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Thematic Review Series: Lipid Posttranslational Modifications. Prelamin A, Zmpste24, misshapen cell nuclei, and progeria—new evidence suggesting that protein farnesylation could be important for disease pathogenesis
Prelamin A undergoes multistep processing to yield lamin A, a structural protein of the nuclear lamina. Prelamin A terminates with a CAAX motif, which triggers farnesylation of a C-terminal cysteine (the C of the CAAX motif), endoproteolytic release of the last three amino acids (the AAX), and methylation of the newly exposed farnesylcysteine residue. In addition, prelamin A is cleaved a second time, releasing 15 more residues from the C terminus (including the farnesylcysteine methyl ester), generating mature lamin A. This second cleavage step is carried out by an endoplasmic reticulum membrane protease, ZMPSTE24. Interest in the posttranslational processing of prelamin A has increased with the recognition that certain progeroid syndromes can be caused by mutations that lead to an accumulation of farnesyl-prelamin A. Recently, we showed that a key cellular phenotype of these progeroid disorders, misshapen cell nuclei, can be ameliorated by inhibitors of protein farnesylation, suggesting a potential strategy for treating these diseases.
In this article, we review the posttranslational processing of prelamin A, describe several mouse models for progeroid syndromes, explain the mutations underlying several human progeroid syndromes, and summarize recent data showing that misshapen nuclei can be ameliorated by treating cells with protein farnesyltransferase inhibitors.
). This lipid modification, generally called protein prenylation, is important for facilitating the binding of proteins to membrane surfaces. Two broad classes of prenylated proteins exist. The Rab GTPases, important for trafficking of proteins to specific membrane compartments within cells, are geranylgeranylated at a pair of C-terminal cysteines (
). A more abundant group of proteins terminate with a C-terminal CAAX motif and are either farnesylated or geranylgeranylated at a C-terminal cysteine (the C of the CAAX motif), then further modified by endoproteolytic processing and methylation of the C-terminal prenylcysteine (
). Examples of prenylated CAAX proteins include regulatory proteins such as the Ras and Rho proteins, several nuclear lamins (prelamin A, lamin B1, and lamin B2), and the yeast mating pheromone a-factor.
In this article, we focus largely on prelamin A, a farnesylated CAAX protein that undergoes multistep processing to yield mature lamin A, a structural protein of the nuclear lamina (
). In addition to the aforementioned CAAX motif modifications, prelamin A undergoes an additional endoproteolytic cleavage reaction that produces mature lamin A; this cleavage step releases 15 amino acids from the C terminus of prelamin A, including the farnesylcysteine methyl ester. Hence, mature lamin A is not farnesylated.
Interest in prelamin A and its posttranslational processing has recently increased with the recognition that certain progeroid syndromes can be caused by mutations that lead to an intracellular accumulation of farnesylated and carboxyl methylated prelamin A (
). Remarkably, at least one of the cellular phenotypes associated with these progeroid disorders, misshapen cell nuclei, can be reversed or ameliorated by inhibitors of protein farnesylation, suggesting a possible strategy for treating these disorders.
In this article, we review the posttranslational processing of prelamin A, describe a mouse model for human progeroid syndromes, explain the mutations underlying several human progeroid syndromes, and summarize recent data indicating that the misshapen nuclei in certain progeroid syndromes can be ameliorated by treating cells with protein farnesyltransferase inhibitors (FTIs).
All of the enzymes responsible for the processing of mammalian prelamin A were initially identified in Saccharomyces cerevisiae, an organism that does not express prelamin A. In yeast, these processing enzymes are essential for the biogenesis of a farnesylated mating pheromone, a-factor, from its precursor protein (
). Parallels between the posttranslational processing of the a-factor in yeast and prelamin A in mammals are both informative and intriguing. Therefore, we will begin this review by “backing up” to yeast and reviewing the biogenesis of yeast a-factor.
