Defining Substrate Requirements for Cleavage of Prelamin A by the Zinc Metalloprotease ZMPSTE24

Proteases play important roles in diverse biological processes relevant to human health and disease and defining their substrate specificity is an essential component to understanding molecular mechanisms of proteolysis. ZMPSTE24 is a zinc metalloprotease that is critical to the proteolytic maturation of lamin A, an intermediate filament protein and component of the nuclear lamina. When ZMPSTE24-dependent processing of prelamin A is disrupted by mutations in either the substrate or the protease, accumulation of uncleaved prelamin A causes a spectrum of genetic disorders including the premature aging disease Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid disorders mandibuloacral dysplasia (MAD-B) and restrictive dermopathy (RD). Prelamin A is the only known mammalian substrate for ZMPSTE24, however, the basis of this specificity remains unclear. To begin to define the sequence requirements for ZMPSTE24 recognition, I have performed a comprehensive mutagenesis scan of the eight residues flanking the cleavage site, from amino acid residue T643 to N650 (TRSY↓LLGN). Mutants were tested in an in vivo humanized yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. Substitutions C-terminal to the cleavage site were generally more disruptive than N-terminal substitutions, although R644 shows a modest bias for positively charged residues. Of particular note is L647 in the P1’ position C-terminal to the cleavage site, of which mutation to arginine has long been known to be “uncleavable.” Here I show that charged residues, aromatics, and proline disrupt cleavage, while hydrophobic residues are well-tolerated. L648 at P2’ shows a similar pattern with several key differences. I have generated a heat map of cleavage efficacy for ZMPSTE24 and positions P1’ and P2’ have emerged as critical for ZMPSTE24 substrate recognition, with a strong preference for hydrophobic residues. Hydrophobic residues at these two positions are evolutionarily conserved. I have shown that the 8 residues flanking the deduced ZMPSTE24 cleavage site in prelamin A sequences from amphibians, birds, fish, and mammals can promote cleavage when placed in the context of human lamin A, albeit with varying efficiency. Taken together, my results provide mechanistic insights into the recognition of prelamin A by ZMPSTE24 and begin to define a consensus motif with potential predictive value to identify other ZMPSTE24 substrates