Martin O. Bergo

Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect

Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24-deficient mice (Zmpste24−/−) have indicated a role for Zmpste24 in the processing of CAAX-type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24−/− mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures—akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24−/− mice. Zmpste24−/− mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24−/− mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.

Targeted Inactivation of the Isoprenylcysteine Carboxyl Methyltransferase Gene Causes Mislocalization of K-Ras in Mammalian Cells

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt−/−). Lysates fromIcmt−/− cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such asN-acetyl-S-geranylgeranyl-l-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt−/− cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt−/− cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt−/− cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.

The C-terminal Polylysine Region and Methylation of K-Ras Are Critical for the Interaction between K-Ras and Microtubules

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.

Blocking the secretion of hepatic very low density lipoproteins renders the liver more susceptible to toxin-induced injury.

Recently, we generated mice lacking microsomal triglyceride transfer protein (MTP) in the liver (Mttp Δ/Δ) and demonstrated that very low density lipoprotein secretion from hepatocytes was almost completely blocked. The blockade in lipoprotein production was accompanied by mild to moderate hepatic steatosis, but the mice appeared healthy. Although hepatic MTP deficiency appeared to be innocuous, we hypothesized that a blockade in very low density lipoprotein secretion and the accompanying steatosis might increase the sensitivity ofMttp Δ/Δ livers to additional hepatic insults. To address this issue, we compared the susceptibility ofMttp Δ/Δ mice andMttp flox/flox controls to hepatic injury fromEscherichia coli lipopolysaccharides, concanavalin A, andPseudomonas aeruginosa exotoxin A. At baseline, neither theMttp Δ/Δ nor theMttp flox/flox mice had elevated serum transaminases or histologic evidence of hepatic inflammation. After the administration of the toxins, however, theMttp Δ/Δ mice manifested higher levels of transaminases and, unlike the Mttp flox/floxmice, developed histologic evidence of hepatic inflammation. The toxic challenge induced tumor necrosis factor-α to a similar extent inMttp Δ/Δ andMttp flox/flox mice, but other parameters of injury (e.g. chemokine transcript levels and lipid peroxides) were disproportionately increased in theMttp Δ/Δ mice. Our results suggest that blocking lipoprotein secretion in the liver may increase the susceptibility of the liver to certain toxic challenges.

N-Myristoyltransferase 1 Is Essential in Early Mouse Development

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a β-galactosidase marker gene. β-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/–) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/– mice yielded no viable homozygotes (Nmt1–/–), and heterozygotes were born at a less than predicted frequency. Nmt1–/– embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1–/– embryos. To explore this concept, we generated Nmt1–/– embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1–/– ES cells, but the total NMT activity levels were reduced by ∼95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1–/– ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.

Mammalian Farnesylated Protein-Converting Enzyme 1

Publisher Summary This chapter discusses the activity, specificity and structural chemistry of mammalian farnesylated protein-converting enzyme 1. Zmpste24 is the mammalian ortholog for Ste24p, an integral membrane metalloproteinase from Saccharomyces cerevisiae. Zmpste24 has also been referred to as farnesylated protein-converting enzyme 1, a-factor converting enzyme and prenyl protein-specific endoprotease 1. The identification and characterization of Zmpste24 has been linked to the characterization of yeast Ste24p. In yeast, Ste24p is required for the maturation of the mating pheromone a-factor. The production of mature a-factor from its precursor peptide requires a series of processing steps which include farnesylation of a C-terminal cysteine, endoproteolytic release of the C-terminal three amino acids, carboxyl methylation of the farnesylcysteine and removal of the N-terminal 21 amino acids of the protein. Human ZMPSTE24 is expressed in a wide variety of tissues, including skin, colon, intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, muscle, liver, lung, placenta, brain and heart. The mouse transcript is also expressed in all tissues, with the highest expression levels in liver and kidney. Immunofluorescence studies in HEK-293 cells transiently transformed with a hemagglutinin-tagged human ZMPSTE24 construct revealed an endoplasmic reticulum-like staining pattern similar to that of calnexin.