POSTTRANSLATIONAL MODIFICATIONS OF CAAX PROTEINS
CAAX proteins terminate with the amino acids CAAX, in which the C is a cysteine, the A residues are usually aliphatic amino acids, and the X can be one of many different residues (
). First, a 15 carbon farnesyl or a 20 carbon geranylgeranyl lipid is added to the thiol group of the cysteine by cytosolic protein prenyltransferases, either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (
). Prelamin A terminates in CSIM and therefore is farnesylated; lamin B1 and lamin B2 are also farnesylated. Next, the last three amino acids (i.e., the AAX of the CAAX motif) are removed by a prenylprotein-specific endoprotease in the endoplasmic reticulum (ER) membrane (
). After endoproteolysis, the newly exposed isoprenylcysteine is methylated by an ER membrane prenylprotein-specific methyltransferase, isoprenylcysteine carboxyl methyltransferase (Icmt, designated Ste14p in yeast) (
). For example, in yeast, Ras proteins that do not undergo endoproteolysis or carboxyl methylation are not properly localized to the plasma membrane (Fig. 2).
PROCESSING OF MATING PHEROMONES IN S. CEREVISIAE
The discovery of protein prenylation and carboxyl methylation arose from the structural analysis of the mating pheromones secreted by several relatively obscure heterobasidiomycetous jelly fungi (Tremella mesenterica, Tremella brasiliensis, and Rhodosporidium torluloides) (
). All of the subsequent breakthroughs in understanding the CAAX processing machinery were made through the analysis of mating-defective mutants in S. cerevisiae. The mating process in S. cerevisiae (Fig. 3)involves two haploid cell types, MATa and MATα, which secrete the diffusible signaling molecules, a-factor and α-factor, respectively (
). The interaction of these mating pheromones with their cognate receptors leads to the formation of diploid yeast (Fig. 3). Despite the similarity in the function of a-factor and α-factor, the two pheromones are structurally and biochemically distinct. α-Factor is a nonlipidated peptide that is secreted by the classical secretory pathway, whereas a-factor is farnesylated and carboxyl methylated and is exported from yeast by a nonclassical secretion mechanism (
) have provided a detailed view of the a-factor biogenesis pathway (Fig. 4). Despite the small size of a-factor (the precursor and mature forms are 36 and 12 amino acids, respectively), the a-factor biogenesis pathway is complex, comprising three major events: C-terminal processing of the CAAX motif of the a-factor precursor (Fig. 4, steps 1–3), proteolytic removal of the N-terminal extension (steps 4 and 5), and secretion of mature bioactive a-factor (step 6) via an ATP binding cassette transporter, Ste6p.
IDENTIFICATION OF ENZYMES RESPONSIBLE FOR THE C-TERMINAL PROCESSING OF a-FACTOR
A major breakthrough in the CAAX processing field was the discovery of the RAM1 gene, which was identified in independent mutant screens as a suppressor of hyperactive Ras (supH) and as a mutant that was completely defective in mating and in the production of a-factor (ste16) (
). This commonality was underscored by the presence of a CAAX motif in both proteins. Ultimately, both Ras and a-factor were shown to be prenylated and methylated, and RAM1, along with a second gene, RAM2, was shown to encode different subunits of a heterodimeric enzyme, protein farnesyltransferase (
Ras and a-factor also require the same gene, STE14, for carboxyl methylation (Fig. 4, step 3). STE14 encodes an ICMT. This gene was initially identified by characterizing a sterile strain of yeast, ste14Δ, that lacked the capacity to produce a functional a-factor (
), demonstrated that Ste14p is indeed an ICMT and that it is the sole enzyme for methylating CAAX proteins in yeast.
