Requirements for Efficient Proteolytic Cleavage of Prelamin A by ZMPSTE24

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.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.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

Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe

Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions1-7. How local chromatin interactions govern higher-order folding of chromatin fibers and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis8 to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes9. Our analyses of wild type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form “globules”. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains9-11, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fiber compaction at centromeres and promotes prominent interarm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions

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

<p>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.</p

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

<p>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 (<i>Rce1</i>, <i>Icmt</i>, and <i>Zmpste24</i>; 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).</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032120#pone-0032120-g002" target="_blank">Figure 2</a>.</p

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

<p>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.</p

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

<p>Within the terminal 41 amino acids (residues 624–664) shown here to be needed for efficient cleavage (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032120#pone-0032120-g002" target="_blank">Figure 2</a>), 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) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032120#pone-0032120-g005" target="_blank">Figure 5</a>). The identity of the nine amino acids (SPRTQSPQN) upstream of the CAAX motif appears not to be critical for cleavage (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032120#pone-0032120-g003" target="_blank">Figure 3</a>). Placing the cleavage site at a further distance from the CAAX motif was found to inhibit cleavage by ZMPSTE24 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032120#pone-0032120-g004" target="_blank">Figure 4</a>), 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032120#pone-0032120-g001" target="_blank">Figure 1</a>).</p