block the enzymatic activity of purified Ste24p.Biochem. Biophys. Res. Commun. 2008; 374: 365-368Crossref PubMed Scopus (42) Google Scholar) were performed by mixing 5 μg of membranes from Δste24Δrce1 yeast that overexpressed mouse ZMPSTE24, 8 μg of membranes from Δste24Δrce1 yeast overexpressing Ste14p, a farnesylated a-factor peptide ([YIIKGVFWDPA(farnesyl)CVIA], 5 μM; EZBioLab, Westfield, IN), and 20 μM S-adenosyl-L-[methyl-14C]methionine (55 Ci/mol; GE Healthcare) in 100 mM Tris-HCl, pH 7.5, in a final volume of 60 μl. Reactions were performed in the presence and absence of GGTI-298 and lopinavir. After incubating the reactions at 30°C for 30 min, the reactions were stopped with 50 μl of 1 M NaOH/1% SDS. The reactions were then spotted on a pleated filter paper, and [14C]methanol was allowed to diffuse into scintillation fluid for 2.5 h. The amount of radioactivity released was quantified by scintillation counting (11.Clarke S. Vogel J.P. Deschenes R.J. Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.Proc. Natl. Acad. Sci. USA. 1988; 85: 4643-4647Crossref PubMed Scopus (294) Google Scholar). We tested whether commonly used GGTIs would lead to an accumulation of prelamin A in human fibroblasts. In our initial experiments, we observed an accumulation of prelamin A in cells treated with GGTI-298, a peptidomimetic inhibitor of GGTase-I. Indeed, the amount of prelamin A accumulation with 10 μM GGTI-298 was close to the amount of prelamin A that accumulates with selective FTIs (PB-43 and ABT-100) (Fig. 1A). When GGTI-298 was combined with an FTI (PB-43), the amount of prelamin A in cells did not increase beyond that in cells treated with FTI alone (Fig. 1A). We also observed prelamin A accumulation with another peptidomimetic GGTI, GGTI-2147 (Fig. 1B). We considered the possibility that the prelamin A accumulation in GGTI-treated cells was due to the fact that these compounds inhibited FTase; however, this was not the case. An FTI (ABT-100), but not a GGTI (GGTI-298), led to an accumulation of nonfarnesylated HDJ-2 (a CaaX protein that is normally farnesylated by FTase) (Fig. 1C). Also, the GGTI, but not the FTI, led to the accumulation of nonprenylated RAP1A (a CaaX protein that is normally geranylgeranylated by GGTase-I). Finding an accumulation of prelamin A in both FTI- and GGTI-treated cells raised the possibility that prelamin A might normally undergo prenylation by both FTase and GGTase-I. To test this possibility, prelamin A levels were assessed in both FTase-deficient hepatocytes and GGTase-I–deficient hepatocytes [isolated from liver-specific Pggt1b knockout mice (Pggt1bfl/flAlbCre+) and liver-specific Fntb knockout mice (Fntbfl/flAlbCre+), respectively] (37.Yang S.H. Chang S.Y. Tu Y. Lawson G.W. Bergo M.O. Fong L.G. Young S.G. Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases.J. Lipid Res. 2012; 53: 77-86Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). As expected, we observed a substantial amount of prelamin A accumulation in FTase-deficient liver cells. However, when GGTase-I–deficient cells were examined, no prelamin A was detected (Fig. 2). These results implied that the accumulation of prelamin A in GGTI-treated cells could not be caused by an inhibition of GGTase-I activity and, instead, must be due to an effect of the GGTI on other enzymes in the lamin A biogenesis pathway. The prelamin A in GGTI-treated cells displayed a slightly faster electrophoretic mobility than nonfarnesylated prelamin A in FTI-treated fibroblasts (Fig. 1AndashC). Previously, we showed that lopinavir, a potent inhibitor of ZMPSTE24, leads to an accumulation of farnesyl–prelamin A in cells (23.Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24.Coffinier C. Hudon S.E. Farber E.A. Chang S.Y. Hrycyna C.A. Young S.G. Fong L.G. HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 13432-13437Crossref PubMed Scopus (108) Google Scholar), and we further showed that the farnesyl–prelamin A in lopinavir-treated cells has a faster electrophoretic mobility than nonfarnesylated prelamin A in FTI-treated cells (23.Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24.Coffinier C. Hudon S.E. Farber E.A. Chang S.Y. Hrycyna C.A. Young S.G. Fong L.G. HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 13432-13437Crossref PubMed Scopus (108) Google Scholar). To characterize the prelamin A in GGTI-treated cells, we compared the electrophoretic mobilities of prelamin A in GGTI-, FTI-, and lopinavir-treated cells (Fig. 3A). The electrophoretic mobility of prelamin A in GGTI- and lopinavir-treated cells was similar, and both migrated faster than prelamin A in FTI-treated cells (Fig. 3A). The more rapid electrophoretic mobility of prelamin A in GGTI-treated cells suggested that a GGTI might inhibit ZMPSTE24 activity. Indeed, this was the case. Like lopinavir, a GGTI potently inhibited the enzymatic activity of recombinant ZMPSTE24 against a farnesylated CaaX peptide substrate (Fig. 3B). The inhibition of ZMPSTE24 by a GGTI would be expected to lead to an accumulation of farnesyl–prelamin A (23.Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24.Coffinier C. Hudon S.E. Farber E.A. Chang S.Y. Hrycyna C.A. Young S.G. Fong L.G. HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 13432-13437Crossref PubMed Scopus (108) Google Scholar). To determine whether the prelamin A in GGTI-treated cells is farnesylated, we performed metabolic labeling experiments with a farnesol analog, 8-anilinogeraniol (AG) (38.Troutman J.M. Roberts M.J. Andres D.A. Spielmann H.P. Tools to analyze protein farnesylation in cells.Bioconjug. Chem. 2005; 16: 1209-1217Crossref PubMed Scopus (43) Google Scholar), in the presence or absence of either an FTI or a GGTI. After entering cells, AG is converted to anilinogeranyl diphosphate (AGPP) and used as a substrate by FTase for protein prenylation. The incorporation of AG into prelamin A can then be detected by Western blotting with an AG-specific monoclonal antibody (23.Coffinier C. Hudon S.E. Lee R. Farber E.A. Nobumori C. Miner J.H. Andres D.A. Spielmann H.P. Hrycyna C.A. Fong L.G. et al.A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.J. Biol. Chem. 2008; 283: 9797-9804Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 38.Troutman J.M. Roberts M.J. Andres D.A. Spielmann H.P. Tools to analyze protein farnesylation in cells.Bioconjug. Chem. 2005; 16: 1209-1217Crossref PubMed Scopus (43) Google Scholar, 41.Yang S.H. Andres D.A. Spielmann H.P. Young S.G. Fong L.G. Progerin elicits disease whether or not it is farnesylated.J. Clin. Invest. 2008; 118: 3291-3300Crossref PubMed Scopus (88) Google Scholar, 42.Yang S.H. Chang S.Y. Andres D.A. Spielmann H.P. Young S.G. Fong L.G. Assessing the efficacy of protein farnesyltransferase inhibitors in mouse models of progeria.J. Lipid Res. 2010; 51: 400-405Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The AG-specific antibody detected trace amounts of prenylated prelamin A in wild-type human fibroblasts (43.Fong L.G. Vickers T.A. Farber E.A. Choi C. Yun U.J. Hu Y. Yang S.H. Coffinier C. Lee R. Yin L. et al.Activating the synthesis of progerin, the mutant prelamin A in Hutchinson-Gilford progeria syndrome, with antisense oligonucleotides.Hum. Mol. Genet. 2009; 18: 2462-2471Crossref PubMed Scopus (34) Google Scholar); the accumulation of this small amount of prenylated prelamin A was eliminated by incubation with an FTI (Fig. 4). The prelamin A that accumulates in GGTI-treated cells is farnesylated, as judged by AG incorporation into the prelamin A and consistent with its ability to inhibit ZMPSTE24. In control experiments, we found, as expected, that the prelamin A in ZMPSTE24-deficient cells is farnesylated and that this farnesylation can be blocked with an FTI (Fig. 4). The nonfarnesylated prelamin A in mevinolin-treated fibroblasts is typically found in the nucleoplasm (44.Lutz R.J. Trujillo M.A. Denham K.S. Wenger L. Sinensky M. Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina.Proc. Natl. Acad. Sci. USA. 1992; 89: 3000-3004Crossref PubMed Scopus (148) Google Scholar), but much of the farnesyl–prelamin A in ZMPSTE24-deficient fibroblasts is found at the nuclear rim or in folds of the nuclear membrane (35.Toth J.I. Yang S.H. Qiao X. Beigneux A.P. Gelb M.H. Moulson C.L. Miner J.H. Young S.G. Fong L.G. Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.Proc. Natl. Acad. Sci. USA. 2005; 102: 12873-12878Crossref PubMed Scopus (227) Google Scholar, 45.Yang S.H. Bergo M.O. Toth J.I. Qiao X. Hu Y. Sandoval S. Meta M. Bendale P. Gelb M.H. Young S.G. et al.Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation.Proc. Natl. Acad. Sci. USA. 2005; 102: 10291-10296Crossref PubMed Scopus (236) Google Scholar), likely reflecting the role of the hydrophobic farnesyl lipid in targeting prelamin A to the nuclear membrane (46.Hennekes H. Nigg E.A. The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties.J. Cell Sci. 1994; 107: 1019-1029Crossref PubMed Google Scholar). Thus, we predicted that we would find most of the nonfarnesylated prelamin A in FTI-treated fibroblasts in the nucleoplasm and much of the farnesyl–prelamin A in GGTI-treated fibroblasts at the nuclear rim (or in nuclear membrane folds). Indeed, confocal immunofluorescence microscopy revealed an accumulation of prelamin A in nuclear membrane folds in GGTI-treated fibroblasts, whereas nearly all of the prelamin A in FTI-treated fibroblasts was in the nucleoplasm (Fig. 5). As expected, farnesylated prelamin A in human ZMPSTE24-deficient fibroblasts was at the nuclear rim or in nuclear membrane folds (Fig. 5) (35.Toth J.I. Yang S.H. Qiao X. Beigneux A.P. Gelb M.H. Moulson C.L. Miner J.H. Young S.G. Fong L.G. Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.Proc. Natl. Acad. Sci. USA. 2005; 102: 12873-12878Crossref PubMed Scopus (227) Google Scholar). In the current study, we show that commonly used inhibitors of GGTase-I lead to an accumulation of prelamin A by inhibiting ZMPSTE24. Four lines of evidence support this view. First, the prelamin A in GGTI-treated cells displayed a faster electrophoretic mobility than the prelamin A in FTI-treated cells and comigrated with the farnesyl–prelamin A in cells treated with lopinavir, a ZMPSTE24 inhibitor. Second, a GGTI inhibited the enzymatic activity of ZMPSTE24 in a biochemical assay with potency similar to that of lopinavir. Third, metabolic labeling studies showed that the prelamin A that accumulates in GGTI-treated cells is indeed farnesylated. Fourth, the prelamin A in FTI- and GGTI-treated cells exhibited different localization patterns within the cell nucleus. The nonfarnesylated prelamin A in FTI-treated fibroblasts was located mainly in the nucleoplasm, but a significant amount of the farnesyl–prelamin A in GGTI-treated cells was targeted to the nuclear membrane. At the drug concentrations that we used, the GGTI did not inhibit FTase. Only the FTI, and not the GGTI, was capable of inhibiting the farnesylation of HDJ-2, whereas only the GGTI led to the accumulation of nonprenylated RAP1A. The ability of GGTIs to inhibit ZMPSTE24 cannot be considered unexpected, given what is known about the structure of GGTIs and the substrate specificities of ZMPSTE24. Most GGTIs, including all of the compounds used in this study, are peptidomimetics that were specifically designed to resemble a CaaX motif. Also, the enzymatic activity of the yeast ortholog of ZMPSTE24, Ste24p, is highly sensitive to the structure of the substrate's CaaX motif. Ste24p efficiently cleaves the –aaX from an a-factor substrate terminating in CAMQ, but it is incapable of cleaving an otherwise identical a-factor substrate terminating in CTLM (33.Boyartchuk V.L. Ashby M.N. Rine J. Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.Science. 1997; 275: 1796-1800Crossref PubMed Scopus (305) Google Scholar, 34.Trueblood C.E. Boyartchuk V.L. Picologlou E.A. Rozema D. Poulter C.D. Rine J. The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.Mol. Cell. Biol. 2000; 20: 4381-4392Crossref PubMed Scopus (81) Google Scholar). Mammalian ZMPSTE24 shares these CaaX-box specificities (18.Leung G.K. Schmidt W.K. Bergo M.O. Gavino B. Wong D.H. Tam A. Ashby M.N. Michaelis S. Young S.G. Biochemical studies of Zmpste24-deficient mice.J. Biol. Chem. 2001; 276: 29051-29058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Given that the enzymatic activity of ZMPSTE24 depends on the structure of the CaaX motif, the finding that some CaaX peptidomimetics inhibit ZMPSTE24 is not particularly surprising. The ability of GGTIs to inhibit ZMPSTE24 may explain the GGTI-induced accumulation of prelamin A in the experiments by Varela et al. (32.Varela I. Pereira S. Ugalde A.P. Navarro C.L. Suarez M.F. Cau P. Cadinanos J. Osorio F.G. Foray N. Cobo J. et al.Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging.Nat. Med. 2008; 14: 767-772Crossref PubMed Scopus (306) Google Scholar). In their experiments, a GGTI, when added to cells with an FTI, led to substantial amounts of prelamin A accumulation. When they treated cells with an FTI alone, little or no prelamin A was apparent, a peculiar finding that raises questions about the activity of their FTI and the level of FTase inhibition that was achieved. In any case, our studies showing that GGTIs inhibit ZMPSTE24 provide a plausible explanation for the prelamin A accumulation when a GGTI is added to cells. Nevertheless the possibility that prelamin A undergoes alternate prenylation remains intriguing. In the end, a definitive understanding of prelamin A alternate prenylation will require direct biochemical assays and genetic studies in which the CaaX prenyltransferases are inactivated with genetic rather than pharmacologic approaches. Some studies with tissue-specific knockout mice have already been reported (37.Yang S.H. Chang S.Y. Tu Y. Lawson G.W. Bergo M.O. Fong L.G. Young S.G. Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases.J. Lipid Res. 2012; 53: 77-86Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 47.Lee R. Chang S.Y. Trinh H. Tu Y. White A.C. Davies B.S. Bergo M.O. Fong L.G. Lowry W.E. Young S.G. Genetic studies on the functional relevance of the protein prenyltransferases in skin keratinocytes.Hum. Mol. Genet. 2010; 19: 1603-1617Crossref PubMed Scopus (32) Google Scholar), and they suggest that alternate prenylation of prelamin A, if it occurs in vivo, may not be particularly efficient. Our studies showing that GGTIs inhibit ZMPSTE24 are relevant to efforts to develop GGTIs as anticancer agents. GGTIs inhibit the growth of tumor cell lines in culture, including KRAS–transformed cells, and have been shown to be efficacious in human tumor cell xenograft models (48.Lobell R.B. Omer C.A. Abrams M.T. Bhimnathwala H.G. Brucker M.J. Buser C.A. Davide J.P. deSolms S.J. Dinsmore C.J. Ellis-Hutchings M.S. et al.Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.Cancer Res. 2001; 61: 8758-8768PubMed Google Scholar). Also, inactivation of Pggt1b (the gene encoding the β-chain of GGTase-I) inhibits tumor cell growth in vivo and ameliorates disease phenotypes in a mouse model of KRAS–induced lung cancer (49.Sjogren A.K. Andersson K.M. Liu M. Cutts B.A. Karlsson C. Wahlstrom A.M. Dalin M. Weinbaum C. Casey P.J. Tarkowski A. et al.GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer.J. Clin. Invest. 2007; 117: 1294-1304Crossref PubMed Scopus (93) Google Scholar). These observations have prompted interest in GGTase-I inhibitors as anti-cancer therapies, and clinical trials are now underway. In light of our current findings, companies developing GGTIs as anticancer agents will likely want to test whether their compounds inhibit ZMPSTE24 and elicit an accumulation of farnesyl–prelamin A. In both humans and mice, ZMPSTE24 deficiency, and the accompanying accumulation of farnesyl–prelamin A, leads to severe progeroid disorders (9.Bergo M.O. Gavino B. Ross J. Schmidt W.K. Hong C. Kendall L.V. Mohr A. Meta M. Genant H. Jiang Y. et al.Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.Proc. Natl. Acad. Sci. USA. 2002; 99: 13049-13054Crossref PubMed Scopus (347) Google Scholar, 17.Pendás A.M. Zhou Z. Cadiñanos J. Freije J.M.P. Wang J. Hultenby K. Astudillo A. Wernerson A. Rodríguez F. Tryggvason K. et al.Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice.Nat. Genet. 2002; 31: 94-99Crossref PubMed Scopus (422) Google Scholar). Thus, it is conceivable that GGTI-mediated ZMPSTE24 inhibition could lead to unwanted complications. The authors thank Drs. David Frost from Abbott Pharmaceuticals for ABT-100 and Michael Gelb from the University of Washington for PB-43. protein farnesyltransferase protein farnesyltransferase inhibitor protein geranylgeranyltransferase-I protein geranylgeranyltransferase inhibitor zinc metalloprotease Ste24p ortholog

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