Interestingly, the genes encoding CAAX endoprotease enzymes (Fig. 4, step 2) were never identified in standard genetic screens for sterile yeast defective in a-factor production. In hindsight, the failure to identify the endoproteases by simple genetic screens was entirely predictable, as we now know that two functionally redundant CAAX endoproteases exist in yeast, Ste24p and Rce1p, and both are capable of participating in the cleavage of the AAX from a-factor (
). Thus, a mutation in a single gene could not be expected to yield a sterile phenotype. The key to discovering RCE1 and AFC1 (another name for STE24) rested on experiments performed by Boyartchuk, Ashby, and Rine (
) with an a-factor substrate that could be cleaved by Ste24p but not by Rce1p. They produced yeast expressing a mutant form of a-factor terminating in CAMQ rather than with the wild-type CVIA motif and demonstrated that the yeast remained capable of producing mature a-factor. The mutant yeast expressing the CAMQ version of a-factor were then mutagenized, and an autocrine arrest selection strategy was used to isolate sterile mutants (
). This approach resulted in the identification of a sterile yeast mutant, and the responsible gene, STE24, was cloned by complementation. STE24 encodes a 453 amino acid protein, Ste24p, with multiple predicted transmembrane helices as well as an HEXXH (H, His; E, Glu) motif characteristic of a group of zinc-dependent metalloproteases (
). Mutating either of the conserved histidines in the HEXXH domain blocked the ability of Ste24p to complement the mating defect of STE24-deficient yeast (ste24Δ) expressing the CAMQ form of a-factor (
STE24-deficient yeast expressing wild-type a-factor secreted reduced amounts of a-factor but were not sterile, suggesting the existence of another gene capable of processing a-factor. The residual CAAX endoprotease activity in ste24Δ yeast membranes was insensitive to the zinc chelator 1,10-o-phenanthroline, suggesting that the remaining endoprotease was not a zinc metalloprotease (
). To isolate the remaining endoprotease, ste24Δ yeast were mutagenized and screened for a mutation that blocked residual a-factor production. This approach led to the identification of RCE1, which encodes a 329 amino acid protein that is also predicted to have multiple transmembrane helices (
). Yeast lacking RCE1 (rce1Δ) manifest a modest decrease in a-factor production, and membranes from rce1Δ yeast had moderately decreased CAAX endoprotease activity in in vitro assays with farnesylated a-factor peptide substrates. The residual CAAX endoprotease activity in rce1Δ yeast was sensitive to o-phenanthroline, consistent with Ste24p being a zinc metalloprotease (
). In this assay, the production of mature a-factor can be visualized by a zone of growth inhibition (halo) in a lawn of yeast with an α-mating type. The single mutants (ste24Δ and rce1Δ) have modestly reduced a-factor production and therefore yield slightly smaller halos, whereas the double mutant (ste24Δrce1Δ) fails to carry out CAAX endoproteolysis, resulting in the complete inability to produce mature a-factor and the complete absence of a halo (Fig. 5).
Ste24p and Rce1p have different abilities to cleave a-factor mutants with different CAAX motifs. An a-factor mutant with the CAMQ sequence motif can only be cleaved by Ste24p, whereas other a-factor mutants, such as one terminating in CTLM, are cleaved solely by Rce1p (Fig. 5) (
). Notably, when the CAAX motif for mammalian prelamin A, CSIM, is tested in the a-factor assay, it is cleaved mainly by Rce1p (Fig. 5). The sequence specificities of Ste24p and Rce1p in the context of a-factor have been examined by Rine and colleagues (
). However, a complete picture of the specificities of these two enzymes has not yet emerged, either for a-factor or for any other CAAX protein, and it is not clear whether any additional structural features, aside from the sequence of the CAAX motif, influence enzyme specificity.
A SECOND ROLE FOR STE24 IN THE BIOGENESIS OF a-FACTOR
A surprising discovery, and one quite relevant to this review, was our finding that one of the a-factor-processing enzymes, Ste24p, actually plays dual roles in a-factor maturation. As shown in Fig. 4, Ste24p mediates both the C-terminal CAAX endoproteolysis (step 2) and the first N-terminal cleavage (step 4) (
). In addition to the CAAX motif modifications on the C terminus of a-factor (farnesylation, endoproteolytic release of the AAX, and methylation), two additional endoprotease steps within the N-terminal portion of the molecule are required to generate mature a-factor. The first of these processing steps, which converts the P1 intermediate to P2, involves the endoproteolytic release of the first seven amino acids of the protein (
). In axl1Δ yeast, the P2 intermediate of a-factor accumulates. A search of the S. cerevisiae sequence database revealed a gene similar to AXL1, designated STE23, and this gene product can also participate in the second N-terminal proteolysis step. Yeast lacking AXL1 have a reduced capacity to produce a-factor, whereas mutants lacking both AXL1 and STE23 (axl1Δste23Δ) are completely sterile (
). The apparent conflict posed by reports indicating different roles in a-factor biogenesis was quickly resolved by additional genetic and biochemical studies, which showed that Ste24p actually has dual roles in a-factor processing. Along with Rce1p, Ste24p is capable of releasing the AAX from a-factor; Ste24p also clearly cleaves the first seven amino acids from the protein (
). Whether farnesylation is required for substrate recognition and specificity, or simply facilitates the recruitment of the a-factor intermediate to membranes containing Ste24p, has not yet been determined. However, the N-terminal cleavage reaction mediated by Ste24p does not require prior carboxyl methylation, because N-terminal cleavage occurs normally in yeast lacking STE14 (
). The precise sequences and structures within the a-factor required for the two N-terminal cleavage reactions have not yet been determined, but with the experimental systems that have been developed, it should be possible to obtain that type of information.
