ZMPSTE24 missense mutations that cause progeroid diseases decrease prelamin A cleavage activity and/or protein stability

Abstract

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor (prelamin A) undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS), and the related progeroid disorders mandibuloacral dysplasia type B (MAD-B) and restrictive dermopathy (RD). Here, we report the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All mutations show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this humanized yeast system can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.

Keywords: Lamin A processing; Progeria disease; Saccharomyces cerevisiae; Ubiquitin-proteasome system.

Fig. 1.

The prelamin A biogenesis pathway. The four steps of prelamin A post-translational processing shown here are described in the text. The lipid farnesyl (a 15-carbon-long isoprenoid lipid) and the carboxyl methyl group (O-CH₃) are indicated. The enzymes that mediate CAAX processing are shown: farnesyltransferase (FTase), the proteases ZMPSTE24 and Ras-converting enzyme (RCE1) and the isoprenylcysteine carboxylmethyl transferase (ICMT). It should be noted that although step 2 in CAAX processing can be carried out redundantly for prelamin A either by ZMPSTE24 or RCE1, step 4 of prelamin A processing is solely mediated by ZMPSTE24. When ZMPSTE24 is absent, processing is blocked at step 4 and not step 2, as RCE1 is present (Varela et al., 2008; C.A.H., E.-T.H. and S.M., unpublished data).

Fig. 2.

Prelamin A is processed to mature lamin A by human ZMPSTE24 in yeast. (A) Schematic of the humanized yeast system. The prelamin A model substrate contains amino acids 431-664 from the C terminus of human LMNA (referred to as LMNA_CT) fused to a 10His-3myc epitope tag. The substrate is expressed from the PRC1 promoter (P_PRC1) and is chromosomally integrated into a ste24Δ strain background, resulting in strain SM6158. Full-length human ZMPSTE24 with an N-terminal 10His-3HA epitope tag is expressed from the PGK1 promoter (P_PGK1) on a CEN URA3 plasmid (pSM2677; Barrowman et al., 2012b). (B) Lysates from ste24Δ strains expressing wild-type (WT) prelamin A (lanes 1 and 3), uncleavable prelamin A (lane 2, L647R) or mature lamin A (lane 4, MAT) and human ZMPSTE24 (lanes 2, 3 and 4) or vector alone (lane 1) were analyzed for prelamin A processing by SDS-PAGE and western blotting. Prelamin A (preLA) and mature lamin A (mLA) were detected with anti-myc antibodies; ZMPSTE24 was detected with anti-HA antibodies. Strains in lanes 1-4 are SM6158/pRS316, SM6177/pSM2677, SM6158/pSM2677 and SM6178/pSM2677, respectively.

Fig. 3.

Cleavage of prelamin A in yeast, as in mammalian cells, requires farnesylation of the CAAX motif and is diminished when carboxyl methylation is lacking. (A) Prelamin A processing is blocked when farnesylation is absent. Prelamin A processing in ste24Δ strains expressing the indicated LMNA_CT (wild type or C661S) and ZMPSTE24 (wild type and H335A) alleles was analyzed by SDS-PAGE and western blotting, as in Fig. 2. (B) The efficiency of prelamin A processing is reduced in a ste14Δ mutant strain. Prelamin A processing in strains expressing the indicated ZMPSTE24 alleles was analyzed. Strains are ste24Δ only (lanes 1 and 2) or a ste24Δste14Δ double mutant (lane 3). Strains in lanes 1-3 are SM6158/pSM2677, SM6158/pSM2673 and SM6187/pSM2677, respectively. preLA, prelamin A; mLA, mature lamin A; WT, wild type.

Fig. 4.

