Abstract
The integral membrane zinc metalloprotease ZMPSTE24 plays a key role in the proteolytic processing of farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A. Failure of this processing step results in the accumulation of permanently farnesylated forms of prelamin A which cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS), as well as related progeroid disorders, and may also play a role in physiological aging. ZMPSTE24 is an intriguing and unusual protease because its active site is located inside of a closed intramembrane chamber formed by seven transmembrane spans with side portals in the chamber permitting substrate entry. The specific features of prelamin A that make it the sole known substrate for ZMPSTE24 in mammalian cells are not well-defined. At the outset of this work it was known that farnesylation is essential for prelamin A cleavage in vivo and that the C-terminal region of prelamin A (41 amino acids) is sufficient for recognition and processing. Here we investigated additional features of prelamin A that are required for cleavage by ZMPSTE24 using a well-established humanized yeast system. We analyzed the 14-residue C-terminal region of prelamin A that lies between the ZMPSTE24 cleavage site and the farnesylated cysteine, as well 23-residue region N-terminal to the cleavage site, by generating a series of alanine substitutions, alanine additions, and deletions in prelamin A. Surprisingly, we found that there is considerable flexibility in specific requirements for the length and composition of these regions. We discuss how this flexibility can be reconciled with ZMPSTE24's selectivity for prelamin A.
Highlights
Membrane associated proteases are important in a variety of physiological and medically relevant processes through their cleavage of specific cellular substrates [1, 2]
We previously developed an in vivo humanized yeast assay that recapitulates ZMPSTE24-mediated processing of prelamin A in Saccharomyces cerevisiae [30, 51]
The yeast strain used in this assay lacks endogenous STE24, and instead expresses epitope-tagged human ZMPSTE24 integrated in the genome, and a plasmid-borne form of prelamin A that contains amino acids 431–664 encoded by the LMNA gene (Fig 1D)
Summary
Membrane associated proteases are important in a variety of physiological and medically relevant processes through their cleavage of specific cellular substrates [1, 2]. Defining the critical features for substrate recognition is key to understanding how specific proteases function and for developing therapeutic strategies for diseases in which they are involved. Our studies are focused on ZMPSTE24, an integral membrane zinc metalloprotease important for human health and longevity [3,4,5]. Substrate requirements for prelamin A cleavage by ZMPSTE24
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