Recently, a histidine-tagged version of Ste24p was detergent-solubilized and purified to homogeneity from yeast and shown to be the sole protein needed to mediate both the C- and N-terminal a-factor cleavage reactions (
). Given that fact that Ste24p is a membrane protein, the retention of enzymatic activities after purification was remarkable. Both of the activities were blocked by o-phenanthroline and reactivated by zinc (
). In each case, the mammalian gene complements the yeast mutant, as judged by a conventional a-factor halo assay and yeast mating tests. In this regard, our studies of human ZMPSTE24 deserve special mention because we were able to demonstrate its ability to complement both the C- and N-terminal endoproteolytic processing reactions of a-factor (
). This point is illustrated in Fig. 7, in which complementation of ste24Δrce1Δ yeast with human ZMPSTE24 is shown by three separate assays (metabolic labeling, halo, and mating). Mouse Zmpste24 also fully complements the a-factor production defect in ste24Δrce1Δ yeast (see Fig. 13A below) (
). The complementation of the mutant yeast was fascinating, particularly because signaling molecules resembling a-factor have never been identified in mammals. Thus, mammalian Zmpste24 retains the capacity to process a yeast substrate after nearly 800 million years of evolution. The ability of the mammalian enzyme to process yeast a-factor has facilitated the functional analysis of mutant forms of ZMPSTE24 in humans (
). Currently, the STE24 designation is used exclusively by bioinformatics databases and the scientific community. Different names have also been applied to the mammalian gene Zmpste24. We cloned the first full-length mammalian version of Zmpste24 and initially designated it HsSTE24 (for Homo sapiens STE24) (
). The official name for the human and mouse genes has since been changed to Zmpste24 (to reflect the fact that this enzyme is an ortholog of Ste24p and is a zinc metalloprotease). One group came up with entirely new names for Ste24p/Zmpste24 and Rce1, calling them FACE-1 and FACE-2, for farnesylated protein-converting enzymes 1 and 2, respectively (
). Those names were less than ideal choices, in our opinion, because it was well established at the time that at least one of the endoprotease activities, now recognized to be Rce1, processes both farnesylated and geranylgeranylated proteins, not just farnesylated proteins.
PHYSIOLOGIC IMPORTANCE OF RCE1 AND ICMT IN MAMMALS AND MICE
Deciphering the physiologic importance of CAAX processing in mammals has been greatly aided by the development of Rce1 and Icmt knockout mice (
). Very rarely, Rce1−/− mice were born alive, but they were runted and lived for only a few weeks. Rce1 deficiency had no significant effect on hematopoiesis, and the major organ systems of Rce1−/− embryos were histologically normal late in gestation (
). Because endoproteolysis is a prerequisite for carboxyl methylation, we predicted that Ras proteins from Rce1−/− fibroblasts would not be methylated. Indeed, this was the case. When we labeled Rce1+/+ and Rce1−/− fibroblasts with l-[methyl-3H]methionine, the Ras proteins from Rce1+/+ fibroblasts contained a 3H-labeled farnesylcysteine methyl ester, whereas the Ras proteins from Rce1−/− fibroblasts did not (
) generated a monoclonal antibody specific for the C terminus of lamin B1, but the antibody did not bind unless the protein had undergone both farnesylation and endoproteolytic processing. Of note, the monoclonal antibody did not detect lamin B1 in Rce1−/− fibroblasts, indicating that Rce1 is solely responsible for the processing of that protein. Because Rce1 has so many protein substrates, it was not surprising that biochemical studies revealed a substantial accumulation of uncleaved and unmethylated CAAX protein substrates in Rce1−/− fibroblasts (
In Rce1+/+ fibroblasts, the Ras proteins are located along the inner surface of the plasma membrane, as judged by cell transfection experiments with green fluorescent protein (GFP)-Ras fusion constructs (
), similar to what is seen in the yeast mutant (Fig. 2), underscoring the importance of endoproteolysis (and subsequent methylation) for proper membrane targeting.
