ORIGINAL PAPER Different prelamin A forms accumulate in human fibroblasts: a study in experimental models and progeria S. Dominici,1 V. Fiori,2 M. Magnani,1 E. Schena,3 C. Capanni,4 D. Camozzi,3 M.R. D’Apice,5 C. Le Dour,6,7 M. Auclair,6,7 M. Caron,6,7 G. Novelli,5,8 C. Vigouroux,6,7,9 N.M. Maraldi,3 G. Lattanzi4 1Institute of Biochemistry “G. Fornaini” University of Urbino, Urbino, Italy; 2Diatheva Fano, Italy; 3Laboratory of Cell Biology, Istituto Ortopedico Rizzoli, Bologna, Italy; 4IGM-CNR, Unit of Bologna, c/o IOR, Bologna, Italy; 5Dept. of Biopathology and Diagnostic Imaging, University of Rome Tor Vergata, Rome, Italy; 6UPMC Université Paris, France; 7INSERM, UMR_S893Eq9, Paris, France; 8Fondazione Livio Patrizi, Rome, Italy; 9AP-HP, Hôpital Tenon, Service de Biochimie et Hormonologie, Paris, France ©2009 European Journal of Histochemistry Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystro- phies feature accumulation of prelamin A, the precursor pro- tein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to charac- terize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylat- ed form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step. Key words: prelamin A, FTI-277, AFCMe, HIV-protease inhibitors, anti-prelamin A antibodies, laminopathies. Correspondence: Giovanna Lattanzi, IGM-CNR, Unit of Bologna, c/o IOR, via di Barbiano 1/10, 40136 Bologna, Italy E-mail: lattanzi@jolly.bo.cnr.it Paper accepted on February 19, 2009 European Journal of Histochemistry 2009; vol. 53 issue 1 (January-March): 43-52 Llamina amin A is which a major plays component different of and the not nuclear yet completely understood roles in nuclear enve- lope and chromatin dynamics (Broers et al., 2006; Vlcek and Foisner, 2007). It is encoded by the LMNA gene on chromosome 1q21, which is tran- scribed as different splicing products including lamin A, lamin C, lamin A Δ 10 and lamin C2 (Arora et al., 2004). Lamin A is translated as a precursor protein har- bouring a C-terminus CaaX sequence, which is typi- cal of farnesylated proteins (Corrigan et al., 2005). In prelamin A, the CaaX sequence is made up of cysteine, serine, isoleucine and methionine (CSIM) (Corrigan et al., 2005). Methionine directs farnesy- lation at the cysteine residue by the enzyme protein farnesyltransferase. Following cysteine farnesyla- tion, the SIM sequence is removed by the endopro- tease ZMPSTE24. This proteolytic reaction is thought to be also carried out by the endoprotease RCE1, which could replace ZMPSTE24 (Rusinol and Sinensky, 2006). The cysteine residue is then methylated by the methyltransferase Icmt and a final proteolytic cleavage, performed by ZMP- STE24, removes a further 15 C-terminal residues to produce mature lamin A (Rusinol and Sinensky, 2006). Thus, at least four prelamin A species are transiently formed during prelamin A maturation, including unprocessed prelamin A, which is not far- nesylated (preLA-CSIM), farnesylated full-length prelamin A (preLA- farnesyl-CSIM), farnesylated prelamin A lacking the SIM sequence (preLA-far- nesyl-C), and carboxymethylated-farnesylated prelamin A (preLA-farnesyl-C-CH3). The knowledge of which prelamin A form is actu- ally more represented in normal and pathological cells is of utmost importance. In fact, all prelamin 43