The prelamin A endoprotease catalyzes the terminal reaction of lamin A maturation in mammalian cells. This is an endoproteolytic removal of 15 amino acid residues from the carboxy terminus of the lamin A precursor, prelamin A. The enzyme requires a farnesylated, carboxy terminal, methylated cysteinyl peptide as a substrate. It also recognizes a hexapeptide, RSY yields LLG, cleaving this sequence where indicated. It has generally been assumed that farnesylation acts as an anchor for proteins to membrane lipid bilayers. However, these results on the prelamin A endoprotease are consistent with an alternative hypothesis: that farnesylation acts, in conjunction with specific amino acid sequences, to mediate protein heterodimer formation. To gain further insight into the mechanism of farnesylated protein recognition by another protein, we are proposing to characterize, in more detail, the mechanism of these interactions between prelamin A and the endoprotease, particularly the farnesyl binding domain(specific aim 2). Such studies will require the development of more efficient and higher capacity purification methods for the enzyme(specific aim 1). We are also proposing to gain further insight into the biological significance of this unusual protein processing pathway. The RSYLLG cleavage sequence is unique among mammalian proteins in the Swiss-Prot data base. We will determine the substrate specificity of the endoproteolytic cleavage site, to test the hypothesis that this reaction is unique to prelamin A processing (specific aim 3). We have shown that this enzyme is regulated both by expression of the prelamin A substrate and the proliferation status of cultured cells. We propose to further characterize the mechanisms of these regulatory responses, an undertaking which requires molecular cloning and recombinant expression of the enzyme(specific aim 4). To achieve these ends, we are proposing the following specific aims: 1) Scale-up of prelamin A endoprotease preparation, possibly through the use of affinity chromatography; 2) Characterization of the farnesyl binding domain and active site of the prelamin A endoprotease through affinity labeling, peptide mapping and sequencing; 3) Characterization of the amino acid substrate specificity of the enzyme through the development of a simpler assay and screening of multiple peptide substrates;4) Molecular cloning and recombinant expression of the prelamin A endoprotease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Physiological Chemistry Study Section (PC)
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Ikeda, Richard A
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East Tennessee State University
Schools of Medicine
Johnson City
United States
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Corrigan, Douglas P; Kuszczak, Danuta; Rusinol, Antonio E et al. (2005) Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24. Biochem J 387:129-38