The Rce1 knockout had functionally important consequences. Rce1−/− fibroblasts grew slightly more slowly than Rce1+/+ fibroblasts and were less susceptible to oncogenic transformation by activated forms of Ras (
). Also, we generated mice homozygous for a conditional Rce1 allele and then showed that Cre-mediated inactivation of Rce1 in fibroblasts reduces transformation by mutationally activated forms of Ras (
) and to our surprise found that this gene defect caused a more severe phenotype than Rce1 deficiency. Icmt−/− embryos were viable until E10.5, but they all died by E12.5. One study contended that Icmt might be particularly important for the development of the liver (
). Icmt deficiency led to a substantial accumulation of unmethylated Icmt protein substrates within cells (i.e., proteins susceptible to methylation in the presence of recombinant Icmt and the methyl donor S-adenosylmethionine) (
) of geranylgeranylated Rab proteins. Primary Icmt−/− fibroblasts clearly grow more slowly than wild-type fibroblasts.
The Ras proteins in Icmt−/− fibroblasts were not methylated, resulting in a very subtle retardation in electrophoretic mobility on SDS-polyacrylamide gels. Also, the Ras proteins were mislocalized in Icmt−/− fibroblasts, as judged by transfection experiments with GFP–Ras fusion constructs (Fig. 8)(
We produced mice that are homozygous for a conditional Icmt allele (Icmtfl/fl). Cre-mediated inactivation of Icmt mislocalized Ras proteins within cells, changed the steady-state concentration of both Rho and Ras proteins, and reduced the ability of mutationally activated Ras proteins to transform cultured fibroblasts (
) have shown that the endoprotease and methyltransferase steps are also important for the proper targeting of some CAAX proteins in cells. We believe that these findings suggest a valuable lesson: whenever CAAX proteins are implicated in human disease, it is important to consider whether the disease might conceivably be treated by “attacking” the posttranslational modifications, thereby interfering with the localization and function of these proteins within cells.
NUCLEAR LAMINS: ABUNDANT MAMMALIAN CAAX PROTEINS
Lamin A, lamin C, lamin B1, and lamin B2 are intermediate filament proteins and are among the most abundant CAAX proteins in mammalian cells (
). These proteins are key structural components of the nuclear lamina, a filamentous meshwork that lies beneath the inner nuclear membrane. Each of the lamin proteins contains an N-terminal globular domain, a central helical rod domain, and a C-terminal globular domain (
). The lamin monomers dimerize to form parallel coiled-coil homodimers, which then associate head-to-tail to form strings and ultimately higher order 10 nm thick filaments that form the meshwork. It is unclear whether the different lamins form heterodimers. The nuclear lamins interact with a variety of integral membrane proteins of the inner nuclear membrane and also with transcription factors, as well as with heterochromatin itself (
). Thus, these proteins have many roles besides simply forming a passive scaffold for the nuclear envelope.
Prelamin A, lamin B1, and lamin B2, but not lamin C, contain CAAX motifs and undergo the usual CAAX motif modifications (isoprenylation, endoproteolysis, and methylation) (Fig. 9). In the case of prelamin A, the release of the AAX is likely a redundant function of Zmpste24 and Rce1 (
). The last 15 amino acids of the protein (including the farnesylcysteine methyl ester) are clipped off and degraded, leaving behind mature lamin A (Fig. 9). This second proteolytic processing step is carried out by Zmpste24 (
Prelamin A and lamin C are products of the same gene (Lmna) and result from alternative splicing. The lamin C transcript terminates at the end of exon 10; the prelamin A transcript requires splicing from mid-exon 10 to exon 11, and then to exon 12 (
), but we presume that continuities between the ER and the nuclear envelope mean that Zmpste24 may also be located along the inner nuclear membrane.] Noncleavable prelamin A mutants, which cannot undergo the second processing step, localize to the nuclear envelope (
). Prelamin A mutations that abrogate farnesylation (i.e., mutants in which the C of the CAAX motif is changed to another amino acid such as serine) do not prevent targeting to the nucleus, but the mutant protein remains largely in the nucleoplasm and fails to reach the nuclear envelope (
Why does nature go to the trouble of modifying the C terminus of prelamin A, given that this portion of the molecule is simply clipped off and degraded? The most obvious explanation is that each of the posttranslational modifications render the C terminus of CAAX proteins more hydrophobic, facilitating the initial targeting of the protein to the inner nuclear membrane (
). Several groups have evaluated mutant prelamin A constructs lacking the last 18 amino acids of the protein (i.e., “mature lamin A” constructs). These constructs are defective in their ability to be targeted to the nuclear envelope (
FPLD is characterized by loss of adipose tissue in the extremities after puberty, increased adipose tissue in the neck and face, increased fasting and postprandial insulin levels, acanthosis nigricans, hypertriglyceridemia, reduced insulin-mediated glucose disposal, frank diabetes mellitus after age 20, and a heightened susceptibility to coronary artery disease (
). Crystallographic studies have revealed that most FPLD mutations are confined to a small region on the surface of the C-terminal globular domain of lamin A/C, whereas mutations causing muscular dystrophy are located throughout the lamin A/C protein core and its surface (
), who investigated lamin A/C structure by nuclear magnetic resonance. FPLD is not the only laminopathy characterized by loss of adipose tissue; this phenotype is also a prominent feature of MAD and HGPS.
