Requirements for efficient proteolytic cleavage of prelamin A by ZMPSTE24

Abstract

Background: The 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 findings: Here 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/significance: Our 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.

Figure 1. Assessing ZMPSTE24-mediated cleavage of the tail from prelamin A in cells blocked at different steps of CAAX processing.

A) Schematic of the post-translational processing pathway for lamin A. Lamin A is synthesized as a precursor, prelamin A, and undergoes CAAX processing (farnesylation, AAX'ing, and carboxylmethylation; steps 1–3), followed by ZMPSTE24-mediated cleavage of the farnesylated, carboxylmethylated tail (step 4). B) An NIH 3T3 stable cell line expressing GFP-lamin A was treated with and without farnesyltransferase inhibitor (FTI). Lysates from these cells were compared to lysates prepared from NIH 3T3 stable cell line expressing GFP-lamin A with a mutation of the CAAX cysteine to serine (C661S) on an 8% SDS-PAGE gel (step 1 block). Lysates prepared from WT MEFs and MEFs containing knockouts of the indicated genes (Rce1, Icmt, and Zmpste24; S. Young, UCLA) were run on 8% gels and probed with antibodies to detect endogenous lamin A. The forms of lamin A are indicated: M (mature), PLA (prelamin A), and PLA(NF) (prelamin A, non-farnesylated).

Figure 2. The last 41 amino acids of prelamin A are sufficient for efficient cleavage of prelamin A by ZMPSTE24.

A) Schematic representation of full length prelamin A, and the deletion set analyzed here. Each deletion consists of GFP fused to the NLS of prelamin A (residues 416–423) and sequentially shorter C-terminal sequences (the cleaved tail encompasses the 15 residues 647–661; prior AAX'ing removes 662–664). B) Analysis of ZMPSTE24-mediated cleavage of the prelamin A deletion constructs. The deletion constructs were transiently transfected into HEK293A cells, and lysates were prepared 24 hours post-transfection. Proteins were resolved on 12% SDS-PAGE gels. For each deletion, mature (MAT) and uncleavable (UC; L647R) versions were included in the analysis to serve as markers for the migration of cleaved and uncleaved versions of each deletion, respectively.

Figure 3. Replacing the nine C-terminal tail residues immediately preceding the CAAX motif does not significantly inhibit cleavage by ZMPSTE24.

Within the GFP-51mer, nine amino acids immediately upstream of the CAAX motif (underlined) were replaced with a nine amino acid long HA epitope and transiently transfected into HEK293A cells. A construct including an uncleavable (UC; L647R) version of the HA swap construct was generated and included as a marker for migration for cleavage inhibition, in addition to FTI treatment. The NF (non-farnesylated) as well as the uncleaved and the cleaved forms are indicated.

Figure 4. Increasing the spacing between the CAAX motif and the tail cleavage site affects prelamin A cleavage by ZMPSTE24.

In order to increase the linear distance between the tail cleavage site and the CAAX cysteine, the nine amino acid sequence upstream of the CAAX motif (SPRTQSPQN) was duplicated within the GFP-51mer. A version of this construct bearing the uncleavable (UC; L647R) mutation was also generated as a marker for migration of uncleaved species. Constructs were transiently transfected into HEK293A cells and analyzed as in Figure 2.

Figure 5. Mutagenesis of the residues surrounding the tail cleavage site reveals a contextual requirement and evidence for a consensus motif for the cleavage of prelamin A by ZMPSTE24.

The indicated residues upstream and downstream of the cleavage site (between Y646 and L647) within the GFP-51mer were mutated to either alanine (A) or arginine (R) and assayed for cleavage by ZMPSTE24 by transient transfection into HEK293A cells. The R644C mutation was also included based on its reported presence in atypical progeria and other diseases as discussed in the text. Amino acid changes that do and do not impact cleavage are indicated. Gel analysis of mutants that impair cleavage is shown.

Figure 6. Summary of the critical features found in this study for ZMPSTE24-mediated cleavage of the prelamin A tail.

Within the terminal 41 amino acids (residues 624–664) shown here to be needed for efficient cleavage (Figure 2), three new residues in addition to L647 (L647R = UC) were identified in this study that contribute to an apparent sequence requirement in the region of the cleavage site (R644, L648, and N650) (Figure 5). The identity of the nine amino acids (SPRTQSPQN) upstream of the CAAX motif appears not to be critical for cleavage (Figure 3). Placing the cleavage site at a further distance from the CAAX motif was found to inhibit cleavage by ZMPSTE24 (Figure 4), suggesting that a critical length from the farnesyl-cysteine may be tolerated by ZMPSTE24. Farnesylation is critical for further processing and cleavage by ZMPSTE24, while carboxylmethylation only modestly contributes to cleavage efficiency (Figure 1).