TY - JOUR
T1 - Site specificity determinants for prelamin A cleavage by the zinc metalloprotease ZMPSTE24
AU - Babatz, Timothy D.
AU - Spear, Eric D.
AU - Xu, Wenxin
AU - Sun, Olivia L.
AU - Nie, Laiyin
AU - Carpenter, Elisabeth P.
AU - Michaelis, Susan
N1 - Publisher Copyright:
© 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.
PY - 2021/1/1
Y1 - 2021/1/1
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.RA120.015792
DO - 10.1074/jbc.RA120.015792
M3 - Article
C2 - 33293369
AN - SCOPUS:85102793840
SN - 0021-9258
VL - 296
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
M1 - 100165
ER -