Several laminopathies exhibit a recessive inheritance pattern. Homozygosity for a LMNA R298C mutation causes some cases of Charcot-Marie-Tooth neuropathy type IIA (
), a motor and sensory neuropathy characterized by muscle weakness, secondary foot deformities, a slight reduction in nerve conduction velocities, with the loss of large myelinated fibers and axonal degeneration. MAD is caused by homozygosity for R527H (
). MAD is a progeroid syndrome characterized by postnatal growth retardation, lipodystrophy, insulin resistance, stiff joints, alopecia, abnormalities in the cranial sutures, osteolysis of the digits, shortening of the clavicle, micrognathia, and reduced ossification of alveolar bone.
Some but not all lamin A/C missense mutations cause abnormalities in the shape and structure of the nuclear envelope, resulting in misshapen nuclei (
). However, the mechanisms by which the lamin A/C mutations cause such a diverse collection of human diseases remain fairly mysterious. Presumably, the explanation relates to abnormalities in heterochromatin organization, the binding of nuclear factors and transcription factors (
). The nuclei from Lmna−/− fibroblasts exhibited exaggerated nuclear deformations, compared with Lmna+/+ control fibroblasts, when subjected to biaxial strain. Also, the nuclear envelope in Lmna−/− fibroblasts was unequivocally more fragile than in Lmna+/+ fibroblasts, as judged by experiments in which a 70 kDa dextran was microinjected into cell nuclei. Perhaps most intriguingly, the Lmna−/− fibroblasts exhibited significantly more cell death than Lmna+/+ fibroblasts when subjected to prolonged cyclic biaxial strain (
). Dual labeling with propidium iodide and an FITC-conjugated annexin V antibody revealed that the decrease in cell viability was caused by increased numbers of both necrotic and apoptotic cells. Lammerding et al. (
) characterized mice lacking a functional lamin B1 (Lmnb1−/−). Homozygous Lmnb1−/− mice survived embryonic development but died at birth with defects in lung and bone. Of note, Lmnb1−/− fibroblasts displayed grossly misshapen nuclei, impaired differentiation, increased polyploidy, and premature senescence (
). Lmna−/− mice have reduced subcutaneous fat, but this could be attributable to stress or reduced nutrition rather than to a bona fide lipodystrophy, as they had no apparent abnormalities in insulin, glucose, or triglyceride metabolism (
An intriguing finding was that emerin, a nuclear envelope protein that binds to lamins A/C, was mislocalized to the ER in Lmna−/− fibroblasts, indicating that lamin A or C (or both) is required for its proper targeting to the nuclear envelope (
). However, a group in France transfected both lamin A and lamin C cDNA constructs into lamin A/C-deficient fibroblasts, and they concluded that both lamin A and lamin C were capable of promoting the targeting of emerin to the nuclear envelope (
) showed that the processing of prelamin A to lamin A is completely dependent on protein prenylation. The same group has also actively investigated the enzymatic activity responsible for clipping off the last 15 amino acids from farnesyl-prelamin A (
). They found that the prelamin A endoprotease was present in crude nuclear extracts and that the enzyme was entirely specific for a prelamin A peptide containing an S-farnesylated cysteine methyl ester (
). At the time, this hypothesis seemed extremely far-fetched, given that prelamin A had no obvious structural similarities to yeast a-factor. In any case, to approach this issue experimentally, we generated Zmpste24−/− mice (
), with an accumulation of prelamin A and a complete absence of mature lamin A (Fig. 10). With an antibody specific for the C terminus of prelamin A (the segment of prelamin A that is normally cleaved off), we observed a striking accumulation of a 74 kDa prelamin A in Zmpste24−/− cells. In extracts from wild-type cells, prelamin A is virtually undetectable, for the simple reason that processing of prelamin A to mature lamin A is an extremely efficient process, rapidly converting the prelamin A to mature lamin A. Western blots of Zmpste24−/− extracts with an antibody against the N terminus of lamin A/C revealed lamin C and prelamin A (74 kDa) but absolutely no mature lamin A.
