Molecular and biochemical analyses of prelamin A and a-factor proteolytic processing by the novel zinc metalloprotease ZMPSTE24 and its yeast homolog, Ste24p

Lamin A is an intermediate filament-type protein that forms part of a dense network on the inner nuclear membrane known as the nuclear lamina. Lamin A is initially synthesized as prelamin A, terminating in a CaaX motif (C is cysteine, a is an aliphatic amino acid, and X is any residue) signaling a series of well characterized post-translational modifications. CaaX processing occurs by three sequential enzymatic reactions: (1) covalent attachment of farnesyl lipid to the cysteine residue by farnesyltransferase, (2) endoproteolysis of AAX residues by ZMPSTE24 or Ras converting enzyme 1 (RCE1), (3) methylation of the newly exposed prenylated cysteine by isoprenylcysteine carboxyl methyltransferase (ICMT). For proper localization of mature lamin A to the nuclear envelope a second cleavage event must occur where ZMPSTE24 removes an additional 15 amino acids from the C-terminus of prelamin A. A similar post-translational modification pathway is paralleled in yeast with the mating pheromone a-factor and the zinc metalloprotease, Ste24p. Mutations to the LMNA and ZMPSTE24 genes result in numerous diseases, many of which are characterized as laminopathies, such as Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD). RD and HGPS, two of the most severe laminopathies, result in death at a very young age due to a LMNA mutation causing an accumulation of farnesylated-prelamin A in the nucleus, respectively. It is evident that ZMPSTE24 protease activity is necessary for human health, but the mechanism by which it functions remains uncharacterized. The work presented in this dissertation focuses on the characterization of mechanistic aspects of the integral membrane protease human ZMPSTE24 and its yeast homolog, Ste24p. First, we biochemically characterized known ZMPSTE24 disease mutants. Each ZMPSTE24 mutant was expressed in yeast and the protease activity in crude membranes was measured using an in vitro coupled proteolysis radioactivity assay. The amount of methylation serves as an indirect measure of ZMPSTE24 endoproteolysis activity. We demonstrated for the first time that the level of protease activity of each ZMPSTE24 mutant correlated with the severity of the disease associated with the mutation. Secondly, we investigated the activity and substrate binding properties of Ste24p, the ZMPSTE24 yeast homolog, using a-factor precursors. Using the crystal structures as a guide, I generated a Ste24p mutant library through site-directed mutagenesis. The activities of the mutants were screened with the coupled proteolysis radioactivity assay. I used photoaffinity labeling (PAL) to elucidate enzyme-substrate interactions using a series of four Ste24p substrate analogs. We have concluded that the C-terminal CaaX activity of ZMPSTE24 disease mutation correlates to the severity of the disease. We have also determined some of the same homologous mutations in yeast follow the same pattern. In our analysis of other Ste24p mutations we have identified important regions for catalysis and substrate binding verified by activity assays and photoaffinity labeling screens. We have also determined that ZMPSTE24 and RCE1 both have the capabilities to cleave a prelamin A peptide and full length prelamin A in mouse embryonic fibroblasts. This research has provided a solid foundation for future work on this project to determine the mechanism by which this unique protease ZMPSTE24 functions