The farnesyl transferase inhibitor (FTI) lonafarnib improves nuclear morphology in ZMPSTE24-deficient fibroblasts from patients with the progeroid disorder MAD-B.

Several related progeroid disorders are caused by defective post-translational processing of prelamin A, the precursor of the nuclear scaffold protein lamin A, encoded by LMNA. Prelamin A undergoes farnesylation and additional modifications at its C-terminus. Subsequently, the farnesylated C-terminal segment is cleaved off by the zinc metalloprotease ZMPSTE24. The premature aging disorder Hutchinson Gilford progeria syndrome (HGPS) and a related progeroid disease, mandibuloacral dysplasia (MAD-B), are caused by mutations in LMNA and ZMPSTE24, respectively, that result in failure to process the lamin A precursor and accumulate permanently farnesylated forms of prelamin A. The farnesyl transferase inhibitor (FTI) lonafarnib is known to correct the aberrant nuclear morphology of HGPS patient cells and improves lifespan in children with HGPS. Importantly, and in contrast to a previous report, we show here that FTI treatment also improves the aberrant nuclear phenotypes in MAD-B patient cells with mutations in ZMPSTE24 (P248L or L425P). As expected, lonafarnib does not correct nuclear defects for cells with lamin A processing-proficient mutations. We also examine prelamin A processing in fibroblasts from two individuals with a prevalent laminopathy mutation LMNA-R644C. Despite the proximity of residue R644 to the prelamin A cleavage site, neither R644C patient cell line shows a prelamin A processing defect, and both have normal nuclear morphology. This work clarifies the prelamin A processing status and role of FTIs in a variety of laminopathy patient cells and supports the FDA-approved indication for the FTI Zokinvy for patients with processing-deficient progeroid laminopathies, but not for patients with processing-proficient laminopathies.

Abolishing the prelamin A ZMPSTE24 cleavage site leads to progeroid phenotypes with near-normal longevity in mice.

Prelamin A is a farnesylated precursor of lamin A, a nuclear lamina protein. Accumulation of the farnesylated prelamin A variant progerin, with an internal deletion including its processing site, causes Hutchinson-Gilford progeria syndrome. Loss-of-function mutations in ZMPSTE24, which encodes the prelamin A processing enzyme, lead to accumulation of full-length farnesylated prelamin A and cause related progeroid disorders. Some data suggest that prelamin A also accumulates with physiological aging. Zmpste24-/- mice die young, at ∼20 wk. Because ZMPSTE24 has functions in addition to prelamin A processing, we generated a mouse model to examine effects solely due to the presence of permanently farnesylated prelamin A. These mice have an L648R amino acid substitution in prelamin A that blocks ZMPSTE24-catalyzed processing to lamin A. The LmnaL648R/L648R mice express only prelamin and no mature protein. Notably, nearly all survive to 65 to 70 wk, with ∼40% of male and 75% of female LmnaL648R/L648R mice having near-normal lifespans of 90 wk (almost 2 y). Starting at ∼10 wk of age, LmnaL648R/L648R mice of both sexes have lower body masses than controls. By ∼20 to 30 wk of age, they exhibit detectable cranial, mandibular, and dental defects similar to those observed in Zmpste24-/- mice and have decreased vertebral bone density compared to age- and sex-matched controls. Cultured embryonic fibroblasts from LmnaL648R/L648R mice have aberrant nuclear morphology that is reversible by treatment with a protein farnesyltransferase inhibitor. These novel mice provide a model to study the effects of farnesylated prelamin A during physiological aging.

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

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.