An FTI completely blocked the processing of prelamin A to mature lamin A (
). In our initial experiments with Icmt−/− fibroblasts, prelamin A processing appeared to be fully blocked. That result was consistent with the earlier biochemical studies from Sinensky and colleagues (
) indicating that the C-terminal methyl ester was essential for the endoproteolytic processing reaction. However, our recent experiments with several immortalized Icmt−/− cell lines have shown that the defect in prelamin A processing is only partial (
). We invariably observe an increase in the amount of prelamin A in Icmt−/− cell lines, as judged by Western blots with an antibody against the C terminus of prelamin A (Fig. 12). Thus, the prelamin A processing in Icmt−/− fibroblasts is clearly abnormal. However, when Western blotting is performed with an antibody against the N-terminal portion of lamin A/C, we always observe some mature lamin A. Thus, Icmt deficiency causes a partial but not a complete blockade in the processing of prelamin A to mature lamin A.
Prelamin A processing in Rce1−/− fibroblasts was normal (
), in which the CAAX motif (CAIM) is similar to that in prelamin A (CSIM). Second, the prelamin A CAAX motif is actually a better substrate for yeast Rce1p than yeast Ste24p (Fig. 5), and the specificities of the mouse Zmpste24 enzymes resemble those of yeast Ste24p (
) have presented evidence that recombinant ZMPSTE24, produced in insect cells, is capable of carrying out both the release of the AAX from a short prelamin A peptide and the release of the 15 additional amino acids. The ability of Zmpste24 to clip the AAX from prelamin A parallels the ability of Ste24p to cleave the AAX from a-factor (
). However, we suspect that Zmpste24's ability to act as a CAAX endoprotease beyond prelamin A is very restricted. We tested whether recombinant Zmpste24 would be capable of processing some or all of the CAAX protein substrates that accumulate in Rce1−/− fibroblasts (
). Whole cell extracts from Rce1+/+ and Rce1−/− fibroblasts were incubated with recombinant Icmt, S-adenosyl-l-[methyl-14C]methionine, and either recombinant mouse Rce1 or Zmpste24. We noted significantly increased carboxyl methylation when recombinant Rce1 was added to extracts from Rce1−/− fibroblasts, reflecting the processing of accumulated Rce1 substrates (
), indicating that Zmpste24 has little if any ability to process the substrates that accumulate in Rce1−/− fibroblasts.
Zmpste24−/− cells contain a striking accumulation of prelamin A. One would expect that prelamin A would be farnesylated in Zmpste24−/− cells, because farnesylation precedes the endoproteolytic cleavage step mediated by Zmpste24. Indeed, several lines of evidence suggest that it actually is farnesylated. First, the electrophoretic migration of prelamin A in human and mouse Zmpste24−/− fibroblasts is retarded when cells are grown in the presence of an FTI (
As explained in more detail below, FTIs have recently been used to improve the nuclear shape in progeroid disorders characterized by an accumulation of farnesylated prelamin A at the nuclear envelope (
). The rationale is that inhibition of farnesylation would mislocalize farnesyl-prelamin A away from the nuclear envelope, thereby attacking the presumptive cause of the misshapen nuclei. However, we suggest that this may not be the only pharmacological strategy. As noted previously, methylation appears to be critical for the membrane attachment of farnesylated proteins (