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Defining substrate requirements for cleavage of farnesylated prelamin A by the integral membrane zinc metalloprotease ZMPSTE24.

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.

SUBMITTER: Wood KM

PROVIDER: S-EPMC7735620 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Defining substrate requirements for cleavage of farnesylated prelamin A by the integral membrane zinc metalloprotease ZMPSTE24.

Wood Kaitlin M KM Spear Eric D ED Mossberg Otto W OW Odinammadu Kamsi O KO Xu Wenxin W Michaelis Susan S

PloS one 20201214 12

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 unusua ...[more]

PMID: 33315887

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Project description:BackgroundThe proteolytic maturation of the nuclear protein lamin A by the zinc metalloprotease ZMPSTE24 is critical for human health. The lamin A precursor, prelamin A, undergoes a multi-step maturation process that includes CAAX processing (farnesylation, proteolysis and carboxylmethylation of the C-terminal CAAX motif), followed by ZMPSTE24-mediated cleavage of the last 15 amino acids, including the modified C-terminus. Failure to cleave the prelamin A "tail", due to mutations in either prelamin A or ZMPSTE24, results in a permanently prenylated form of prelamin A that underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) and related progeroid disorders.Methodology/principal findingsHere we have investigated the features of the prelamin A substrate that are required for efficient cleavage by ZMPSTE24. We find that the C-terminal 41 amino acids of prelamin A contain sufficient context to allow cleavage of the tail by ZMPSTE24. We have identified several mutations in amino acids immediately surrounding the cleavage site (between Y646 and L647) that interfere with efficient cleavage of the prelamin A tail; these mutations include R644C, L648A and N650A, in addition to the previously reported L647R. Our data suggests that 9 of the 15 residues within the cleaved tail that lie immediately upstream of the CAAX motif are not critical for ZMPSTE24-mediated cleavage, as they can be replaced by the 9 amino acid HA epitope. However, duplication of the same 9 amino acids (to increase the distance between the prenyl group and the cleavage site) impairs the ability of ZMPSTE24 to cleave prelamin A.Conclusions/significanceOur data reveals amino acid preferences flanking the ZMPSTE24 cleavage site of prelamin A and suggests that spacing from the farnesyl-cysteine to the cleavage site is important for optimal ZMPSTE24 cleavage. These studies begin to elucidate the substrate requirements of an enzyme activity critical to human health and longevity.

Dataset Information

Defining substrate requirements for cleavage of farnesylated prelamin A by the integral membrane zinc metalloprotease ZMPSTE24.

Publications

Defining substrate requirements for cleavage of farnesylated prelamin A by the integral membrane zinc metalloprotease ZMPSTE24.

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