ZMPSTE24 disease mutants show diminished prelamin A cleavage and for some alleles dramatically decreased protein levels. Lysates from strain SM6158 (ste24Δ myc-LMNA_CT) transformed with plasmids expressing the indicated HA-ZMPSTE24 alleles or vector only were analyzed by SDS-PAGE and western blotting. (A) Average (mean) percentage of prelamin A cleavage for each ZMPSTE24 variant was calculated from four independent experiments, with s.d. shown as error bars. For comparison, wild-type ZMPSTE24 cleavage was set to 100%; P<0.005 for all mutants compared with wild type. ^§Catalytically dead mutants. (B) ZMPSTE24 proteins were detected with α-HA antibodies and the ZMPSTE24 levels were normalized to the loading control Sec61, with wild-type ZMPSTE24 set to 100%. The average (mean) and s.d. are shown for the same four experiments as in (A). P<0.05 for all mutants compared with wild type, except N265S and Y399C, which were not considered to be significantly different from wild type. We note that the multiple banding pattern seen here for ZMPSTE24 occurs not only in our yeast system, but also for endogenous ZMPSTE24 in mammalian cells (Pendas et al., 2002) and when ZMPSTE24 is expressed in other heterologous expression systems (Clark et al., 2017; E.P.C. and L.N., unpublished observations). The different mobilities might simply reflect distinct SDS-binding patterns for this protein or an, as yet, unknown modification. preLA, prelamin A; mLA, mature lamin A; WT, wild type.

Fig. 5.

Blocking the ubiquitin/proteasome-dependent degradation of mutant ZMPSTE24 proteins enhances prelamin A cleavage for some ZMPSTE24 disease variants. (A,B) Examining the effects of blocking ubiquitylation. Strains SM6158 (ste24Δ myc-LMNA_CT) or SM6184 (ste24Δdoa10Δ myc-LMNA_CT) expressing the indicated ZMPSTE24 variants were analyzed by SDS-PAGE and western blotting using (A) α-HA and (B) α-myc antibodies. ZMPSTE24 protein levels were normalized against the loading control Sec61 (not shown). The doa10Δ mutant strain is designated as ‘Δ’ and the wild-type DOA10 strain as ‘+’. Data shown is mean±s.d. for four independent experiments. P<0.05 for all comparisons between + and Δ for ZMPSTE24 protein levels; P<0.005 for P248L and W340R comparing activity (B). (C,D) Examining the effects of proteasome inhibition. To test the effect of proteasome inhibition on ZMPSTE24 protein levels and activity, strain SM6159 (pdr5Δste24Δ myc-LMNA_CT) expressing the indicated ZMPSTE24 variants was treated with 20 µM bortezomib (+) or DMSO vehicle (−), as described in the Materials and Methods section. (C) HA-ZMPSTE24 proteins detected with anti-HA antibodies were normalized to the loading control Sec61 (not shown) and levels were expressed as the fold change between treated (+) and untreated (−) samples. A representative gel is shown, with the mean±s.d. for three independent experiments shown above; P<0.05 for all comparisons of mutant ZMPSTE24 proteins. (D) Prelamin A cleavage from the same samples shown in (C) was assessed with anti-myc antibodies; P<0.05 for P248L, W340R and L462R compared with wild type. preLA, prelamin A; mLA, mature lamin A; WT, wild type.

Fig. 6.

Comparison of ZMPSTE24 mutants for clearance of the clogger protein. Strain SM6117 (ste24Δ P_GAL-Clogger-HA) transformed with the indicated ZMPSTE24 plasmids was induced to express the clogger protein by addition of galactose, as described in the Materials and Methods section. Lysates were resolved by SDS-PAGE and probed with α-HA antibodies to detect the clogger. The inserted and clogged or cytoplasmic species are indicated, with the percentage clogged/cytoplasmic indicated on the y-axis. Data shown are the mean±s.e.m. for five individual experiments; P<0.05 for vector, P248L, H335A and H339A compared with wild type (WT). ^§Catalytically dead mutants.

Fig. 7.

Location of missense disease alleles in the ZMPSTE24 structure. Positions of the missense disease alleles listed in Table 1 are indicated on a ribbon diagram of the ZMPSTE24 structure (PDB entry 2YPT; Quigley et al., 2013). The yellow ball represents the zinc ion at the catalytic site. Dashed lines indicate the approximate delineation of the lipid bilayer; the ER lumen and nucleoplasm/cytoplasm (NP/CP) are indicated.