Abstract
The integral membrane zinc metalloprotease ZMPSTE24 is important for human health and longevity. ZMPSTE24 performs a key proteolytic step in maturation of prelamin A, the farnesylated precursor of the nuclear scaffold protein lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that prevent cleavage cause the premature aging disease Hutchinson–Gilford progeria syndrome (HGPS) and related progeroid disorders. ZMPSTE24 has a novel structure, with seven transmembrane spans that form a large water-filled membrane chamber whose catalytic site faces the chamber interior. Prelamin A is the only known mammalian substrate for ZMPSTE24; however, the basis of this specificity remains unclear. To define the sequence requirements for ZMPSTE24 cleavage, we mutagenized the eight residues flanking the prelamin A scissile bond (TRSY↓LLGN) to all other 19 amino acids, creating a library of 152 variants. We also replaced these eight residues with sequences derived from putative ZMPSTE24 cleavage sites from amphibian, bird, and fish prelamin A. Cleavage of prelamin A variants was assessed using an in vivo yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. We found that residues on the C-terminal side of the cleavage site are most sensitive to changes. Consistent with other zinc metalloproteases, including thermolysin, ZMPSTE24 preferred hydrophobic residues at the P1’ position (Leu647), but in addition, showed a similar, albeit muted, pattern at P2’. Our findings begin to define a consensus sequence for ZMPSTE24 that helps to clarify how this physiologically important protease functions and may ultimately lead to identifying additional substrates.
Highlights
We previously demonstrated that expression of human ZMPSTE24 together with the C terminus of human prelamin A in a yeast strain deleted for endogenous STE24 provides an ideal model system for studying lamin A maturation in vivo [28, 59]
The initial steps of lamin A biogenesis are mediated by the yeast CAAX processing machinery, resulting in a farnesylated, carboxymethylated prelamin A substrate (Fig. 1A)
Western blotting shows that cleavage of the prelamin A substrate is dependent on the presence of ZMPSTE24, and at steady state, approximately 80–90% of the substrate is in the mature lamin A form (Fig. 1C)
Summary
Apart from the known scissile bond cleaved by ZMPSTE24 in prelamin A (between Y646 and L647) [14, 15, 56], and by Ste24 in yeast a-factor (between T7 and A8) [40, 42], there is limited information about the rules governing substrate cleavage. We used a variation of our previously characterized humanized yeast assay [28, 59] to define the rules of allowable substitutions at each of the eight residues flanking the prelamin A cleavage site in order to gain information about how the enzyme works, to assess potential prelamin A disease alleles, as well as to eventually help to identify other putative ZMPSTE24/Ste24 substrates.
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